WO2016112849A1 - New use of i-type interferon and mesenchymal stem cell in preparation of antineoplastic drug - Google Patents

Abstract

Provided are mesenchymal stem cells (MSCs) pre-treated by I-type interferon (IFN) or over-expressing the I-type IFN, and use of the MSCs in the preparation of antineoplastic drug. Also provided is a use of the I-type IFN in the preparation of a composition for improving the expression of MHC I in the MSCs, reducing the immunosuppression ability of the MSCs, improving the antitumor immune response of organism, inhibiting the DNA combining ability of STAT1 in the MSCs and reducing the expression of inducible nitric oxide synthase (iNOS) in the MSCs.

Description

New use of type I interferon and mesenchymal stem cells in the preparation of antitumor drugs Technical field

The present invention belongs to the field of immunology, and more particularly, the present invention relates to a novel use of type I interferons and mesenchymal stem cells in the preparation of antitumor drugs.

Background technique

Mesenchymal Stem Cells (MSCs) play an important role in the treatment of immune disorders due to their immunomodulatory effects. More importantly, the immunosuppressive effect of MSCs is the stimulation of inflammatory factors. Moreover, the core molecules that mediate the immunosuppressive effects of MSCs are species-differentiated, inducible nitric oxide synthase (iNOS) in mouse MSCs, and indoleamine-2,3 in human MSCs. - Dioxygenase. In mouse MSCs, the production of iNOS is mainly dependent on co-stimulation of IFNγ and TNFα or IL-1, of which IFNγ is essential.

It is well known that the interferon-type I interferon (IFNα and IFNβ) and type II interferon (IFNγ) and TNFα synergistically induce mouse macrophages to express iNOS. However, for MSCs, type I interferon IFNα or IFNβ is not a substitute for IFNγ and TNFα to induce iNOS expression.

Interferon (IFN), first discovered by British scientist Isaacs in 1957 using the chicken chorioallantoic membrane to study the phenomenon of influenza virus interference, is a cytokine that inhibits cell division, regulates immunity, and is antiviral. Anti-tumor and other effects. Its essence is protein, and its type can be divided into several types such as α, β, γ, and ω.

IFN can induce cell resistance to viral infection. It is the most important anti-viral infection and anti-tumor biological product by interfering with viral gene transcription or translation of viral protein components to prevent or limit viral infection. However, in the clinical application, type I interferon involves the problem of large side effects and short half-life, which limits its application value.

Summary of the invention

The object of the present invention is to provide a novel use of type I interferons and mesenchymal stem cells in the preparation of antitumor drugs.

In a first aspect of the invention, there is provided a mesenchymal stem cell which is a type I interferon-pretreated mesenchymal stem cell or a mesenchymal stem cell overexpressing type I interferon.

In a preferred embodiment, the pre-processing comprises:

The type I interferon is mixed with the mesenchymal stem cells such that the final concentration of the type I interferon is 200-10000 U/ml; preferably 300-8000 U/ml; more preferably 500-5000 U/ml; for example 800U /ml, 1000U/ml, 15000U/ml, 2500U/ml.

In another preferred embodiment, the type I interferon-overexpressing mesenchymal stem cells are transformed with a polynucleotide encoding a type I interferon.

In another preferred embodiment, the type I interferon is selected from the group consisting of: IFNα or IFNβ; or the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells, or umbilical cord, fat, placenta, gums, etc. Mesenchymal stem cells.

In another preferred embodiment, the type I interferon is IFNα, and the amino acid sequence thereof is shown in GenBank Accession No. NP_034633.2.

In another preferred embodiment, the type I interferon is IFNβ and the amino acid sequence thereof is shown in GenBank Accession No. AAA37891.1.

In another aspect of the invention, there is provided the use of the mesenchymal stem cell for the preparation of a medicament for inhibiting tumors.

In a preferred embodiment, the tumor is melanoma.

In another aspect of the present invention, a pharmaceutical composition for inhibiting a tumor comprising any of the mesenchymal stem cells described above is provided.

