CN109646678A - SUN2 albumen, its pharmaceutical applications and drug - Google Patents

Abstract

The present invention relates to SUN2 protein, its pharmaceutical use and medicine. Specifically, the present invention relates to the use of an agent that reduces the level of SUN2 protein or reduces or loses the activity of SUN2 protein involved in cytoskeletal rearrangement in the preparation of a medicament for the treatment of diseases that benefit from increased M1 macrophages, or in the preparation of The use in the M1 polarization of macrophages or the depolarization of tumor-associated macrophages, or the use in the preparation of drugs for enhancing inflammatory response or anti-tumor immunity. The present application enhances immune response and anti-tumor immunity by reducing SUN protein level and/or SUN2 protein activity, so as to achieve prevention and treatment of diseases that benefit from increased M1 macrophages.

Description

SUN2 albumen, its pharmaceutical applications and drug

Technical field

The present invention relates to field of biotechnology, and in particular to SUN2 albumen, its pharmaceutical applications and drug.

Background technique

Macrophage participates in the first line of defence of composition human antibody external world danger signal, participates in stable state and maintains, metabolism is adjusted Multiple physiology courses such as control.By microenvironment, the monocyte polarization in the circulatory system is macrophage.Macrophage-shaped State and vdiverse in function, all plays vital effect in the innate immunity and acquired immunity.Macrophage is broadly divided into Two classes, i.e., proinflammatory M1 type and anti-inflammatory M2 type.In the stimulation of GM-CSF or Th1 cell factor such as interferon and LPS Under, macrophage polarization is M1 type macrophage.M1 type macrophage can produce such as IL-6, IL-12, IL-23 and TNF The proinflammatory cytokine of α inhibits the proliferation of peripheral cell and damages adjacent tissue.M1 type macrophage is in inflammatory reaction as a result, It plays a significant role in antineoplastic immune.On the contrary, under the stimulation of M-CSF or Th2 cell factor such as IL-4 and IL-13, Macrophage polarization is M2 type macrophage.M2 inhibits inflammatory reaction and promotes wound reparation and organization healing.M1-M2 type macrophage In vivo by precision control, a variety of diseases and inflammation are related with the polarized imbalance of macrophage for the polarization of cell.

Macrophage not only plays a role in immune response and tissue damage reparation, also plays in tumour generation to pass Important role, wherein tumor-associated macrophage (TAMs) plays a significant role in inflammation and cancer conversion process.In tumour Early stage occurs, tumor-associated macrophage has the rush inflammation phenotype of M1 type macrophage, and advanced stage, tumour phase occurs in tumour Close the phenotype that macrophage conversion shows M2 type macrophage, enhancing immunosupress reaction.Generally, tumour correlation macrophage is thin Born of the same parents promote tumour growth, in most entity tumors, such as: breast cancer, oophoroma, cervical carcinoma, all exist in prostate cancer compared with More tumor-associated macrophages, and it is poor related with the transfer of related neoplasms and prognosis.

Nuclear membrane all plays in cellular physiological processes as the interruption for separating nucleus and cell rest part Important function, maintenance and Germinal Vesicle Migration including cytoskeleton hardness.Nucleus and cytoplasm pass through connection cytoskeleton With the LINC compound conductive mechanical power of cell nucleus and skeleton.The compound is by having the SUN structural domain and KASH guarded in evolution The albumen of structural domain forms.SUN albumen is II type memebrane protein, and structure is by N-terminal structural domain, transmembrane domain and conservative SUN structural domain composition.The N-terminal structural domain of SUN albumen is located in nucleus, with lamina and chromobindins phase interaction With.SUN structural domain is located in the inner cavity of nucleus bilayer nuclear membrane, and in conjunction with the KASH structural domain of KASH albumen, the two forms different Source tripolymer.SUN-KASH compound is the important component that mechanical force conduction is carried out on nuclear membrane, to cell migration and polarization All play an important role.In human body, the mutation of SUN albumen and KASH albumen with include lamin syndrome, mutual aid The diseases such as imbalance, senium praecox, congenital agyria are related.The regulation of element recent studies have shown that SUN2 may be interfered.

Summary of the invention

First aspect present invention provides a kind of reduction SUN2 protein level or SUN2 albumen is made to participate in cytoskeleton rearrangement The purposes of reduced activity or the reagent of forfeiture in the drug that the disease that M1 type macrophage increases is benefited from preparation treatment.

Second aspect of the present invention provides a kind of reduction SUN2 protein level or SUN2 albumen is made to participate in cytoskeleton rearrangement Reduced activity or the reagent of forfeiture promote the M1 polarization of macrophage in preparation or remove the polarized drug of tumor-associated macrophage In purposes.

Third aspect present invention provides a kind of reduction SUN2 protein level or SUN2 albumen is made to participate in cytoskeleton rearrangement The purposes of reduced activity or the reagent of forfeiture in the drug of preparation enhancing inflammatory reaction or antineoplastic immune.

In one or more embodiments, the reagent for reducing SUN2 protein level includes:

(1) reagent of CK2 kinase expression level is improved；

(2) reagent of E3 ubiquitin ligase expression is improved；With

(3) inhibit SUN2 gene expression or reduce the reagent of its expression.

In one or more embodiments, the reagent for improving CK2 kinase expression includes TLR2, TLR4, TLR7 and The agonist of TLR9.

In one or more embodiments, the reagent for improving CK2 kinase expression includes MALP-2, LPS, R-848 With CpG DNA.

In one or more embodiments, the reagent for improving E3 ubiquitin ligase expression is E3 ubiquitinbond The expression vector of enzyme.

In one or more embodiments, the E3 ubiquitin ligase is β TrCP1 or β TrCP2.

In one or more embodiments, the reagent for inhibiting SUN2 gene expression or reducing its expression is to use In the siRNA of SUN2 gene.

In one or more embodiments, the siRNA is as shown in SEQ ID NO:6.

In one or more embodiments, the reduced activity for making SUN2 albumen participate in cytoskeleton rearrangement or forfeiture Reagent act on SUN2 gene, deposit the SUN2 albumen of the coded by said gene in its transmembrane domain and/or SUN structural domain SUN2 albumen is being caused to participate in the reduced activity of cytoskeleton rearrangement or the mutation of forfeiture.

It is described to benefit from the disease that M1 type macrophage increases as tumour in one or more embodiments.

In one or more embodiments, the tumour is tumour relevant to tumor-associated macrophage.

In one or more embodiments, the tumour is solid tumor, including but not limited to breast cancer, oophoroma, palace Neck cancer, melanoma and prostate cancer.

Fourth aspect present invention provides SUN2 gene or SUN2 albumen and benefits from M1 macrophage in preparation or screening treatment Purposes in the drug of the disease increased.

In one or more embodiments, the SUN2 gene or SUN2 albumen behaviour SUN2 gene or people's SUN2 egg It is white.

In one or more embodiments, the nucleotides sequence of the SUN2 gene is classified as Serial No. in Genebank The sequence of NM_001199580；The SUN2 protein sequence is the sequence of Serial No. AAT90500.1 in Genebank.

In one or more embodiments, the drug are as follows: nucleic acid molecules, carbohydrate, lipid, small molecule chemical combination Object, albumen (including antibody) or interference slow virus.

Fifth aspect present invention provides a kind of pharmaceutical composition, described pharmaceutical composition containing be reduced SUN2 protein level or SUN2 albumen is set to participate in the reagent of cytoskeleton rearrangement miopragia or forfeiture.

In one or more embodiments, the reagent for reducing SUN2 protein level includes:

(1) reagent of CK2 kinase expression level is improved；

(2) reagent of E3 ubiquitin ligase expression is improved；With

(3) inhibit SUN2 gene expression or reduce the reagent of its expression.

In one or more embodiments, the reagent for improving CK2 kinase expression includes TLR2, TLR4, TLR7 and The agonist of TLR9.

In one or more embodiments, the reagent for improving CK2 kinase expression includes MALP-2, LPS, R-848 With CpG DNA.

In one or more embodiments, the reagent for improving E3 ubiquitin ligase expression is E3 ubiquitinbond The expression vector of enzyme.

In one or more embodiments, the E3 ubiquitin ligase is β TrCP1 or β TrCP2.

In one or more embodiments, the reagent for inhibiting SUN2 gene expression or reducing its expression is to use In the siRNA of SUN2 gene.

In one or more embodiments, the siRNA is as shown in SEQ ID NO:6.

In one or more embodiments, the reagent effect for destroying SUN2 albumen and participating in cytoskeleton rearrangement function In SUN2 gene, having the SUN2 albumen of the coded by said gene in its transmembrane domain and/or SUN structural domain leads to SUN2 Albumen participates in the mutation of cytoskeleton rearrangement miopragia or forfeiture.

Sixth aspect present invention provides a kind of method of inflammatory reaction and antineoplastic immune for enhancing tumor patient, the side Method includes the step for reducing object SUN2 protein level or making its SUN2 albumen participation cytoskeleton rearrangement miopragia or forfeiture Suddenly.

Seventh aspect present invention provides a kind of tumor therapeuticing method, and the method includes reducing the SUN2 of object in need Protein level or make its SUN2 albumen participate in cytoskeleton rearrangement miopragia or lose the step of.

In one or more embodiments, the SUN2 protein level for reducing object in need includes:

(1) it is horizontal to improve object CK2 kinase expression；

(2) object E3 ubiquitin ligase expression is improved；And/or

(3) inhibit object SUN2 gene expression or reduce its expression.

In one or more embodiments, improving object CK2 kinase expression level includes giving to improve CK2 kinase expression Reagent, including but not limited to TLR2, TLR4, the agonist of TLR7 and TLR9.

In one or more embodiments, the reagent for improving CK2 kinase expression includes MALP-2, LPS, R-848 With CpG DNA.

In one or more embodiments, the raising E3 ubiquitin ligase expression includes giving to improve E3 ubiquitin The reagent of ligase expression, the including but not limited to expression vector of E3 ubiquitin ligase.

In one or more embodiments, the E3 ubiquitin ligase is β TrCP1/2.

In one or more embodiments, the inhibition SUN2 gene expression or to reduce its expression include giving and pressing down SUN2 gene expression processed or the reagent for reducing its expression, the including but not limited to siRNA of SUN2 gene.

In one or more embodiments, the siRNA is as shown in SEQ ID NO:6.

