CN1150035C - Recombination plasmid and application in disease prevention and control - Google Patents
Recombination plasmid and application in disease prevention and control Download PDFInfo
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- CN1150035C CN1150035C CNB00132196XA CN00132196A CN1150035C CN 1150035 C CN1150035 C CN 1150035C CN B00132196X A CNB00132196X A CN B00132196XA CN 00132196 A CN00132196 A CN 00132196A CN 1150035 C CN1150035 C CN 1150035C
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- recombination plasmid
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Abstract
The present invention relates to the field of biomedicine, more specifically to a recombination plasmid of carrying human hepatocyte growth factor genes and application to the prevention and the treatment of diseases. Human hepatocyte growth factor genes are inserted into pUDK plasmid carriers for obtaining the recombination plasmid pUDKH. After being transferred to the ischemia positions of rat limbs and rabbit limbs by local intramuscular injection or a gene gun method, the recombination plasmid can be effectively expressed for obviously promoting blood vessel formation. The recombination plasmid is coated on the wound surface of a rabbit ear or is transferred to the wound position by the gene gun method; the recombination plasmid has the efficacy of preventing and treating scars. Therefore, the recombination plasmid has good application prospects in the prevention and the treatment of disc deficiency diseases and pathological scars.
Description
Technical field
The present invention relates to biomedical sector, relate to the recombiant plasmid and the application in ischemic diseases, the control of pathologic cicatrix thereof of carrier's liver cell growth factor gene specifically.
Background technology
Blood vessel infraction property disease comprises periphery infraction property angiopathy, coronary artery infraction and cerebral arteries infraction, is a class disease the most serious in the cardio-cerebrovascular, the serious threat human health.At present this disease is not still had effective drug treatment, and mainly rebuild the function tremulous pulse, but danger is bigger by means of surgical operation, case fatality rate and complication height, prognosis is very poor.The new way of the blood vessel infraction that therefore seeks treatment property disease is the current problem that presses for solution.Gene therapy is a kind of new treatment pattern of rising in recent years, promptly shift the gene of effective angiogenic growth factor in ishemic part by suitable carriers, promote the formation of neovascularity, can be in local collateral circulation, foundation " molecule bridging " mechanism of forming of ischemia.At present, the existing both at home and abroad experimentation with VEGF (VEGF) gene therapy vascular occlusive disease has entered the research of II phase II bed (Lancet, 1996 abroad; 348:370).
The formation of cicatrix is the product and the symbol of wound healing.Yet over-drastic scar hyperplasia then is a kind of morbid state performance, is a kind of important complication of the traumatology department.Not only influence outward appearance, and influence body function, canceration also can take place late period in cicatrix.At present, the treatment main armrest art of people after to scar hyperplasia, pressurization, method such as freezing all are not ideal means, so the research of prevention of scar has extremely important meaning.At present, domestic and international still useless biological engineering method prepares the report of external used medicine prevention of scar.
HGF (patent No. JP209449/89, JP88592/90, JP200898/90) be a multi-functional somatomedin, important regulatory role has been taken place for multiple histiocytic division, travel motion, form, and (HGF is a kind of short vascular endothelial cell growth factor, have effect (J Cell Biol, 1992 that short endothelial cell proliferation and new vessels generate; 119:629).HGF also stimulates the Skeletal Muscle Cell secretion of VEGF except that direct effect.HGF also can activate immobilized muscle satellite cell, stimulates the regeneration of body Endoskeleton flesh, prevents the generation of amyotrophy and myofibrosis, so HGF can promote the formation of ishemic part new vessels net, improves the motor function of limbs.In addition, HGF can suppress the generation of transforming growth factor (TGF-β) and activity (NatureMed, 1999 that strengthen collagenase significantly; 5:226), thereby can effectively reduce the level of TGF-β in the tissue, suppress the hyperplasia of collagen fiber, have the effect of control fibrosis lesion.Scar tissue essence is Fibrotic granulation tissue, is that the collagen deposition that enriches is accompanied the result of collagen configuration disorder, also belongs to fibrosis lesion.
It is exactly the cell that how therapeutic gene is imported effectively target tissue that gene therapy is used for the clinical greatest problem that faces, make it with suitable horizontal expression, reach therapeutic effect, promptly how to set up a safety, efficient, practical, gene transfer system repeatably.At present, gene transfer method can be divided into non-virus-mediated and virus-mediated two big classes substantially, and the former is safer than the latter.Exposed plasmid DNA carrier is a kind of novel non-virus carrier that development in recent years is got up, and is used for the research of gene vaccine and gene therapy.Studies have shown that, recombiant plasmid direct injection or heeling-in at skeletal muscle, cardiac muscle or blood vessel, or are applied in the wound surface, exogenous gene can be transferred to local organization, and the sustainable respective egg white matter that gives expression to.It is reported that an intramuscular injection sometimes can continuous expression respective egg white matter 3-6 month.Enter the plasmid DNA of cell, can be not and chromosomal integration, and in the nuclear exoplasm, duplicate.
Summary of the invention
Given this, we have made up the eukaryon expression plasmid pUDKH of human hepatocyte growth factor.The exposed plasmid DNA of local intramuscular injection or DNA is wrapped on the gold grain and with particle gun is transferred to the ischemic muscle part can obviously promote the formation of limbs acute ischemia position neovascularity.In addition, recombiant plasmid directly is applied in wound surface, or with the sub-wound of particle gun method transferring plasmid, but accelerating wound healing prevents or reduces the formation of pathologic cicatrix.Has potential potential applicability in clinical practice.