In another aspect of the invention, there is provided a use of a type I interferon for the preparation of a composition for increasing the expression of MHC I in a mesenchymal stem cell; or for the preparation of a reduced immunosuppressive ability of a mesenchymal stem cell (ie, a transfer chamber) A composition in which a mesenchymal stem cell exerts an immune promoting effect or an anti-tumor immune response of the body.

In another aspect of the invention, there is provided a use of a type I interferon for preparing a composition for inhibiting DNA binding ability of STAT1 in mesenchymal stem cells; or for reducing inducible nitric oxide synthesis in mesenchymal stem cells A composition for the expression of an enzyme.

In another aspect of the invention, the use of any of the foregoing, wherein the type I interferon is selected from the group consisting of: IFNα or IFNβ.

Other aspects of the invention will be apparent to those skilled in the art from this disclosure.

DRAWINGS

Figure 1. IFNα alone or in combination with TNFα does not stimulate nitric oxide production in MSCs.

(A) Bone marrow-derived macrophages or MSCs, stimulated with various combinations of cytokines of IFNγ (10 ng/ml), TNFα (10 ng/ml) or IFNα (2500 U/ml) for 24 hours, and assayed for culture by Greiss reagent The content of nitric oxide in the Qing; Control is PBS. The figure in the figure is means±SD.

(B) MSCs were stimulated with IFNγ (10 ng/ml), IFNα (2500 U/ml) or IFNβ (2500 U/ml) for 12 hours, and then the expression of MHC class I molecule K ^b D ^b was detected by flow cytometry. Data are 1 representative result from 3 independent replicates.

Figure 2. IFNα can attenuate the immunosuppressive effects of MSCs.

(A) MSCs were co-cultured with CD3 antibody-activated splenocytes and treated with L-NMMA (1 mM) or IFNα (2500 U/ml) for 48 hours. Nitric oxide in the culture supernatant was determined by the Greiss method, and the level of cell proliferation was measured by ³ H-TdR incorporation. Control is the PBS treatment group.

(B) MSCs of different densities were co-cultured with CD3 antibody-activated splenocytes (Spl) and treated with IFNα (2500 U/ml) or IFNβ (2500 U/ml) for 48 hours. The level of cell proliferation was measured by ³ H-TdR incorporation. The figure in the figure is means±SD. Data are 1 representative result from 2 independent replicate experiments. Control is the PBS treatment group.

Figure 3. IFNα inhibits IFNγ and TNFα-mediated iNOS production in MSCs.

AC and MSCs were stimulated with various cytokine combinations of IFNγ (10 ng/ml), TNFα (10 ng/ml) or IFNα (2500 U/ml) for 24 hours, and the mRNA (A) and protein expression levels of iNOS were detected (B left). And the content of nitric oxide in the supernatant (B right); Control is the PBS treatment group. The figure in the figure is means±SD. Data are 1 representative result from at least 3 independent replicate experiments.

Figure 4. IFNα does not affect the expression of most of the cytokines, chemokines, and the like detected by the present inventors. MSCs were treated with IFNγ (10 ng/ml) and TNFα (10 ng/ml) or IFNγ (10 ng/ml), TNFα (10 ng/ml) and IFNα (2500 U/ml) for 24 hours. The supernatant was detected by Bio-Plex; Control was PBS treatment group. The figure in the figure is means±SD. Data are 1 representative result in at least 2 independent replicate experiments. The abscissa below each column map applies to all graphs in the column as the abscissa.

Figure 5. IFNα treatment did not affect IFNγ receptor expression in MSCs.

(A) MSCs were treated with IFNy (10 ng/ml) and TNFα (10 ng/ml) or IFNγ (10 ng/ml), TNFα (10 ng/ml) and IFNα (2500 U/ml) for 24 hours. Total RNA collected was detected using Affymetrix mouse 4302.0 expression profile chip; Control was treated with PBS. The ordinate on the left is applied to all the maps in the row as the ordinate.

(B) MSCs were treated with IFNγ (10 ng/ml) and TNFα (10 ng/ml) or IFNγ (10 ng/ml), TNFα (10 ng/ml) and IFNα (2500 U/ml), and IFNγ was detected by flow cytometry. The level of expression of the body. The values in the figure are means ± SEM; Control is the PBS treatment group.