It is described so that SUN2 albumen is participated in cytoskeleton rearrangement miopragia or lose packet in one or more embodiments Include: donation makes the gene institute in the reagent for participating in cytoskeleton rearrangement function for destroying SUN2 albumen of SUN2 gene The SUN2 albumen of coding exists in its transmembrane domain and/or SUN structural domain causes SUN2 albumen to participate in cytoskeleton rearrangement The mutation of miopragia or forfeiture.

Detailed description of the invention

Fig. 1: LPS stimulation inducing macrophage nuclear morphology changes.The variation of nucleus size after A:LPS stimulation；B:DAPI Dyeing observation nucleus；C: the spacing of Electronic Speculum detection nucleus bilayer nuclear membrane；D: atomic force microscope observation nucleus.

Fig. 2: the variation of macrophage differentiation and polarized SUN1/2 protein level in the process.The source A-B:THP-1 it is huge Protein expression level in phagocyte；C: flow cytometry n F4/80⁺The expression of SUN2 after PEM cell LPS stimulation；D: SUN2 and LaminA/C after fluorescence microscope detection LPS stimulation in macrophage；The macrophage quilt in the source E:THP-1 After MALP2, polyI:C, R848 or cpDNA stimulation, the protein level of SUN1/2；F: SUN1/2 egg during macrophage activation The ideograph that white level is lowered；The protein level of SUN1/2 after G:PEM cell M1 or M2 polarization；H: derived from bone marrow monocyte exists The protein level of SUN1/2 after GM-CSF or M-CSF stimulation.

Fig. 3: CK2 alpha-beta TrCP mediates SUN1/2 to degrade and influence nucleus micromechanical power.The PEM cell of A:LPS stimulation The protein level of SUN1/2 after being handled with MG132；The ubiquitination level of SUN2 after the PEM cell of B:LPS stimulation；C: β TrCP1/2 The immunoblotting assay of SUN1/2 after the cell of knockout is handled with MG132；D: the ubiquitination level of SUN2 after transfection β TrCP；E: People, orangutan, rat, mouse and chicken SUN2 β TrCP binding motif alignment；Mode of the F:SUN2 in conjunction with β TrCP Figure；G: the SUN2 of wild type and SA mutant ubiquitination level；H: the macrophage in the source THP-1 of expression SUN2SA mutant is thin The nucleus size of born of the same parents；I: atomic force microscope detects the nucleus for having transfected wild type or SA mutant SUN2；J:LPS stimulation Macrophage with CK2 inhibitor TBB handle after SUN2 immunoblotting；The vitro kinase of K:CK2 α phosphorylation SUN2 is tested； The macrophage in the source L:THP-1 is with TBB treated average cell core size；M: atomic force microscope detects the source THP-1 Macrophage with TBB treated nucleus.

Fig. 4: SUN protein regulation nucleus micromechanical power and macrophage function.A: wild type and SUN1/2^-/-It knocks out PEM cell LPS stimulation after average cell core size；B: wild type and SUN1/2^-/-It is mechanical after the MEF cell LPS stimulation of knockout Force characteristic；C: wild type and SUN1/2^-/-The endocytosis of the PEM cell of knockout；D: wild type and SUN1/2^-/-The PEM cell of knockout Middle M1 type macrophage marker molecule；Il6, Il1b and Nos2: and M2 type macrophage marker molecule (Arg1, Mrc1 and Retnla transcriptional level)；E: wild type and SUN1/2^-/-The metaboilic level of the PEM cell of knockout；ECAR and OCR)；F:M0, The benchmark ECAR and OCR of M1 and M2 type macrophage are horizontal；G: FCM analysis macrophage activation marker molecule CD86 (M1 Type) and CD206 (M2 type)；F4/80 after H: transfection siSUN1/2 or control siRNA⁺CD206⁺The ratio of macrophage.

Fig. 5: it knocks out SUN albumen and changes nucleus, cytoskeleton and cellular morphology.A: siSUN1/2 or control have been transfected The immunofluorescence of F- actin after the macrophage LPS stimulation of siRNA；B: wild type or SUN1/2^-/-In the PEM of knockout Triton X-100 is solvable and insoluble component in beta-actin level；The protein mediated macrophage activation process of C:SUN The ideograph of middle cytoskeleton variation.

Fig. 6: knocking out SUN albumen enhances TLR cell pathway.A: wild type or SUN1/2^-/-The PEM cell LPS of knockout is stimulated The immunoblotting of pIKK β afterwards, pJNK, pERK and I κ B α s, beta-actin is as negative control；B: wild type or SUN1/2^-/- The transcriptional level of cell factor after the PEM cell LPS stimulation of knockout；C: the SUN2 of wild type or Del TM mutant turns Il6 The horizontal rescue experiment of record；D: wild type or SUN1/2^-/-The PEM cell of knockout with different TLR agonists handle after TNF α with The generation of IL-6；E:HEK293T cell transfecting is unloaded or MyD88, IRAK1, TRAF6, IKK β or p65 and graded doses After SUN1/2, the activity of fluorescent reporter gene；F:HEK293T knocks out the activation of NF- κ B after SUN1/2.

Fig. 7: it knocks out SUN1/2 and macrophage is promoted to break up to M1.A: wild type and knockout SUN1/2^-/-PEM cell born of the same parents Gulp down experiment；B: chamber technique detects THP-1 cell and knocks out the transfer ability after SUN1/2；C: cell flow cytometer showed macrophage swashs Marker molecule living, i.e. the marker molecule CD206 of marker molecule CD86 and the M2 macrophage of M1 macrophage；D: wild type and SUN1/2^-/-In the PEM cell of knockout in M1 macrophage Il6, Il1b and Nos2 transcriptional level, in M2 macrophage The transcriptional level of Arg1, Mrc1 and Retnla.

Fig. 8: SUN1/2 enhancing mouse inflammation and antitumor reaction are knocked out.A: wild type and knock-out mice LPS are post-stimulatory Survival rate；The generation of mouse lung cell factor, ELISA detect TNF α, IL-1 β and IL-6 after B:LPS stimulation；C: wild type And SUN1^flox/floxLysM^cre/creSUN2^-/-Tumor load of the knock-out mice tail vein injection B16-F10 after ten days；D: wild type And SUN1^flox/floxLysM^cre/creSUN2^-/-Knock-out mice lung immunohistochemical analysis；E: wild type PyMT and SUN1^{flox/ flox}LysM^cre/creSUN2^-/-The tumor size of PyMT mouse；F: wild type PyMT and SUN1^flox/floxLysM^cre/creSUN2^-/- The tumour immunity histochemical analysis of PyMT mouse.

Specific embodiment

The application has studied the variation of karyomorphism and mechanical force characteristic in macrophage differentiation/polarization process.As a result It was found that the expression of SUN2 can change under the stimulation of macrophage differentiation/polarized signal, dynamic response cell weight The function of the process of structure and the reconstruct regulation macrophage by regulating cell skeleton.Knocking out SUN albumen can promote M1 type huge The activation of phagocyte, inhibit class M2 type tumor-associated macrophage activation, thus greatly enhance immune response and it is antitumor It is immune.

Therefore, the application enhances inflammatory reaction and antitumor by reducing SUN protein level and/or SUN2 protein active It is immune, realize the prevention and treatment for benefiting from the disease that M1 type macrophage increases.

In the application, SUN2 gene and SUN2 albumen can be SUN2 gene and SUN2 albumen from separate sources, example Different Individual such as from different plant species or from same species.For example, SUN2 gene can be the SUN2 gene of people or mouse.? In certain embodiments, the nucleotides sequence of the SUN2 gene from people is listed in the Serial No. NM_ in Genebank 001199580.In certain embodiments, SUN2 gene described herein includes the homologous sequence of SUN2, i.e., with The nucleotide sequence of sequence shown in Serial No. NM_001199580 is compared in Genebank, in definite molecular length, sequence Column show at least about 50%, preferably at least about 75%, more preferably at least about 80%, more preferably at least about 85%, especially it is good at least About 90%, most preferably at least about 95%, the DNA sequence dna of for example, at least about 98% sequence similarity.It is furthermore preferred that the homologous sequence It is classified as the SUN2 gene from mouse.Log in number GenBank:AY682988.1 of people's SUN2 albumen in Genebank or AAT90500.1。

In the application, reducing SUN protein level includes the level and SUN2 protein expression for making SUN2 albumen in macrophage Unaffected wild type macrophage compared to reduce at least 30%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even do not express SUN2 albumen completely.

SUN protein level can be reduced by one or more of mode:

(1) it is horizontal to improve CK2 kinase expression；

(2) E3 ubiquitin ligase expression is improved；And/or

(3) inhibit SUN2 gene expression or reduce its expression.

The expression of CK2 kinases can be improved by the way that the reagent of CK2 kinase expression can be improved.This kind of reagent includes but not It is limited to TLR2, TLR4, one of TLR7 and TLR9 or any are a variety of.For example, this kind of reagent include but is not limited to MALP-2, LPS, R-848 and CpG DNA.It in certain embodiments, can also be by being transferred to kinase c K2 (such as CK2 α, nucleosides in cell Acid sequence is shown in that GenBank:J02853.1, amino acid sequence are shown in GenBank:AAA56821.1) expression vector and its table is provided Up to level.

The expression of E3 ubiquitin ligase can be improved by the way that the reagent of E3 ubiquitin ligase expression can be improved.This Class reagent includes but is not limited to: the expression vector of E3 ubiquitin ligase, the aforementioned reagent and CK2 that can improve CK2 kinase expression The expression vector of kinases.In certain embodiments, E3 ubiquitin ligase is β TrCP1 or β TrCP2.Therefore, in these implementations It, can (β TrCP1 nucleic acid sequence be shown in that GenBank:AF101784.1, protein sequence are shown in by giving β TrCP1 or β TrCP2 in scheme GenBank:AAD08702.1；β TrCP2 nucleic acid sequence is shown in that GenBank:AF176022.1, protein sequence are shown in GenBank: AAF04528.1) expression vector improve the expression of ubiquitin ligase.Technological means well known in the art also can be used Engineered cells genome, to realize the high level expression of E3 ubiquitin ligase.

Technology well known in the art can be used to inhibit the expression of SUN2 gene or reduce its expression.For example, can be used RNAi technology interferes the expression of SUN2 gene, or knocks out SUN2 gene using homologous recombination technique.It, can in these embodiments The inhibition or reduction are realized by giving corresponding reagent.For example, the siRNA of SUN2 gene can be used (for example, SEQ ID SiRNA shown in NO:6) lower the expression of SUN2 gene.Alternatively, SUN2 gene full genome is knocked out using targeting vector. Therefore, this kind of reagent includes but is not limited to siRNA and targeting vector.

In the application, the activity for reducing SUN2 albumen includes dropping the activity of SUN2 albumen compared with wild type SUN2 albumen Low at least 30%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even completely It is inactive.

In general, its activity can be made to reduce and introducing mutation in SUN2 albumen.Herein, the activity of SUN2 albumen is outstanding It refers to that it participates in the function of cytoskeleton rearrangement.Therefore, in certain embodiments, in the transmembrane domain of SUN albumen and/or The mutation for causing the structural domain to participate in cytoskeleton rearrangement miopragia or forfeiture is introduced in SUN structural domain；Especially preferably It is that the mutation is introduced in SUN structural domain.Mutation can be 1 or it is several it is even more (such as 10 or more, 20 with It is upper, 30 or more) insertion, deletion or substitution of a amino acid.Preferably participate in amino acid necessary to cytoskeleton rearrangement It mutates, leads to the miopragia or forfeiture.In certain embodiments, the SUN structural domain of SUN2 albumen lacks completely. It can make the gene institute by donation in the reagent for participating in cytoskeleton rearrangement function for destroying SUN albumen of SUN gene The SUN albumen of coding exists in its transmembrane domain and/or SUN structural domain causes SUN albumen to participate in cytoskeleton rearrangement function The mutation that can weaken or lose.The sequence of SUN2 gene can be changed in this kind of reagent, and the SUN2 albumen for causing it to encode exists above The mutation, to have the activity or loss of activity weakened.For example, the SUN2 that can be mutated by homologous recombination technique SUN2 gene in gene replacement wild-type cell, so as to cause its expression except weak activity or inactive SUN2 albumen.

SUN protein level reduces and/or SUN2 protein active weakens or forfeiture facilitates macrophage and breaks up to M1 type, is swollen The depolarising of tumor associated macrophages.Therefore, by reducing SUN protein level and/or SUN2 protein active, inflammatory reaction can be enhanced And antineoplastic immune, realize the prevention and treatment for benefiting from the disease that M1 type macrophage increases.Benefit from M1 type macrophage The disease increased includes but is not limited to tumour, tumour especially relevant to tumor-associated macrophage.In certain embodiments In, the tumour is solid tumor, including but not limited to breast cancer, oophoroma, cervical carcinoma, melanoma and prostate cancer.

Therefore, provided herein is it is previously described reduce SUN2 protein level or make SUN2 albumen participate in cytoskeleton rearrangement Reduced activity or forfeiture reagent preparation treatment benefit from the disease that M1 type macrophage increases drug in purposes.

Above-mentioned function based on SUN2, the present invention also provides CK2 kinases and/or its gene, E3 ubiquitin ligase and/or its Gene and/or SUN2 gene and/or SUN2 albumen are treated previously described M1 macrophage of benefiting from preparation or screening and are increased Disease drug in purposes.This kind of purposes includes the drug that preparation or screening can lower SUN2 gene expression, and/or preparation Or screening can inhibit SUN2 albumen to participate in the active drug of cytoskeleton rearrangement.In general, in this this kind of application, CK2 kinases and/or Its gene, E3 ubiquitin ligase and/or its gene and/or SUN2 gene and/or SUN2 albumen are used as target spot.It can be used for treating The previously described drug for benefiting from the disease that M1 macrophage increases can be nucleic acid molecules, carbohydrate, lipid, small point Sub- compound, albumen (such as antibody) or interference slow virus.Therefore, the present invention also provides a kind of screening treatment it is previously described by The method of the drug of the disease increased beneficial to M1 macrophage, this method include the body for making drug to be screened and expressing SUN2 albumen The step of system's contact, wherein the drug that can reduce SUN2 protein level in the system is the disease benefiting from M1 macrophage and increasing The drug candidate of disease.The system of expression SUN2 albumen can be the cell of various expression SUN2 albumen, including but not limited to naturally The cell of SUN2 albumen is expressed, and is genetically engineered and expresses the cell of SUN2 albumen.

Therefore, it is also provided herein a kind of pharmaceutical composition, the composition, which contains, as described herein reduces SUN2 albumen water Reduced activity that is flat or making SUN2 albumen participation cytoskeleton rearrangement or the reagent of forfeiture.The reagent can be nucleic acid molecules, One of carbohydrate, lipid, small molecule compound, albumen (such as antibody) or interference slow virus are a variety of.For example, described Nucleic acid molecules can be siRNA, targeting vector and CpG DNA etc..The small molecule compound can be R848 etc..

Pharmaceutically acceptable carrier or excipient can also be contained in pharmaceutical composition.For example, in preparation pharmaceutical composition When, usually the reagent is mixed with excipient, or with figuration dilution agent or Bao Ke with capsule or anther sac, nano particle shape In carrier existing for formula.When excipient plays diluent, it can be solid, semisolid or fluent material as figuration The medium of agent, carrier or active constituent.Therefore, pharmaceutical composition can be tablet, pill, pulvis, solution, syrup, go out Bacterium injects solution etc..The example of suitable excipient includes lactose, glucose, sucrose, sorbierite, mannitol, starch, crystallite fibre Tie up element, polyvinylpyrrolidone, cellulose, water, nano particle etc..Pharmaceutical composition can also contain other ingredients, such as wet Agent, emulsifier, preservative (such as methyl hydroxybenzoate and propyl ester and sweetener.

In general, active constituent of the pharmaceutical composition containing therapeutically effective amount, so that the amount of application of pharmaceutical composition is enough Change the transcription or translation of SUN2 gene, or changes the expression or activity of SUN2 albumen enough.Specific dosage is contemplated that administration The factors such as approach, patient health situation, within the scope of these are all skilled practitioners technical ability.

Herein, object is usually mammal, especially people.

The present invention will be hereafter illustrated in a manner of specific embodiment.The experiment side of actual conditions is not specified in the following example Method, usually according to normal condition such as Sambrook, " molecular cloning: lab guide " (New York, United States: cold spring harbor laboratory Publishing house, 1989) condition described in, or carry out according to the normal condition proposed by manufacturer.For the usage and dosage of reagent, remove It is non-to be otherwise noted, otherwise used according to conventional usage and dosage.

Basic experiment method:

1, the clone of target gene: for required target gene design primer, with the method for PCR by the target gene from It clones and in the cDNA of plasmid comprising this target gene or Hela cell, there are two different for target gene fragment both sides band Restriction enzyme site.Whether the size with the method detection PCR product of agarose gel electrophoresis is in the same size with required target gene, And the segment is recycled using Ago-Gel DNA QIAquick Gel Extraction Kit.It is added and PCR product both sides in glue recovery product The corresponding enzyme of restriction enzyme site and suitable buffer carry out double digestion, 37 DEG C of overnight incubations.Meanwhile with the double enzymes of identical enzyme Cut the segment carrier to be connected.The segment after digestion is recycled using common DNA product purification kit, utilizes fine jade Sepharose DNA QIAquick Gel Extraction Kit recycles the carrier after digestion, has then been connect segment with carrier with T4 ligase Come, and the connection product is converted into DH5 α.Using resistance screening, the clone of verifying and sequencing acquisition target gene.

2, the rite-directed mutagenesis of plasmid: designing pair of primers near mutational site, and there is 15-20bp at the 5 ' ends of this two primers Reverse complemental region, mutational site should be located at this reverse complemental region centre.The size in the incomplementarity region at 3 ' ends is at least For 10bp.Using this pair of primers, plasmid is carried out by Inverse PCR amplification by the method for PCR.It is purified using common DNA product Kit recycles the product of PCR, and DpnI, 37 DEG C of incubation 2h, by the plasmid template of methylation are added in recovery product Removal.Then, the sample after DpnI being handled directly converts DH5 α.Gram of target gene is obtained using resistance screening and sequencing It is grand.

3, the expression and purification of albumen: prokaryotic expression plasmid is taken to convert BL21,37 DEG C of culture 12h.It is first inoculated with, that is, is chosen It is cloned into the LB culture medium that 7.5ml contains antibiotic, in 37 DEG C, 220rpm shake culture 10-12h.Then expand culture again, Above-mentioned 7.5ml bacterium solution is all added in the antibiotic TB culture medium of 750ml, 37 DEG C, 220rpm shake culture to OD600 For 0.6-0.8 (about 3-4h).At this point, shaking table temperature is adjusted to 16 DEG C, after bacterium solution is cooled to 16 DEG C completely, according to 1:2000 Ratio be added IPTG (final concentration of 0.4mM), continue shake culture 16-18h.Finally use the high speed refrigerated centrifuge of Beckman Machine collects thallus, i.e., 4 DEG C, 6000rpm is centrifuged 10min, abandons supernatant to the greatest extent, protein purification or preservation are carried out after thallus is weighed In -80 DEG C.

GST column affinity chromatography:

Lysis buffer: 20mM HEPES pH 7.0,500mM NaCl, 1mM DTT；

Washing buffer: 20mM HEPES pH 7.0,500mM NaCl, 1mM DTT；

Elution buffer: 20mM HEPES pH 7.0,500mM NaCl, 10mM GSH, 1mM DTT；

Suitable beads is taken to pour into chromatographic column, with a large amount of ddH₂O rinses beads, and generally (CV is represented for 10CV × 5 Column volume, similarly hereinafter).It is balanced with lysis buffer: 10CV × 5.By what is obtained after the beads balanced immigration bacterial cell disruption In supernatant, in Cool Room 4 DEG C Mix 1-2h.500g, 4 DEG C of centrifugation 2min collect supernatant and flow through liquid.50ml washing buffer is added Liquid, 500g, 4 DEG C of centrifugation 2min are repeated twice,.Beads is poured into chromatographic column, continues to be washed once with washing buffer.With washing De- buffer elution: eluent is collected with EP pipe in 0.5CV × 10 respectively.SDS-PAGE electroresis appraisal.