Figure 6. IFNα inhibits the binding activity of STAT1 in MSCs.

(A) MSCs were harvested after treatment with various cytokines (TNFα (10 ng/ml), IFNγ (10 ng/ml), IFNα (2500 U/ml) for 24 hours to collect total protein. STAT1 and p65 were incubated and enriched with agarose microbeads ligated to their respective binding site oligonucleotide sequences, and then detected by western blotting.

(B) MSCs were first transfected with STAT1 transcriptionally active reporter plasmid-2×SIE luciferase plasmid, then treated with various cytokines and their combinations, and finally STAT1 was detected by luciferase assay kit at different time points. Transcriptional activity. The ordinate on the left is applied to all the maps in the row as the ordinate.

(C) MSCs were treated with a combination of various cytokines (TNFα (10 ng/ml), IFNγ (10 ng/ml), IFNα (2500 U/ml), and mRNA levels of IL-6 and iNOS were measured at different time points. The values in the figure are means ± SEM. Data are 1 representative result from 3 independent replicates.

Figure 7. IFN[alpha] pretreated MSCs significantly inhibited the growth of mouse melanoma in vivo.

(A) B16-F0 melanoma cells (1×10 ⁶ /25 μl) were co-injected with IFNα (0.25 μg/g mouse body weight) treated MSCs, MSC-GFP or MSC-IFNα (1×10 ⁶ /25 μl). Leg muscles of C57BL/6 mice. After 12 days, the tumor was excised from the legs of the sacrificed mice and weighed.

(B) MSCs were pretreated with IFNα (2500 U/ml) for 24 hours, and then co-injected into the leg muscles of C57BL/6 mice by the method of A with B16-F0 melanoma cells. MSCs (2 × 10 ⁶ ) pretreated with IFNα for 24 hours were injected into the tumor site every 3 days. After 14 days, the tumor was stripped from the legs of the sacrificed mice and weighed. The values in the figure are means ± SEM. Data are 1 representative result from 2 independent replicate experiments.

detailed description

The inventors have intensively studied for the first time that type I interferon can inhibit the growth of tumor by inhibiting the expression of iNOS by mesenchymal stem cells (MSCs). The invention provides a new important mechanism for the application of type I interferon and MSCs in anti-tumor, and proposes a new clinical treatment route.

To determine whether type I interferons have signal transduction in MSCs, the inventors first tested the expression of genes regulated by type I interferons on MSCs. The results showed that type I interferon could up-regulate the expression of MHC I on MSCs, and the effect was not significantly different from that of type II interferon. However, type I interferons can eliminate the immunosuppressive effects of MSCs. In view of the same regulatory effects of IFNα and IFNβ on MSCs, IFNα was used in subsequent experiments. Analysis of the signaling pathway revealed that IFNα did not affect IFNγ receptor expression, phosphorylation of STAT1 and NF-κB, and their entry into the nucleus. Luciferase assays and specific oligonucleotide binding experiments indicate that IFNα inhibits the binding capacity of STAT1, but not NF-κB. Therefore, IFNα reduces the expression of iNOS by inhibiting the binding ability of STAT1, thereby reducing the immunosuppressive ability of MSCs. The exertion of immunosuppression of MSCs is also dependent on the high level of chemokines expressed by them. With chemokines, MSCs can recruit immune cells such as T cells to their surroundings, thereby exerting their powerful immunosuppressive effects. However, microbead-based multi-channel cytokine results show that IFNα does not affect the expression of chemokines in MSCs. Therefore, the regulation of IFNs on MSCs is mainly regulated by the regulation of iNOS expression, but there is no expression of chemokines. Significantly affected. More importantly, the inventors demonstrated in vivo tumor growth experiments that IFNα pretreated MSCs can inhibit the development of B16-F0 melanoma by down-regulating the expression of nitric oxide.

Based on the novel findings of the present inventors, the present invention provides an MSCs which are pre-treated with type I interferon MSCs, or MSCs that overexpress type I interferons. Preferred are type I interferon-pretreated MSCs, which are simple to prepare, do not require transgenic manipulation, do not involve insertion of foreign genes, and do not present a safety problem when administered.