4, luciferase reporter gene is tested: luciferase reporter gene experiment is first to examine using fluorescein as substrate Survey the activity of firefly luciferase, after the activity of renilla luciferase is detected as substrate using coelenteron fluorescein, and rear The continuous substrate that renilla luciferase is added joined the substance for inhibiting firefly luciferase catalysis luciferin simultaneously, make subsequent Detection is only measured to the activity of renilla luciferase, realizes the detection of luciferase reporter gene.Firefly luciferase Luciferin can be catalyzed and be oxidized to oxyluciferin, during luciferin oxidation, bioluminescence can be issued.Sea pansy Luciferase can be catalyzed coelenteron fluorescein and be oxidized to coelenteramide, can also send out during the oxidation of coelenteron fluorescein Bioluminescence out.It may then pass through fluor tester and be also referred to as Chemiluminescence Apparatus or liquid flashing determining instrument and measure above-mentioned luciferase and urge The bioluminescence discharged in the oxidation process of change.Firefly luciferase is catalyzed the luminous most strong emission wavelength of luciferin 560nm.The most strong emission wavelength that renilla luciferase is catalyzed coelenteron fluorescein luminescence is 465nm.Pass through luciferase and its bottom This bioluminescence system of object, can extremely sensitive, the efficient expression for detecting gene.Usually interested genetic transcription Controlling element is cloned in the upstream or other places appropriate of firefly luciferase, is built into reporter plasmid, then turns Cell is contaminated, lytic cell after appropriate stimulation or processing measures uciferase activity.Thorn is judged by the height of uciferase activity Swash the influence of front and back or different stimulated to interested controlling element.Renilla luciferase is more used as the interior of transfection efficiency Ginseng, to eliminate the difference of cell quantity and transfection efficiency.

Experimental procedure: culture HEK293FT cell (or other aim cells), and be inoculated in 24 orifice plates, grow 12-24 Hour (80% convergence degree).It is glimmering with the luciferase reporter plasmid and sea pansy of lipo2000 transfection expression firefly luciferin Light element enzyme internal reference reporter plasmid and required destination gene expression plasmid.After 24-48 hours, cell cracking is added Liquid (5X lysis buffer is diluted with water to 1X, is purchased from promega), the every hole of 24 orifice plates adds 120 μ l, and the every hole of 48 orifice plates adds 60 μ l. The plate is put into -80 DEG C of jelly 1h.Plate is taken out from -80 DEG C, after room temperature is melted, cell pyrolysis liquid is sucked out, is transferred in EP pipe.Often Pipe lysate takes 5 μ l to be added in 384 orifice plates.The reaction substrate of 10 μ l firefly luciferases is added, surveys the glimmering of reporter gene Light value.The reaction substrate of 10 μ l is added, surveys the fluorescent value of renilla luciferase.The ratio of reporter gene and sea pansy fluorescein fluorescence value Value is the relative fluorescence signal value of each sample.

In the experiment that detection compound influences intracellular reporter gene expression, Flag is transfected in HEK293FT cell The RIG-I (residue 1-238) and IFN β of label or the reporter gene of ISRE, luciferase reporter gene.After 12 hours, disappear Change cell, be seeded in 384 orifice plates with 10000 cells/every hole density, the cell culture medium of 50 μ l is added.After 7 hours, The compound that addition AlphaScreen experiment screening obtains, final concentration of 10 μM of compound.After 37 DEG C of culture 15h, detect glimmering Light element enzymatic activity.

5, immunoblotting: protein sample is prepared according to requirement of experiment, 100 DEG C are denaturalized 10 minutes, and 13200rpm is centrifuged 2 points Clock takes the supernatant of equivalent to be added in the loading hole of SDS-PAGE glue.Protein sample voltage when being concentrated in glue is 80V, is being separated Voltage is 120V when in glue.After electrophoresis, remove gel, membrane-transferring device be installed in the following order: (cathode), filter paper, gel, Nitrocellulose filter, filter paper, (anode).Membrane-transferring device is put into Cool Room 4 DEG C, 100V constant pressure transfers 1h.After the completion of transferring film, take out Film is immersed in 5% skim milk with TBST buffer preparation, is incubated on shaking table at room temperature by nitrocellulose filter 1h.Film, 5min × 3 time are washed with TBST buffer solution.

It is added and uses the diluted primary antibody of 5%BSA solution to scale, be incubated overnight on the shaking table of Cool Room 4 DEG C.Use TBST Buffer solution washes film, 10min × 3 time.It is added to scale with the diluted secondary antibody of 5% milk, is incubated on shaking table at room temperature 40-60min.Film, 10min × 3 time are washed with TBST buffer solution.Chromogenic substrate is covered on nitrocellulose filter, room temperature is aobvious Color 2 minutes.It is shot with LAS4000 cold light/bioluminescence image analyzers.

The preparation of required reagent: it 5%BSA solution: weighs 5g BSA powder and is dissolved in 1 × PBS solution of 100ml, add 0.02% Sodium azide is stored in 4 DEG C.10 × Western blot transferring film buffer: it weighs 30.3g Tris alkali and 144g is sweet 300ml methanol is added in propylhomoserin, then with deionized water constant volume to 1L.

6, the extracting of RNA: the cell after transfection is discarded into culture medium, and is cleaned cell one time with the PBS of pre-cooling.Six holes 500 μ l Trizol Reagent are added in each hole of plate, and room temperature is handled 5 minutes.Cell pyrolysis liquid is transferred to 1.5ml In Eppendorf pipe.The chloroform of 100 μ l is added in every part of sample, and vortex low speed stands 5 minutes after mixing.4 DEG C, 12000g from The heart 15 minutes, the upper strata aqueous phase of 240 μ l was shifted into new Eppendorf pipe.The isopropanol of 240 μ l, top is added in every part of sample It mixes, is placed at room temperature for 10 minutes.4 DEG C, 12000g is centrifuged 15 minutes, is discarded supernatant, and leaves the RNA precipitate of white.Every part of sample 75% ethyl alcohol of 500 μ l is added in product, is mixed by inversion.4 DEG C, 12000g is centrifuged 5 minutes, abandons supernatant to the greatest extent.Room temperature is dried.It is added appropriate The processed deionized water of DEPC sufficiently dissolves.The concentration of extracted RNA is measured with NanoDrop ND1000,260nm's The ratio of the light absorption value of light absorption value and 280nm should maintain between 1.9-2.0.By extracted RNA be used for reverse transcription or directly Freeze to -80 DEG C and saves.

The preparation of required reagent: phosphate buffered saline solution (PBS): 800ml distilled water dissolves 0.2g KCl, 8g NaCl, 0.24g KH₂PO₄With 1.44g Na₂HPO₄.The pH value for adjusting solution with HCl is settled to 1L to 7.4.High pressure steam sterilization or mistake Filter out bacterium.

7, co-immunoprecipitation: the cell after transfection is discarded into culture medium, and is cleaned cell one time with the PBS of pre-cooling.Add Enter suitable cell lysis buffer solution RIPA (containing protease inhibitors), cracks 30min on ice, by cell pyrolysis liquid in 4 DEG C, most Supernatant is taken after big revolving speed centrifugation 30min.The desired amount of albumin A/G sepharose 4B is taken, is washed 3 times with appropriate lysis buffer.It takes few It measures lysate to analyze in case of Western blot, the pretreated albumin A of 20 μ l/G sepharose 4B and 1 is added in remaining lysate The corresponding antibody of μ g, slowly rotation is incubated overnight on the rotator of Cool Room 4 DEG C.After the completion of immune precipitation, 4 DEG C with The speed of 7000rpm is centrifuged 1min, and sepharose 4B is centrifuged to EP tube bottom.Supernatant is carefully sucked, sepharose 4B is split with 300 μ l Solution buffer is washed 2 times, then is washed 2 times with 300 μ lPBS.It is eventually adding 2 × SDS sample-loading buffer of 20 μ l, 100 DEG C are boiled 10 minutes. The albumen in conjunction with antibody is analyzed using SDS-PAGE and Western blot

The preparation of required reagent: RIPA buffer: 150mM NaCl, 100mM Tris pH 8.0,1%TritonX- 100,5mM EDTA,10mM NaF。

8, cell culture: HEK293, HEK293T, MEF cell culture are in DMEM (Hyclone) culture solution, in culture solution Add 10% serum, 100ug/ml penicillin, 100 μ g/ml streptomysins.Cell is cultivated in 37 DEG C, and gas concentration lwevel is 5%.

9, microscopic

2×10⁵A macrophage is inoculated in the 6 orifice plates with coverslip, after 8 hours, is stimulated with the LPS of 1 μ g/ml. Cell is fixed through the methanol of 4% paraformaldehyde and pre-cooling.Cell is dyed with corresponding antibody or dyestuff, it is total by laser Focusing microscope or fluorescence microscope detect cell.

10, mass spectrum

Sample for Mass Spectrometer Method is that co-immunoprecipitation obtains overall length SUN albumen in cell.Above-mentioned sample 60ug, sample Buffer is 50mM TRIS-HCL pH7.5,0.1mM EGTA, 10mM magnesium acetate, and it is latent to test and analyze protein by mass spectrograph Interaction protein and phosphorylation site.

11, vitro kinase activity is tested

Reaction system is 40ul, including 10ug Flag-CK2 α, GST SUN2 (1-154) albumen, 1mM ATP, buffer For 50mM Tris pH7.5,0.1mM EGTA, 10mM magnesium acetate, remaining volume is supplied with distilled water, and 30 DEG C are incubated for 30 minutes.

The reaction system of kinase activity experiment is 40ul, the kinases including 4ul of 1ug saturation phosphorylation, [γ³²P] ATP1.5uCi, buffer are the TRAF structural domain conduct of 50mM Tris pH7.5,0.1mM EGTA, 10mM magnesium acetate TRAF6 Reaction substrate, final concentration of 1mg/ml, 30 DEG C are incubated for 20 minutes.Reaction product is added isometric 2 × SDS sample-loading buffer and exists 100 DEG C are denaturalized 10 minutes, after SDS-PAGE separation, are detected by autoradiograph.PAGE uses phosphorus screen (GE Healthcare) Exposed overnight, the testing result on FLA-9000 imager (GE Healthcare).

12, the building of MST4 knock out mice

The 4-6 weeks male C57BL/6 mouse (Slac) normally raised.MST4shRNA or control shRNA (500nM) dissolution In the solution that 200 μ l contain 5% glycerol and transfection reagent jetPEI (Polyplus, France, Illkirchcedex), ShRNA intravenous injection daily in continuous 7 days.Animal culture and zoopery are in accordance with Chinese Academy of Sciences's Shanghai school of life and health sciences bioid Learn article related to Institute of Cell Biology Animal Management Committee and animal welfare policy, Customs Assigned Number 081.

13, the foundation of disease mice model

In the experiment that research LPS stimulation influences mouse, it is injected intraperitoneally with LPS (5mg/kg).Establishing mouse In the experiment of septicemia model, with 5 × 10⁴Salmonella typhimurium strain SL13344 is injected intraperitoneally in the standard of CFU/ml (North Connaught, Shanghai, China), Induction of bacterial peritonitis.Mouse model is punctured to establish Cecal Ligation, With the amobarbital intraperitoneal injection of anesthesia mouse of 80mg/kg, in abdomen median incision, find caecum, ligature ileocaecal sphineter, then with Pin puncture ligatures position twice, puts back to enteron aisle, sutures abdomen.The survival rate of mouse is recorded, serum and purpose organ are periodically collected.

Mouse after bacterium infection is put to death with amobarbital after intraperitoneal injection 18 hours, aseptically collects blood Liquid, lung, spleen, kidney and peritoneal lavage fluid.Tissue homogenate, blood and peritoneal lavage fluid are with sterile PBS gradient dilution, respectively Inoculation after 37 DEG C are cultivated 18 hours, counts the clone of each sample on LB agar plate.

14, the adoptive feedback of macrophage in Mice Body

Before LPS is stimulated 48 hours, the clodronate liposome from tail vein to mouse injection or PBS that are suspended by.48 is small When after use flow cytomery (BD FACS Caliblur, USA), Testing index is expressed as with F4/80, sorting macrophage is thin Born of the same parents.

Peritoneal macrophages transfect shMST4-GFP or control GFP plasmid, transfect 48 hours with activating macrophage.Pass through Selected by flow cytometry apoptosis GFP positive cell (BD FACS) then injects mouse, note before LPS stimulation in the method for intravenous injection Cell density is 10 when penetrating⁷.F4/80 antibody is bought from eBioscience.

15, immunohistochemistry

Tissue samples are according to BD Pharmingen^TMIHC Zinc Fixative handbook (handbook number: 550523), uses Zinc agent fixes (BD Biosciences) and carries out paraffin embedding.Histotomy (5 μm of thickness) is fixed by heating, is sliced two It dewaxes 5 minutes in toluene, then uses fresh dimethylbenzene dewaxing instead, share dimethylbenzene and dewax 3 times.Dehydrated alcohol 5 minutes, twice. 90% ethyl alcohol 5 minutes, twice, 70% ethyl alcohol 5 minutes, once.Distilled water 5 minutes, twice.According to different antigen and antibody, It can choose and slice is placed in following antigen retrieval buffers, 10mM sodium citrate, pH6.0 or 1mM EDTA, pH8.0, or Tris, pH10.0,95 DEG C of 10mM are heated 12 minutes, are about slowly cooled to room temperature in 30 minutes.5% skim milk is added Closing 60 minutes.

The all steps since closing, centainly it is noted that the moisturizing of sample, avoids the drying of sample, otherwise easily generate Higher background.Proper proportion dilutes primary antibody, and 4 DEG C slowly shake overnight incubation, recycle primary antibody, and PBST is added, and washs 5 minutes. After exhausting cleaning solution, cleaning solution is added, is washed 5 minutes.It washs 3 times altogether.Horseradish peroxidase is diluted according to proper proportion (HRP) or biotin (Biotin) or alkaline phosphatase (AP) label secondary antibody.Room temperature or 4 DEG C slowly shake on the side shaker It is dynamic to be incubated for one hour.Recycle secondary antibody.PBST cleaning solution is added, slowly shakes washing 5 minutes on the side shaker.Exhaust cleaning solution Afterwards, cleaning solution is added, is washed 5 minutes.It washs 3 times altogether.DAB is selected to carry out subsequent detection.HE dyeing is carried out after DAB dyeing. Finally carry out dehydrated, transparent, neutral resin sealing.

16, real-time quantitative fluorescence PCR: real-time quantitative fluorescence PCR is real using Applied Biosystems company two-step method When PCR (Realtime-PCR) system, detect opposite CT value.Using quantitative fluorescence PCR premixed liquid, (Toyobo company is providedGreen Realtime PCR reagent) configuration reaction system detects and the expression of quantitative objective gene, GAPDH is as internal reference.

17, data are analyzed: being analyzed using SAS data software analysis bag (9.1.3) data, statistical data is averaged Number ± standard deviation.Single factor analysis of variance (ANOVA) and Student ' s t-test are for analyzing continuous variable.Confidence area Between be P < 0.05.

Embodiment 1: the reconstruct of the morphological change and mechanical property of nucleus in macrophage polarization process

1, experiment purpose: whether research macrophage can show cellular machine in cell differentiation and immunoreaction process The change of tool force characteristic.

2, experimental method:

(1) influence of the microscopic LPS to nucleus size

Experimental method is as shown in basic experiment method 9.LPS (Escherichia coli, serotype 055:B5) it is purchased from Sigma)) with PBS solution is prepared, and cell is handled.

(2) influence of the atomic force microscope observation LPS to karyomorphism

Atomic force microscope (Atomic Force Microscope, AFM) is micro- by detection sample to be tested surface and one Extremely weak interatomic interaction force between type force sensitive element studies the surface texture and property of substance.

When detecting karyomorphism using atomic force microscope, macrophage is cultivated in 35mm culture dish to 70% density And it is stimulated with LPS.It is cleaned once in room temperature with PBS buffer solution, then harvests cell with cell scraper, use 6ml hypotonic buffer Liquid (10mM HEPES, 1mM KCl, 1.5mM MgCl₂, 0.5mM dithiothreitol (DTT), protease inhibitors) and it is incubated for 5 minutes on ice, Lytic cell.Using homogenizer by cell homogeneity, 4 DEG C of 700g are centrifuged 5 minutes, collect cell fragment.By cell fragment with low Oxidation buffer cleaning is primary, is centrifuged again.Nucleus is collected, (20mM HEPES, pH 7.8,25mM is resuspended in S buffer KCl,5mM MgCl₂, 0.25M sucrose and 1mM ATP).At room temperature, by the I buffer of 10000 nucleus and 0.5ml (20mM HEPES, pH 7.8,25mM KCl, 5mM MgCl₂With 1mM ATP) in the coverslip for being coated with poly-L- lysine It is upper to stand 30 minutes.The cell sample prepared is placed on objective table, probe is moved to cell with the help of microscope Cell is collapsed after in core region and obtains force-distance curve.It is then true using Hertz model analysis probe approximating curve Determine contact point and calculate Young's Moduli using Slope Method, the elasticity modulus of cell is extrapolated using Slope Method.

(3) influence of the Electronic Speculum detection LPS to nuclear membrane gap

Cell culture when being used for electron microscope experiment, using 2.5% glutaraldehyde cross-linking 1.5 hours, is fixed thin on coverslip Born of the same parents.Then three times using phosphate-buffered (pH 7.4) cleaning of 0.1M, 10 minutes every time.Room temperature decline sample 1% it is molten 1 hour is fixed in liquid then, is cleaned using the phosphate-buffered (pH 7.4) of 0.1M and is dehydrated with the ethyl alcohol of gradient concentration. 812 epoxy resin are mixed with the ratio of 1:1 and 100% acetone infiltrates sample, then are infiltrated sample overnight with 812 epoxy resin. Finally, sample is embedded in 812 epoxy resin, it polymerize 48 hours at 60 DEG C.Ultra-thin section (thickness 70nm) is carried out to sample It is placed in progress Electronic Speculum detection on 100 mesh copper mesh.

3, experimental result and analysis:

LPS (Gram-negative bacteria bacteria lipopolysaccharide) can cause its morphologic variation with stimulating expression of macrophage, be related to thin The aggregation of actin fiber, the formation of filopodia, lamellipodia and cell membrane fold in born of the same parents' migration, cytoskeleton.For Nucleus morphology in this course, the variation of mechanical property and these variations are studied in monocyte/macrophage Function in polarization and atomization, the present embodiment dyes the DNA in cell using Hoechst33342, by LPS After stimulation 5 hours, burnt microwell plate imaging is copolymerized by high intension, the inventors discovered that THP-1 (human monocyte cell line) source The nucleus size of macrophage averagely reduces 25% (Fig. 1, A).Similar knot is also observed in peritoneal macrophage (PEM) Fruit.Meanwhile the above results (Fig. 1, B) is demonstrated using fluorescence microscope.In addition, by electron microscopy, human hair of the present invention The stimulation of existing LPS makes the gap between nuclear membrane increase about 50% (Fig. 1, C).

For further verify this as a result, and detect nucleus in morphologic variation, the present inventor is stimulated with LPS The macrophage in the source THP-1 uses PBS to handle identical cell as negative control as experimental group.Then, separating experiment The nucleus of group and the macrophage of control group detects nucleus using atomic force microscope (AFM).In the macrophage of LPS stimulation In cell core, highest part is significantly reduced compared to the cell that control group PBS is handled.Equally, measure of cell nuclear bomb The elasticity modulus (DMT modulus) of property and the Young's modulus (Young ' s modulus) of hardness are in the cell that LPS is stimulated It is significantly reduced (Fig. 1, D).

The above result shows that shrinkage occurs on the karyomorphism of macrophage under the stimulation of LPS, hardness is reduced, carefully Karyon film spacing becomes larger.

The response inflammatory stimulus signal of embodiment 2:SUN protein level

1, experiment purpose: whether research inflammatory stimulus signal will affect the protein expression level of nuclear membrane Protein S UN.

2, experimental method:

(1) detection of western blotting method detection protein expression level is as shown in basic experiment method 5.

(2) flow cytomery protein expression level

Cell is placed in 5ml round bottom polystyrene tube, and antibody corresponding with that need to detect albumen is incubated for 30 minutes in 4 DEG C.It will Cell room temperature in the PBS buffer solution containing 2% formaldehyde fixes 10 minutes, then 4 DEG C using permeabilization liquid (0.1% saponin(e, 0.1% bovine serum albumin(BSA) Hank balanced salt solution) it is dyed, then detected on flow cytometer.

(3) laser confocal microscope observes protein expression level as shown in basic experiment method 9.