In one aspect, the MSCs are MSCs pretreated with type I interferon; preferably, the pretreatment comprises: mixing type I interferons with mesenchymal stem cells to achieve a final concentration of type I interferon Can be 200-10000U/ml.

The type I interferon is a known protein (polypeptide), which may be a recombinant polypeptide or a synthetic polypeptide.

As a preferred mode of the present invention, the type I interferon is IFNα, which may have the amino acid sequence shown by GenBank Accession No. NP_996753.1. The nucleotide sequence of the polynucleotide encoding IFNα may be GenBank Accession No. NC_000070.6.

In a preferred embodiment of the invention, the type I interferon is IFNβ, which may have the amino acid sequence set forth in GenBank Accession No. AAA37891.1.

Type I interferon variants or biologically active fragments formed by substitution, deletion or addition of one or more amino acid residues but retaining the same function as the type I interferon used in the examples of the invention are also included in the present invention. . Type I interferon variants or biologically active fragments include a replacement sequence for a portion of a conserved amino acid that does not affect its activity or retains its partial activity. Proper replacement of amino acids is a technique well known in the art that can be readily implemented and ensures that the biological activity of the resulting molecule is not altered. These techniques have taught one in the art that, in general, altering a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity. See Watson et al, Molecular Biology of The Gene, Fourth Edition, 1987, The Benjamin/Cummings Pub. Co. P224.

Any biologically active fragment of type I interferon can be used in the present invention. Here, the meaning of the biologically active fragment of type I interferon refers to a polypeptide which still retains all or part of the function of the full-length type I interferon. Typically, the biologically active fragment retains at least 50% of the activity of the full length type I interferon. Under more preferred conditions, the active fragment is capable of maintaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% activity of the full length type I interferon.

Type I interferons modified or modified may also be employed in the present invention, for example, Type I interferons modified or modified to promote their half-life, effectiveness, metabolism, and/or potency of the protein. That is, any variation that does not affect the biological activity of the type I interferon can be used in the present invention.

In the present invention, a polynucleotide sequence encoding an overexpressed type I interferon can be inserted into a recombinant expression vector to transform MSCs. Any plasmid and vector can be used in the present invention as long as it can be replicated and stabilized within the MSCs. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.

Methods well known to those skilled in the art can be used to construct expression vectors containing a type I interferon polynucleotide sequence and a suitable transcriptional/translational control signal. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The transformation vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Based on the novel findings of the present invention, there is also provided the use of a type I interferon-pretreated MSCs or MSCs overexpressing type I interferons for the preparation of a medicament for inhibiting tumors.

The tumor may be various benign or malignant tumors, such as melanoma, liver cancer, breast cancer, gastric cancer, prostate cancer, lung cancer, brain tumor, ovarian cancer, bone tumor, colon, thyroid tumor, mediastinal tumor, small intestine tumor, kidney Tumor, adrenal tumor, bladder tumor, testicular tumor, malignant lymphoma, multiple myeloma, nervous system tumor, etc.

MSCs have chemotaxis, which is capable of accumulating in tumor regions. Therefore, the interferon-pretreated MSCs or MSCs overexpressing type I interferons can achieve anti-tumor function at the tumor site.

Based on the novel findings of the present invention, the use of type I interferons is also provided for preparing a composition for improving the expression of MHC I in mesenchymal stem cells; or for preparing an immunosuppressive ability for reducing mesenchymal stem cells (ie, mobilization room) a composition in which a mesenchymal stem cell exerts an immune action to enhance an anti-tumor immune response; or a composition for inhibiting DNA binding ability of STAT1 in mesenchymal stem cells; or a preparation for reducing inducible type in mesenchymal stem cells Composition for expression of nitric oxide synthase

The present invention also provides a composition comprising an effective amount (e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight) of the interferon-pretreated MSCs or Type I interferon-expressing MSCs, and a pharmaceutically acceptable carrier.

Typically, the cells can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium. Wherein the pH is usually from about 5 to about 8, preferably, the pH is from about 6 to about 8.

As used herein, the term "contains" means that the various ingredients can be used together in the mixture or composition of the invention. Therefore, the terms "consisting essentially of" and "consisting of" are encompassed by the term "contains." As used herein, the term "effective amount" or "effective amount" refers to an amount that can produce a function or activity on a human and/or animal and that can be accepted by a human and/or animal.

As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, having a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the manufacturer. The suggested conditions.

Materials and Method

Reagent

Fluorescently labeled flow antibody and isotype control antibody used in flow cytometry were purchased from eBioscience; antibodies for immunoblotting: antibodies against iNOS, STAT1, p-STAT1, p65, p-p65, GAPDH, respectively, were purchased from Cell Signaling Technology the company. RNA extraction kits and reverse transcription kits were purchased from Tiangen biotech; RIPA lysates were purchased from Millipore; Nucleofector kit V was purchased from Amaxa; and the culture broth components used to culture MSCs were all purchased from Gibco.

Obtainment of MSCs: isolated and purified from mouse bone marrow. The isolated MSCs were cultured in low glucose DMEM medium.

In the present example, the IFNα protein (obtained from PBL assay science) for cell processing is used, and the amino acid sequence thereof is shown as the first position in GenBank Accession No. NP_996753.1. The overexpressed IFNα in MSCs has a polynucleotide sequence as shown in GenBank Accession No. NC_000070.6 or its degenerate sequence. The IFNβ has an amino acid sequence as shown in GenBank Accession No. AAA37891.1.

2. Experimental animals

The experimental mice were purchased from the Slack Shanghai Laboratory Animal Center of the Chinese Academy of Sciences and were raised in the SPF barrier environment of the Laboratory Animal Science Department of Shanghai Jiao Tong University School of Medicine. In all experiments, experiments were performed using mice of gender and age, and all animal procedures were reviewed by animal ethics.

3. Immunoblot analysis

(1) Preparation of protein samples

a) Prepare: ice, pre-chilled PBS, pre-cooled EP tube, pre-chilled centrifuge.

b) Place the experimentally treated cells on ice in time, interrupt the stimulation signal, discard the supernatant, wash it with pre-cooled PBS; add 1 ml of pre-cooled PBS, scrape the cells with cell scraping, and transfer to pre-cooled EP In the tube.

c) Centrifugation: 3000 rpm, 5 min; remove the supernatant.

d) Centrifuge again at 3000 rpm for 2 min to completely remove PBS.

e) Resuspend the cells with appropriate amount of lysate (PI and PMSF when used) (to avoid air bubbles as much as possible), place on ice for 30 min; mix every 10 min.

d) Centrifugation, 1.32X 10 ⁵ rpm, 15 min. Take the supernatant and store at -80 °C for later use.

(2) Immunoblot analysis

a) Experimental preparation: configure a suitable concentration of electrophoresis gel (PAGE); prepare electrophoresis buffer and transfer buffer.

b) Protein sample treatment: Take the sample from -80 ° C, melt on ice, determine the total protein concentration by BCA method, and dilute the total protein of each sample to the same concentration for use; add 3X SDS loading buffer, incubate at 100 ° C After 10 min, cool on ice for 2 min, centrifuge, and mix.

c) Place the prepared electrophoresis gel into the electrophoresis tank, add the electrophoresis buffer, and sample the processed protein sample (50 μg/well).

d) Conventional methods for electrophoresis.

4. Flow cytometry

1X10 ⁶ cells were resuspended in 100 μl of FACS buffer, mixed with fluorescently labeled antibody, and incubated at 4 ° C for 30 min in the dark. After centrifugation, the supernatant was discarded and washed three times with FACS buffer. Finally, the cells were resuspended in 300 μl of FACS buffer and assayed using a FACS Calibur flow cytometer. Data analysis with FCS Express software.

5. Luciferase reporter gene experiment

A 2X SIE plasmid containing a specific binding site for STAT1 (see Wu, TR et al. (2002). SHP-2is a dual-specificity phosphatase involved in Stat1 dephosphorylation at both tyrosine and serine residues in nuclei. J Biol Chem 277, 47572-47580 Transfected into MSCs by Amaxa Nuleofector device, inoculated into 24-well plates and cultured overnight. After treatment with different cytokines, cells were harvested at different time points (6 hr, 12 hr and 24 hr), respectively, and luciferase activity was measured using a luciferase assay kit (Promega).