3, experimental result and analysis:

Since the gap in the case where LPS is stimulated between nuclear membrane broadens, the present inventor guesses that monocyte/macrophage is thin Specifically expressed albumen may respond relevant differentiation/polarized signal access on karyon film, to make the component of nuclear membrane And micromechanical power changes.To verify this it is assumed that the present inventor has detected LPS stimulation in connection nuclear membrane The influence of outer membrane related component Protein S un1/2, Nesprin1/2 and LaminA/C.As shown in Fig. 2 (A), with 1 μ g/ml's LPS is handled THP-1 cell 1 hour or 5 hours, and the protein level of Sun1/2 is remarkably decreased, and Nesprin1/2 and LaminA/ The protein level of C does not have significant change.Obtained in PEM cell similar as a result, and the protein level of Sun2 changes the most Obviously (Fig. 2, B).In the PEM cell for further demonstrating the F4/80 positive of LPS processing by flow cytometer, the egg of Sun2 White level is significantly lower than the control group (Fig. 2, C) of PBS processing.Laser scanning confocal microscopy shows that LPS stimulates THP-1 cell Afterwards, not only the size of nucleus is obviously reduced, and the fluorescence intensity of Sun2 also obviously lowers, and the fluorescence intensity of LaminA/C does not have It substantially change (Fig. 2, D).Since TLRs agonist can also be polarized with the M1 type of inducing macrophage, the present inventor is with a series of The ligand of TLRs handles THP-1 cell, including MALP-2 (TLR6-TLR2), poly (I:C) (TLR3), LPS (TLR4), R-848 (TLR7) or CpG DNA (TLR9), its influence to Sun2 protein level is detected.It is similar to the result of LPS stimulation, removing poly (I:C) under the stimulation of all TLR ligands outside, the protein level of Sun1/2 is lowered, this shows that TLR signal path and macrophage are thin The activation of born of the same parents has universal correlation (Fig. 2, E) independent of specific stimulating factor.The protein level pair of Sun1/2 It is most important in the micromechanical force environment in regulating cell core, thus, Sun1/2 may participate in the process of macrophage activation (Fig. 2, F).

If SUN protein delation participates in the micromechanical force environment in regulating cell core, in the mistake that M1-M2 polarizes and breaks up Cheng Zhong, expression or expression pattern will change.The present invention detects the egg of Sun1/2 in discovery M1 type macrophage White level is lower than M0 or M2 type macrophage (Fig. 2, G).Equally, the monocyte of derived from bone marrow is handled with GM-CSF and M-CSF, It is divided into the macrophage of class M1 type and class M2 type, Sun1/2 shows different expression patterns (Fig. 2, H).

In conclusion nuclear membrane Protein S un1/2 is likely via the micromechanical force environment in regulating cell core, ginseng With the differentiation of regulation macrophage, polarization and activation.

Embodiment 3:CK2 α phosphorylation SUN2 albumen promotes β TrCP degradation SUN albumen

1, experiment purpose: the mechanism of SUN2 protein level variation in research macrophage polarization process.

2, experimental method:

(1) experimental method of the mass spectral analysis of co-immunoprecipitation is as shown in basic experiment method 7 and 10.

(2) shRNA of β TrCP1/2

The target sequence (shRNA) of one section of 21 base of design knocks out the code area of β TrCP1/2, which can form instead To complementary hair fastener shape structure, silencing related gene expression.β TrCP1/2 shRNA and control shRNA (scramble) connection To slow virus carrier pLKO.1-puro.ShRNA (scramble) is control.

People sh β TrCP1/2 oligo (is directed to the code area sh β TrCP1/2)

It is positive:

5'CCGGAAGTGGAATTTGTGGAACATCCTCGAGGATGTTCCACAAATTCCACTTTTTTTG(SEQ ID NO:1)

It is reversed:

5'AATTCAAAAAAAGTGGAATTTGTGGAACATCCTCGAGGATGTTCCACAAATTCCACTT 3'(SEQ ID NO:2)

The forward and reverse DNA chain of 0.01M mixes, and is diluted to 50ul, by reacting as follows:

95℃	5min
		70℃	10min
55℃	30min
		37℃	30min

Annealing is connected as double-strand.Double-stranded DNA is connect with pLKO.1-puro (EcoRI/AgeI double digestion) carrier, converts DH5 α, the positive colony identified extract plasmid after sequence verification.By 293 cell of plasmid transfection, sieved with 2 μ g/ml puromycins The cell for selecting genome stable integration shRNA sequence after 48-72 hours, uses Vivaspin 20ml (Sartorius Stedim Biotech, Aubagne, France) with the centrifugal force of 3000g, it is centrifuged 50min, concentrating virus liquid.

PLKO.1-puro plasmid is from Shanghai Inst. of Life Science, CAS institute biochemistry and RESEARCH ON CELL-BIOLOGY It obtains at institute Ji Hongbin researcher, for the common carrier of commercially available building RNAi, can be bought from Addgene company.

(3) 131/132/136 mutant serine of SUN albumen is alanine (" SA ")

SUN mutant carries out PCR amplification using wild type SUN as template, using primer shown in SEQ ID NO:3-4 in table 1 Afterwards, unmutated wild type SUN2 template is removed using DpnI demethylase, amplification obtains the band for having specific locus mutation There are the carrier segments of SUN2 gene, PCR product is converted into DH5 α competent cell, passes through resistance screening, the positive colony of acquisition.

Table 1: primer sequence

(4) experimental method of CK2 α external kinase activity experiment is as shown in basic experiment method 13.

3, experimental result and analysis:

The mechanism declined for SUN1/2 protein level after research TLRs stimulation.The present inventor has detected the egg of SUN1/2 first White level lowers whether depend on proteasome pathway.Inhibited with the proteasome inhibitor MG132 processing meeting of PEM cell (part) The protein level of SUN1/2 caused by LPS is induced reduces.Further study showed that LPS processing can be continuously increased the ubiquitin of SUN2 Change horizontal (Fig. 3, B).And the transcriptional level of SUN1/2 is not substantially change after LPS processing.This shows that LPS stimulation is to pass through promotion SUN proteins ubiquitin and proteasome degradation pathway cause SUN protein level to be lowered.

Next, the present inventor sets about finding the E3 ubiquitin ligase of degradation SUN albumen.Mass spectral analysis and co-immunoprecipitation Experiment shows that β TrCP1/2 may exist with SUN1/2 and interacts.It is related to SUN protein degradation for verifying β TrCP1/2 Property, the present inventor has transfected the shRNA (sh β TrCP1/2) for β TrCP1/2 in cell, it is real then to carry out immunoblotting It tests.As shown in Fig. 3 (C), the protein level that SUN1/2 can be remarkably reinforced in β TrCP1/2 is knocked out, and MG132 can (part) resistance The aggregation for the SUN1/2 that disconnected β TrCP1/2 is mediated.(expression vector is commercial vectors to cotransfection β TrCP1/2 in cell PCDNA3.1) ubiquitination level of SUN2 is enhanced, its protein level is reduced, and there is dose-dependent effect (Fig. 3, D).This table Bright β TrCP1/2 is the E3 ubiquitin ligase of SUN2 albumen.

Sequence analysis shows, " SSSGYSSSEDD " is potential β-TrCP1/2 binding motif, and dynamic in different lactations There is conservative (Fig. 3, E) in the SUN albumen of object.Mass spectral analysis shows that S131, S132 and S136 are potential phosphorylation sites, By structural simulation it has also been found that having the binding motif of β TrCP1/2 on SUN2 albumen, the two may carry out phosphorus by classical mode Acidification reaction (Fig. 3, F).The phase interaction of β-TrCP1/2-SUN2 whether is mediated for the identification motif of the β-TrCP1/2 of research supposition With, 131/132/136 mutant serine of SUN2 albumen is alanine (" SA ") by the present inventor, to inhibit its phosphorylation, Combination between the two is destroyed, and then inhibits the ubiquitination of SUN2 albumen.It is overexpressed β-TrCP1 and significantly improves the general of SUN2 albumen Elementization is horizontal, but does not influence (Fig. 3, G) to its SA mutant.

To study whether the expression of SUN2 albumen influences intracellular micromechanical force environment, the present inventor is in cell The SA mutant of SUN2 is transfected, which by β-TrCP1/2 ubiquitination and cannot degrade.It is different from cellular control unit, transfection The size of the cell average cell core of SUN2SA mutant is not influenced (Fig. 3, H) by LPS stimulation.In addition, AFM analytical table Bright, the elasticity and hardness for having transfected the nucleus of the cell of SUN2SA mutant, which are much higher than, has transfected empty carrier or wild type SUN2 Cell (Fig. 3, I).The above result shows that in the macrophage of LPS stimulation, the micromechanical force environment of nucleus by The regulation of SUN albumen.

Since the phosphorylation of SUN2 is particularly significant by β-TrCP1/2 identification for it, the present inventor is further studied During SUN2 protein level is lowered in LPS stimulation, the kinases of phosphorylation SUN2 albumen.The present inventor uses protein kinase motif Prediction algorithm, wherein CK2 may phosphorylation SUN2 136 serines, phosphorylation motif S/T-X-X-D/E.Since LPS can With the CK2 in activating macrophage, the present inventor speculates that the activity of CK2 may become the protein level of the LPS SUN1/2 induced Change plays an important role.The present inventor's inhibitors 4 special using CK2,5,6,7- tetrabromo benzotriazole (TBB) block its kinases living Property.The results show that the SUN albumen that 10 μM of TBB can block LPS in the macrophage in the source THP-1 to induce downward (Fig. 3, J).This shows that the kinase activity of CK2 is related to the downward of SUN protein level.External kinase activity experiment also demonstrates CK2 α The SUN2 albumen (Fig. 3, K) of Direct Phosphorylation purification.In addition, similar with SUN2SA mutant, it is flat for the cell of TBB processing Equal nucleus size is not influenced (Fig. 3, L) by LPS stimulation.Equally, after TBB processing, LPS will not influence the elasticity of nucleus And hardness (Fig. 3, M).

In conclusion the proteasome pathway regulation that the protein level of SUN1/2 is mediated by CK2 and β TrcP, egg The variation of white level will affect the micromechanical force environment of nucleus in macrophage polarization process.

Embodiment 4:SUN albumen changes mechanical force microenvironment in nucleus and influences macrophage function

1, experiment purpose: SUN albumen influences the mechanism of macrophage function in research macrophage polarization process.

2. experimental method:

(1) SUN1/2 is knocked out

The siRNA and negative control for knocking out SUN1 and SUN2 are synthesized by Shanghai JiMa pharmacy Technology Co., Ltd. SiRNA sequence is as shown in table 2.

Table 2:siRNA sequence

(2) the marker molecule such as Arg1 of macrophage marker molecule such as Il6, Il1b and Nos2 and M2 type macrophage, The expression of Mrc1 (mannose receptor CD206) and Retnla

Cell extraction total serum IgE method is as described in basic experiment method 6, and AMV reverse transcriptase synthesizes the first chain, and using it as mould Plate carries out PCR amplification with corresponding primer under the action of DNA taq enzyme；Reverse transcription system is shown in Table 3, reverse transcription reaction program 4 are shown in Table, cell sample amplification PCR primer is shown in Table 5, Realtime-PCR reaction system and is shown in Table 6, Realtime-PCR amplification program It is shown in Table 7.