6.MSCs immunosuppressive experiment

³ H-methyl-thymidine method: First, a certain number of MSCs were inoculated in a 96-well plate overnight. The supernatant was aspirated and 2×10 ⁶ mouse spleen cells were inoculated per well (activated with 1 μg/ml anti-CD3 and 1 μg/ml anti-CD28, respectively). After mixing with MSCs for 2-3 days, 1 μCi of ³ H-methyl-thymidine was added per 100 μl of cell culture solution for the last 6 hours. After 6 hours, the plate was placed in a -80 ° C refrigerator, and the cells were repeatedly frozen and thawed twice before the measurement. The cells were transferred to a special filter by vacuum adsorption, scintillation fluid was added, and T cell proliferation was measured using a Wallac Microbeta scintillation counter.

7. Detection of gene expression by real-time PCR

a) RNA extraction: Total RNA in cells was extracted with RNAprep pure Cell/Bacteria Kit (TianGen).

b) Reverse transcription synthesis of cDNA: The RNA extracted in the previous step was reverse transcribed into cDNA using TaqMan Reverse Transcription Reagents.

All reagents of TaqMan Reverse Transcription Reagents were thawed on ice and placed in an RNase-free tube according to the following reaction system.

Thermal cycle parameters: 25 ° C 10 min → 48 ° C 30 min → 95 ° C 5 min.

c) The cDNA obtained above was stored at -20 ° C or diluted to 100 μl with sterile ddH ₂ O for use.

d) Takara SYBR Green reaction system:

SYBR Green PCR Master Mix 5μl

Primers (5μM) 0.5μl each

cDNA 4μl

7900HT进行PCR反应。Place the sampled 384-well plate in the ABI

The 7900HT was subjected to a PCR reaction.

8. Transform MSCs

Preparation of MSCs overexpressing IFNα (MSC-IFNα)

The mouse type I interferon IFNα coding sequence was amplified from the bone marrow-derived DC cell cDNA by PCR, and the primer sequences were: 5'ACGCTCGAGGCCACCATGGCTAG ACTCTGTGC 3' (SEQ ID NO: 1) and 5'ACGTCTAGATCACTCCTTCTCTTCACT CAG 3 '(SEQ ID NO: 2). The resulting fragment was digested with XhoI and XbaI, and the digested product was inserted into the lentiviral shuttle plasmid pLVX-IRES-zsGreen1 (with GFP, purchased from Clontech Laboratories) to obtain plasmid pLVX-IRES-zsGreen1-mIFNα.

Viral packaging was performed using a three-plasmid system, including pMD2G, pSPAX2, and a shuttle plasmid (pLVX-IRES-zsGreen1 or pLVX-IRES-zsGreen1-mIFNα). Three plasmids were transfected into 293FT cells together using Lipofectamine 2000. After 48 hours, the cell culture supernatant was collected and centrifuged at 800 g for 10 min to remove cell debris. The virus was concentrated using an Ultra-15 ultrafiltration tube equipped with an Ultracel-100 membrane and the virus titer was determined. MSCs were infected with viruses with MOI=30, respectively. GFP and GFP together with IFNα-infected MSCs were named MSC-GFP and MSC-IFNα, respectively.

Example 1. IFNα can not induce MSCs to produce iNOS but can upregulate MHC class I molecules

In order to investigate the effect of IFNα on the immunomodulatory effects of MSCs, the inventors first examined whether IFNα can synergize with TNFα to induce nitrogen oxide (NO) in MSCs. Consistent with the literature reports, both IFNα and IFNγ can synergize with TNFα to induce mass production of bone marrow-derived macrophages. Raw NO. However, the inventors have found that unlike the effect of IFNy in MSCs, the combination of IFNα and TNFα does not induce the production of NO by MSCs (Fig. 1A). Almost all types of cells express type I interferon receptors, and the inventors' gene chip data also indicate that MSCs express IFNα receptors: IFNAR1 and IFNAR2.

In order to detect whether the signal of IFNα can be normally transmitted in MSCs, the inventors examined the expression of MHC class I (K ^b D ^b ) by MSCs under the action of IFNs. The results showed that both IFNα and IFNβ up-regulated the expression of K ^b D ^b on MSCs like IFNγ (Fig. 1B). This demonstrates that classical type I interferon signaling is present in mouse MSCs.

Example 2, IFNα attenuates the immunosuppressive effect of MSCs

Since type I interferon does not induce nitric oxide, the core molecule of MSCs that produces its immunomodulatory effects, does IFNα affect the immunomodulatory effects of MSCs? To solve this problem, the present inventors added IFNα to an immunosuppressive system in which MSCs were co-cultured with activated mouse spleen cells (splenocytes) to study their effects. The present inventors have found that, similar to the iNOS inhibitor L-NMMA, the addition of IFNα not only abolished the inhibitory effect of MSCs on the activation of spleen cells, but increased the proliferation of spleen cells compared with the control group (Fig. 2A left panel).

Meanwhile, the inventors examined the amount of NO in the co-culture system and found that IFNα is similar to L-NMMA, and both inhibited NO production by MSCs (Fig. 2A right panel).

Another molecule of the type I interferon family, IFNβ, also abolished the immunosuppressive effects of MSCs (Fig. 2B), which also means that the body's anti-tumor immune response can be improved. IFNα was used in the subsequent examples.

Example 3, IFNα inhibits IFNγ and TNFα-stimulated MSCs to produce iNOS

IFNα can attenuate the immunosuppressive effects of MSCs and reduce the amount of NO in the co-culture system. Therefore, the inventors hypothesized that IFNα may inhibit the expression of iNOS or block the function of iNOS, thereby reducing the production of NO. First, the present inventors stimulated MSCs together with IFNα or IFNγ and TNFα, and collected RNA and protein 24 hours later, and detected the expression of iNOS at mRNA and protein levels by real-time quantitative PCR and Western blotting, respectively. The results showed that IFNα partially inhibited the expression of iNOS at the mRNA level (Fig. 3A), while the inhibition of its protein level was more pronounced (Fig. 3B).

Previous studies by the present inventors have shown that MSCs also need to produce a large amount of chemotaxis when exerting immunosuppressive effects. Factor, recruiting immune cells to migrate to the vicinity of MSCs, so that NO can play a role in inhibiting cell proliferation in a short distance. So, does IFNα also have a role in producing chemokines in MSCs? The present inventors co-stimulated MSCs with IFNγ and TNFα or IFNα, IFNγ and TNFα for 24 hr, and collected supernatants, and detected the expression of chemokines using the Bio-Plex suspension chip system. It was found that IFNα not only significantly inhibited the expression of NO by MSCs but also promoted the expression of IL-6, which had no significant effect on the expression of other chemokines (Fig. 4).

Example 4, IFNα does not affect the expression of IFNγ receptor on MSCs and the expression, phosphorylation level and nuclear importing ability of STAT1 and p65

Next, the inventors focused on the mechanism by which IFNα inhibits iNOS expression of MSCs. It has been reported in the literature that IFNα/β inhibits macrophage activation by down-regulating the expression of IFNγ receptor IFNGRs on the surface of macrophages, making the host susceptible to Listeria monocytogenes. The inventors' chip data analysis showed that the expression of IFNγ receptor IFNGR1/2 was not affected by IFNα, and as a positive control, interferon could up-regulate the expression of H2-D1 and H2-K1 (Fig. 5A).

The inventors obtained similar results by flow cytometry analysis (Fig. 5B).