Table 3: reverse transcription reaction system

Table 4: reverse transcription process

37℃

15min

50℃

5min

95℃

5min

4℃

20min

Table 5: primer sequence

Table 6:Realtime-PCR reaction system

Table 7:PCR reaction system program setting

(3) metaboilic level of macrophage uses the seahorse glycolysis detection kit of Agilent Technologies.Inspection Survey method is by described in product manual.

3, experimental result and analysis:

Further to verify influence of the SUN albumen in nucleus in micromechanical power, the present inventor has detected knockout The nucleus of the cell of Sun1/2 expression.As shown in Fig. 4 (A), the nucleus for knocking out the PEM cell of Sun1/2 compares wild-type cell It is substantially reduced.In addition, LPS stimulation no longer has an impact nucleus size.Equally, the l cell that Sun1/2 is knocked out (MEF) elasticity and hardness of nucleus significantly reduce (Fig. 4, B) than wild type.The above result shows that LPS stimulation changes cell Micromechanical force environment in core depends on SUN albumen.And human intervention adjusts intracellular SUN protein level and can change carefully Micromechanical force environment in karyon.

Since the reconstruct of nucleus can deeply influence cell function and phenotype, and caused by SUN albumen stimulates LPS The change of endonuclear micromechanical force environment is most important, and the present inventor, which speculates that SUN albumen has, influences macrophage function The important function of energy.Sun1/2 is knocked out in PEMs cell, relative to control group, the phagocytosis of cell is enhanced, this shows SUN albumen has negative regulation effect (Fig. 4, C) for the activation of M1 type macrophage.The present inventor analyzes M1 or M2 activation The expression of marker molecule in mouse PEM cell.Knocking out SUN1/2 can be enhanced M1 type macrophage marker molecule such as Il6, Il1b and Nos2, and the marker molecule such as Arg1 of M2 type macrophage, Mrc1 (mannose receptor CD206) and Retnla Expression declines (Fig. 4, D).This illustrates that lacking SUN albumen can promote macrophage to break up to M1 type.

The present inventor then has detected whether SUN1/2 is changing the same of the expression of macrophage polarization special molecular When, also change the metaboilic level of macrophage.Macrophage activation be M1 or M2 type during along with metaboilic level Variation generates lactic acid, has high extracellular acidification rate if the glycolysis level of M1 type macrophage increases；And M2 type macrophage Activation needs the beta-oxidation of fatty acid, thus mitochondria consumption rate (OCR) rises, and consumes carnitine and palmitate to promote rouge Fat acid oxidase (FAO).It is similar to the promotion polarized result of M1 type obtained before, knock out the generation that SUN1/2 changes macrophage It thanks to level, promotes its glycolysis, reduce fatty acid oxidation (Fig. 4, E, F).In addition, no matter in M1 type or M2 type macrophage, It knocks out SUN1/2 and all further increases ECAR level, it is horizontal (Fig. 4, F) to reduce OCR.These results suggest that reducing SUN albumen water The flat rearrangement for promoting macrophage metabolic process, makes it polarize to M1 type, to realize corresponding biological function.

The present inventor detects the influence that SUN albumen generates M1 type and M2 type macrophage.Flow cytometry shows In SUN1/2^-/-In the PEM cell of knockout, M1 type (CD86⁺) macrophage ratio greatly improves, and M2 type (CD206⁺) macrophage is thin Born of the same parents' ratio significantly reduces (Fig. 4, G).(the F4/80 in addition, the M2 type of PEM cell polarizes⁺CD206⁺) blocking is obviously knocked out by SUN1/2 (Fig. 4, H).

These results suggest that SUN albumen is one in the micromechanical force environment of nucleus in macrophage polarization process Important transmitting molecule.Knocking out SUN albumen promotes macrophage to break up to M1 type.

The protein mediated nucleus micromechanical power of embodiment 5:SUN, which changes, influences cytoskeleton and cellular morphology

1, experiment purpose: the influence of SUN protein on cells skeleton and cellular morphology in research macrophage polarization process.

2, experimental method:

Cytoskeleton is observed by laser confocal microscope, and experimental method is as shown in basic experiment method 9

3, experimental result and analysis:

Since SUN albumen is the important component of LINC compound, cytoskeleton and nucleus, therefore, the present inventor are connected Speculate, SUN albumen may affect the rearrangement of cytoskeleton and the reconstruct of nucleus and cellular morphology, and then influence macrophage The polarization and activation of cell.To verify this point, the present inventor passes through cell shape after the detection LPS stimulation of ultrahigh resolution microscope The variation of state.The result shows that the protein expression level of SUN2 reduces after LPS stimulation, nucleus reduces (Fig. 5, A).Meanwhile it knocking out SUN1/2 not only makes the cytoskeleton rearrangement of macrophage, also changes the form of its fold and lamellipodia, promotes it to the type side M1 To differentiation.The level of beta-actin in soluble constituent and insoluble component after Triton X-100 processing is further had detected, To characterize the associated actin of cytoskeleton and intracellular actin, i.e., non-polymeric G- actin and polymerization F- actin.After LPS handles PEM cell, beta-actin level decline in insoluble component after Triton X-100 processing, F- actin, which is presented, significantly removes polymerization state.And SUN1/2^-/-In the PEM cell of knockout, β-flesh moves egg in insoluble component White level further declines (Fig. 5, B).

The above result shows that nuclear membrane Protein S UN1/2 affects the rearrangement of cytoskeleton.Swash in M1 type macrophage In process living, SUN protein expression level is lowered, and is influenced the micromechanical force environment of nucleus, is reduced nucleus, nucleus Intermembrane space becomes larger, and nucleus hardness reduces, and then influences cytoskeleton and cellular morphology (Fig. 5, C, D).

Embodiment 6:SUN albumen negative regulation TLR signal path

1, experiment purpose: regulating and controlling effect of the research SUN albumen to the TLR signal path of macrophage activation

2, experimental method:

(1) experimental method of the expression detection of Tnf α, Il1b, Il6 with embodiment 4, Il1b, the inspection of Il6 expression Survey primer is shown in Table the horizontal detection primer of 5, Tnf alpha expression and is shown in Table 8.

Table 8: primer sequence

(2) the SUN2 plasmid of wild type, mutant DelTM

SUN2 plasmid is provided by research institute, Univ Pennsylvania USA Mark I professor Green.

Deletion mutant DelTM is cloned with wild type SUN2 as template, passes through Takara high-fidelity DNA polymerase PrimeSTAR Amplification obtains the carrier segments with SUN2 gene with missing specific domain, is gone using DpnI Methylase removes unmutated wild type SUN2 template, and pcr amplified fragment is converted DH5 α competent cell, utilizes large intestine bar The characteristics of repair vector that bacterium itself has missing, whole fragment is cyclized to form carrier, by resistance screening, acquisition with original The SUN2 carrier framework of beginning is identical, but the positive colony of the corresponding TM structural domain of SUN2 fragment deletion, uses after sequence verification is correct In experiment.

PCR primer is shown in Table 9, PCR reaction system and is shown in Table 10.PCR amplification program are as follows: 98 DEG C of 2min；PCR cycle: 98 DEG C 10s, 58 DEG C of 10s, 68 DEG C of 1kb/min, 18 circulations；68℃10min.

Table 9: primer sequence

Table 10:PCR reaction system

(3) sensibility of TLR ligand is obtained by the horizontal of detection TNFa and IL-6.It collects cell and culture solution carries out ELISA detects cell factor.The level of TNF α and IL-6 are detected using eBioscience kit in human archeocyte.Source of mouse is thin The level of TNF α and IL-6 are detected using BD Bioscience kit in born of the same parents.Detection method is by described in product manual.

(4) NF- κ B Reporter Assay system is from Shanghai Inst. of Life Science, CAS institute biochemistry and cell It is obtained at biological study institute Wang Chen researcher, plasmid information is referring to (2007, the UXT is a novel such as Sun S andessential cofactor in the NF-κB transcriptional enhanceosome.JCB 178(2): 231-244).Double fluorescein assays are as described in basic experiment method 4.

3, experimental result and analysis:

Since TLR plays a significant role in macrophage activation and function, the present inventor have detected LPS stimulation for Influence of the SUN albumen to TLR4 downstream signaling pathway.As shown in Fig. 6 (A), in wild type PEM cell, LPS stimulation can be lured Lead degradation and the kinase activity (IKK β, JNK, ERK) of I κ B α.And after knocking out SUN albumen, LPS stimulation will aggravate the degradation of I κ B α And kinase activity, this shows SUN1/2 negative regulation TLR4 signal path.Equally, caused by missing SUN1/2 can stimulate LPS to stimulate The expression (Fig. 6, B) of proinflammatory inflammation factor (Tnf α, Il1b, Il6).The present inventor has carried out rescue experiment also further to verify Effect in signal path caused by SUN albumen is stimulated in LPS.The transfected wild-type in the PEM cell of missing SUN1/2 SUN2, Il6 caused by inhibiting LPS to stimulate expression, and the SA mutant that SUN2 is transfected in same cell can further suppress The degradation of LPS induction and the transcription (Fig. 6, C) of Il6.And the mutant DelTM for transfecting missing SUN2 transmembrane region can't change The mRNA level in-site of Il6, it is necessary that this, which illustrates that positioning of the SUN2 on nuclear membrane regulates and controls TLR4 signal path to it,.

The present inventor has detected function of the SUN1/2 in different TLR ligand signal accesses.The result shows that different TLR The level of SUN albumen is different in ligand-mediated signal path, and knocking out SUN1/2 can be enhanced macrophage to except poly (I:C) The sensibility of outer all TLR ligands, wherein the influence of LPS and MALP2 (TLR7) are maximum (Fig. 6, D).

The present inventor further studies the mechanism of SUN protein regulation TLR signal path.The present inventor is thin in HEK293T NF- κ B reporter gene and SUN1/2 have been transfected in born of the same parents.As shown in Fig. 6 (E), it is overexpressed SUN albumen and NF- κ B is obviously inhibited to exist Activation under the stimulation of the activation signals such as MyD88, IRAK1, IKK β, TRAF6 or p65, and this inhibiting effect has dose-dependant Effect.And MyD88 can be promoted by knocking out SUN1/2, IRAK1, IKK β, the NF- κ B of TRAF6 and p65 induction activates (Fig. 6, F).