Example 5, IFNα reduces iNOS expression in MSCs by inhibiting the binding ability of STAT1

The above results indicate that IFNα does not affect the expression of IFNγ receptor and the expression, phosphorylation and nuclear importing ability of STAT1 and p65 on MSCs. In general, activated STAT1 can enter the nucleus and bind to specific recognition sites in the promoter of a specific gene (such as iNOS) to exercise its ability to regulate the transcriptional activity of its gene. Next, the inventors used an oligonucleotide sequence capable of specifically binding to a transcription factor (STAT1: 5'GATCCTTCTGGGAATTCCTAGATC 3' (SEQ ID NO: 3); p65: 5' AGTTG AGGGGACTTTCCCAGG 3' (SEQ ID NO: 4) The linked agarose beads precipitate STAT1 or NF-κB to detect their DNA binding ability, respectively. It was found that the STAT1 binding ability of IFNα-treated MSCs was significantly decreased, while the binding ability of NF-κB was not significantly affected (Fig. 6A).

The results of the luciferase reporter gene assay further confirmed that IFNα can inhibit the transcriptional activity of STAT1 (Fig. 6B).

MSCs stimulated by IFNγ/TNFα and IFNγ/TNFα/IFNα were collected at different time points, total RNA was extracted, and the expression pattern of iNOS and IL-6 in 24 hr was detected by real-time PCR. Results Consistent with previous bio-plex results, IFNα inhibited iNOS expression and promoted IL-6 expression (Fig. 6C).

At the same time, it was found that IFNα inhibited the expression of iNOS very weakly within 6 hr, and the inhibition effect of IFNα on iNOS expression was more significant with time delay (Fig. 6C).

Example 6. IFNα pretreated MSCs inhibit tumor growth

Stromal cells in the tumor microenvironment play an important role in the development of tumors. Previous reports have shown that MSCs can be isolated from tumors, and the regulation mechanism of nitric oxide production is very similar to that of bone marrow-derived MSCs. In vitro experiments have shown that tumor stromal cells can suppress immune responses by producing nitric oxide. In vivo experiments have also demonstrated that inhibition of nitric oxide production by tumor stromal cells can significantly alleviate tumor development.

Therefore, this study first used MSCs (MSC-IFNα) overexpressing IFNα, and co-injected it with B16-F0 melanoma cells in mouse leg muscles; injection of PBS+B16-F0, injection of IFNα+B16-F0, Mice injected with MSC-GFP + B16-F0 served as controls. The results showed that MSC-IFNα significantly inhibited the growth of melanoma (Fig. 7A).

In order to rule out the direct inhibition of tumor growth by IFNα, the inventors also adopted a method of co-injection of MSCs pretreated with IFNα for 24 hours and B16-F0. The results showed that IFNα pretreated MSCs still inhibited tumor growth (Fig. 7B). Therefore, the present inventors have shown that type I interferon can inhibit tumor growth by inhibiting the production of nitric oxide in MSCs and affecting its immunosuppressive effect in the tumor microenvironment.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims (10)

A mesenchymal stem cell characterized in that the mesenchymal stem cell is a type I interferon-pretreated mesenchymal stem cell or a mesenchymal stem cell overexpressing type I interferon. The mesenchymal stem cell according to claim 1, wherein said pretreatment comprises: The type I interferon is mixed with the mesenchymal stem cells such that the final concentration of the type I interferon is 200-10000 U/ml. The mesenchymal stem cell according to claim 1, wherein the type I interferon-derived mesenchymal stem cell is transformed with a polynucleotide encoding a type I interferon. The mesenchymal stem cell according to any one of claims 1 to 3, wherein the type I interferon is selected from the group consisting of IFNα or IFNβ. The use of the mesenchymal stem cells according to any one of claims 1 to 4 for the preparation of a medicament for inhibiting tumors. The use according to claim 5, wherein the tumor is melanoma. A pharmaceutical composition for inhibiting tumors, characterized in that the pharmaceutical composition comprises the mesenchymal stem cells according to any one of claims 1 to 4. The use of type I interferon for the preparation of a composition for increasing the expression of MHC I in mesenchymal stem cells; or for the preparation of a composition for reducing the immunosuppressive ability of mesenchymal stem cells or for enhancing the body's antitumor immune response. Use of a type I interferon for the preparation of a composition for inhibiting the DNA binding ability of STAT1 in mesenchymal stem cells; or for the preparation of a composition for reducing the expression of an inducible nitric oxide synthase in mesenchymal stem cells. The use according to any one of claims 8 or 9, wherein the type I interferon is selected from the group consisting of IFNα or IFNβ.

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