The above result shows that the signal path of SUN albumen corresponding regulating cell skeleton and cellular morphology, makes its expression Lower, by the regulation feedback regulation of micromechanical force environment in active cell core, enhance TLR signal path, this with it is existing It matches about relevant report of the activation of NF-kB and the variation of cytoskeleton.

Embodiment 7: the M1 type polarization that SUN albumen promotes macrophage is knocked out

1, experiment purpose: research SUN albumen is to the polarized regulating and controlling effect of macrophage

2, experimental method:

(1) cell migration ability detects

Macrophage migration ability passes through the cell Transwell detection (Corning, article No. 3422), detection method reference The product description.

(2) the marker molecule such as Arg1 of macrophage marker molecule Il6, Il1b and Nos2 and M2 type macrophage, The expression detection method of Mrc1 (mannose receptor CD206) and Retnla is the same as embodiment 4.

3, experimental result and analysis:

We have detected the phagocytic function of PEM cell first.Relative to control cell, SUN albumen is knocked out in PEM cell Significantly enhance its phagocytic activity and phagocytosis efficiency (Fig. 7, A).In addition, we have detected the migration energy of cell using chamber technique Power, the results showed that knocked out in the macrophage in the source THP-1 SUN1/2 can significantly increase its cell migration ability (Fig. 7, B).By Flow Cytometry, we analyze the PEM cell for knocking out SUN1/2, wherein thin with the M1 type macrophage that CD86+ is characterized Born of the same parents' quantity obviously increases, and significantly reduces (Fig. 7, C) with the M2 type macrophage that CD206+ is characterized.We further have detected M1 And the expression of M2 type macrophage activation key gene, after knocking out SUN1/2, the mark molecule of M1 type macrophage, packet Il6, Il1b and Nos2 expression is included obviously to raise, and the mark molecule of M2 type macrophage, including Arg1, Mrc1 (i.e. sweet dew Saccharide acceptor CD206) and the obvious downward (Fig. 7, D) of Retnla expression.In conclusion macrophage will be promoted by knocking out SUN albumen It polarizes to M1 type.

Embodiment 8: the enhancing inflammatory reaction of SUN albumen and anti tumor immune response are knocked out

1, experiment purpose: research SUN albumen acts on inflammatory reaction and anti tumor immune response.

2, experimental method:

(1) mouse that SUN1/2 is knocked out is provided by Fudan University professor Han Min.Express the breast epithelium of polyomavirus T antigen Cell transgene mouse model (PyMT mouse) is purchased from The Jackson Laboratory company.SUN1/2-/- PyMT hybridization Mouse is obtained by above two mouse hybrid.Animal culture and zoopery are in accordance with Chinese Academy of Sciences's Shanghai school of life and health sciences biology Chemistry article related to Institute of Cell Biology Animal Management Committee and animal welfare policy.

(2) method of the mouse infection shock of LPS induction is as shown in basic experiment method 13.

(3) method of adoptive cellular is as shown in basic experiment method 14.

(4) histochemical analysis method is as shown in basic experiment method 15.

3, experimental result and analysis:

Further to study the function of SUN albumen in vivo and verifying its effect in macrophage, the present inventor is ground Response of the mouse in the infectious shock induced LPS of SUN1/2 knockout is studied carefully.In view of the redundancy of SUN1/2 protein function, We in macrophage by will specifically knock out the mouse (SUN1 of SUN1^flox/floxLysM^cre/cre) and SUN2 knockout Mouse (SUN2^-/-) hybridization obtain mouse (SUN1^flox/floxLysM^cre/creSUN2^-/-).Age and gender are identical one group small It is observed 8 days after mouse injection LPS.Compared to the mouse of wild type, SUN1^flox/floxLysM^cre/creAnd SUN2^-/-Mouse injection It is easier to death after LPS, and SUN1^flox/floxLysM^cre/creSUN2^-/-The survival rate of mouse is minimum (Fig. 8, A).In addition, knocking out SUN1/2^-/-Mouse lung can detecte LPS induction generate pro-inflammatory cytokine (TNF α, IL-1 β and IL-6) (Fig. 8, B).The wild-type mice mean survival time for having injected LPS is 5 days, and SUN1/2 is knocked out in macrophage^-Mouse Mean survival time be only 2 days.This, which shows to knock out SUN1/2 in macrophage, can make the inflammation damnification of LPS induction generation more Seriously.

The present inventor has detected effect of the protein mediated macrophage polarization of SUN in antineoplastic immune.In wild type And SUN1^flox/floxLysM^cre/creSUN2^-/-B16-F10 melanoma cells are injected in the mouse of knockout respectively, in SUN1^{flox /flox}LysM^cre/creSUN2^-/--The quantity of tumour sharply reduces (Fig. 8, C) in the mouse of knockout.SUN1/2^-/-The mouse of knockout Lung is lighter than the control mice of its littermate.Meanwhile histochemical analysis shows SUN1/2^-/-The structure of the lung of the mouse of knockout with just The structure of normal lung is almost the same (Fig. 8, D).And the SUN1/2 that adopted^-/-The quantity of the B16-F10 mouse tumor of macrophage compared Quantity after the B16F10 mouse tumor of wild type macrophage is substantially reduced.

Since SUN albumen promotes macrophage to break up to M1 type, the present inventor guesses that SUN albumen may inhibit the shape of TAMs At (i.e. the class M2 type differentiation of macrophage) and inhibit tumour growth.The present inventor is by PyMT mouse and SUN1^{flox/ flox}LysM^cre/creSUN2^-/-Mouse hybrid obtains SUN1^flox/floxLysM^cre/creSUN2^-/-PyMT hybridizes mouse, the mouse Tumour time of origin obvious postpone, the generation of tumor of breast are also suppressed to relatively great extent (Fig. 8, E).Histochemical analysis shows The state in growth diffusion of tumour in control group mice, and in SUN1^flox/floxLysM^cre/creSUN2^-/-PyMT hybridization is small In mouse, the development of tumour is obviously suppressed (Fig. 8, F).

It can be by promoting the M1 of macrophage to polarize and go TAMs polarization to enhance inflammation in conclusion knocking out SUN1/2 Reaction and antineoplastic immune.

Sequence table

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Claims (10)

1. reducing SUN2 protein level or making the reduced activity of SUN2 albumen participation cytoskeleton rearrangement or the reagent of forfeiture The purposes in the drug for the disease that M1 type macrophage increases is benefited from standby treatment, or promotes the M1 polarization of macrophage in preparation Or purposes in the polarized drug of tumor-associated macrophage is gone, or in preparation enhancing inflammatory reaction or the drug of antineoplastic immune In purposes.

2. purposes as described in claim 1, which is characterized in that the reagent for reducing SUN2 protein level is selected from:

(1) reagent of CK2 kinase expression level is improved；

(2) reagent of E3 ubiquitin ligase expression is improved；With

(3) inhibit SUN2 gene expression or reduce the reagent of its expression.

3. purposes as claimed in claim 2, which is characterized in that

It is described improve CK2 kinase expression level reagent be CK2 kinases expression vector, or be selected from TLR2, TLR4, TLR7 with The agonist of TLR9 is preferably selected from MALP-2, LPS, R-848 and CpG DNA；

The reagent for improving E3 ubiquitin ligase expression is the expression vector of E3 ubiquitin ligase, it is preferable that the E3 Ubiquitin ligase is β TrCP1 or β TrCP2；

The inhibition SUN2 gene expression or to reduce the reagent of its expression be for the siRNA of SUN2 gene or for knocking out The carrier of SUN2 gene；Preferably, the siRNA is as shown in SEQ ID NO:6.

4. purposes as described in claim 1, which is characterized in that the activity for making SUN2 albumen participate in cytoskeleton rearrangement subtracts Reagent that is weak or losing acts on SUN2 gene, ties the SUN2 albumen of the coded by said gene in its transmembrane domain and/or SUN There is the mutation of the reduced activity or forfeiture that cause SUN2 albumen to participate in cytoskeleton rearrangement in structure domain.

5. such as purposes of any of claims 1-4, which is characterized in that the M1 type macrophage of benefiting from increases Disease be tumour, tumour preferably relevant to tumor-associated macrophage is more preferably selected from breast cancer, oophoroma, uterine neck Cancer, melanoma and prostate cancer.

6.CK2 kinases or its gene, E3 ubiquitin ligase or its gene and/or SUN2 gene or SUN2 albumen are being made as target spot The purposes in the drug for the disease that M1 macrophage increases is benefited from standby or screening treatment.

7. purposes as claimed in claim 6, which is characterized in that

The CK2 kinases is CK2 alpha kinase, and nucleotides sequence is classified as the sequence of Serial No. J02853.1 in GenBank, amino Acid sequence is the sequence of Serial No. AAA56821.1 in GenBank；

The E3 ubiquitin ligase is β TrCP1 or β TrCP2, wherein the nucleic acid sequence of β TrCP1 is Serial No. in GenBank The sequence of AF101784.1, protein sequence are the sequence of Serial No. AAD08702.1 in GenBank；The nucleic acid sequence of β TrCP2 For the sequence of Serial No. AF176022.1 in GenBank, protein sequence is the sequence of Serial No. AAF04528.1 in GenBank Column；

The SUN2 gene or SUN2 albumen behaviour SUN2 gene or people's SUN2 albumen；Preferably, the nucleosides of the SUN2 gene Acid sequence is the sequence of Serial No. NM_001199580 in Genebank；The SUN2 protein sequence is sequence in Genebank Number be AAT90500.1 sequence.

8. purposes as claimed in claims 6 or 7, which is characterized in that described to benefit from the disease that M1 macrophage increases be swollen Tumor, tumour preferably relevant to tumor-associated macrophage, is more preferably selected from breast cancer, oophoroma, cervical carcinoma, melanoma And prostate cancer.

9. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition contains the reagent for being reduced SUN2 protein level or makes SUN2 albumen participates in the reagent of cytoskeleton rearrangement miopragia or forfeiture；

Preferably, the reagent for reducing SUN2 protein level is as claimed in claim 3；It is described that SUN2 albumen is made to participate in cell bone Frame resets miopragia or the reagent of forfeiture is as claimed in claim 4.

10. a kind of method for the inflammatory reaction and antineoplastic immune or the tumour for treating the patient for enhancing tumor patient, feature It is, the method includes reducing object SUN2 protein level or its SUN2 albumen is made to participate in cytoskeleton rearrangement miopragia Or the step of losing.