First purpose of the present invention is to provide a kind of recombiant plasmid, this recombiant plasmid portability human hepatocyte growth factor gene.
Second purpose of the present invention is to use the recombiant plasmid treatment periphery infraction property angiopathy of this carrier's liver cell growth factor gene.
The 3rd purpose of the present invention is to use the recombiant plasmid treatment coronary artery infraction and the cerebral arteries infraction property disease of this carrier's liver cell growth factor gene.
The 4th purpose of the present invention is that the recombiant plasmid of using this carrier's liver cell growth factor gene is prevented and treated the optic atrophy that ischemia causes.
The 5th purpose of the present invention is that the recombiant plasmid of using this carrier's liver cell growth factor gene is prevented and treated amyotrophy and myofibrosis.
The 6th purpose of the present invention is the excessive formation of using the recombiant plasmid control cicatrix of this carrier's liver cell growth factor gene.
The 7th purpose of the present invention is that the recombiant plasmid of using this carrier's liver cell growth factor gene promotes wound healing.
The 8th purpose of the present invention is to provide the recombiant plasmid that carries other functional genes except that human hepatocyte growth factor gene, is used for the control of some disease.
Specific embodiments of the present invention is as follows:
1. the structure of the recombiant plasmid pUDKH of carrier's liver cell growth factor gene.As Fig. 1: the pUC18 cloning vehicle with Pvu II enzyme action, is reclaimed the dna fragmentation of 2.3kb.With NruI and PvuII double digestion pcDNA3, reclaim the dna fragmentation of 1kb, this fragment contains cytomegalovirus promoter, multiple clone site and growth hormone terminator.Get these two fragments continuously plasmid pUD (3.3kb) mutually.With AvaII and SspI double digestion pUD, reclaim the dna fragmentation of 2.7kb, in addition with AvrII enzyme action pEGFP-N1, reclaim the dna fragmentation of 1.1kb, this fragment contains complete kanamycin gene; Connect two recovery dna fragmentations and get plasmid pUDK (3.8kb).Then human hepatocyte growth factor coding region cDNA is inserted into the multiple clone site (between two restriction enzyme sites of BamHI and ApaI) of pUDK carrier, obtains the recombinant vector pUDKH of carrier's liver cell growth factor gene.
2. the pilot-scale purification of recombiant plasmid preparation.
The plasmid purification preparation flow comprises: fermentation, centrifugal collecting cell, alkaline denaturation cracking, separation and purification.The centrifugal gained antibacterial in fermentation back adds the resuspended liquid of cell, fully mixing.Slowly add lysate immediately, mixing was put ice bath 10 minutes.Slowly add neutralizer, form white depositions, put ice bath 60 minutes.Centrifugal, obtain cleared lysate.Behind buffer equilibrium separation post Ultrapure100 (QIAGEN company), with sample on the cleared lysate, wash impurity with another kind of buffer then, with special buffer solution elution recombiant plasmid.The isopropanol precipitating plasmid that in eluent, adds 0.7 volume.Institute obtains precipitation with a small amount of 70% washing with alcohol once, adds the source water dissolution precipitation of reducing phlegm and internal heat on a small quantity, promptly gets the pure product of recombiant plasmid after the dialysis.Agarose gel electrophoresis and ultraviolet spectrophotometry are determined the purity and the content of plasmid.The result shows that the super spirial plasmid ratio is greater than 85%, and agarose gel electrophoresis is not seen RNA, spectrophotometric analysis A
260nm/ A
280nmBetween 1.75-1.85.The quality control standard that meets plasmid.
3. observing this recombiant plasmid can be at the former rat skeletal muscle cell of being commissioned to train foster of external transfection effectively.Behind the former primary cultures of rat Skeletal Muscle Cell of pUDK-lacZ transfection, transfection efficiency is 0.057%; And behind this recombiant plasmid of transfection, detected the expression (as Fig. 2, Fig. 3) of HGF in the secretion supernatant of cell.
4. observing this recombiant plasmid can be at external rotaring copolymering NIH 3 T 3 cell effectively.Behind the pUDK-lacZ rotaring copolymering NIH 3 T 3 cell, transfection efficiency is 7.96%; And behind this recombiant plasmid of transfection, detected the expression (as Fig. 4, Fig. 5) of HGF in the secretion supernatant of cell.
5. observe the propagation (as Fig. 6 a, Fig. 6 b) of liver cell growth factor gene expression product energy significant stimulation vascular endothelial cell.
6. observe hepatocyte growth factor the NIH3T3 cell is not had significant stimulation effect (as Fig. 7).
7. in rat limbs acute ischemia model, observe the expression of beta galactosidase behind the localized transfection pUDK-lacZ plasmid; And to behind local intramuscular injection or the recombiant plasmid with the local transfer of particle gun purification, immunohistochemical method detects the expression of hepatocyte growth factor in injection site muscular tissue; And matched group does not see that tangible HGF expresses.
8. observe behind the local pUDK-lacZ of transfer of new zealand rabbit rabbit ear cicatrix model wound plasmid beta galactosidase expression; Behind the local transfer of the wound recombiant plasmid, immunohistochemical method detects the expression of hepatocyte growth factor in shifting local organization; And matched group does not see that tangible HGF expresses.
10. after observing rat limb ischemia model transfer recombiant plasmid, gross anatomy shows the offside showed increased that vessel density is not injected plasmid, H.E. can be observed the formation of a large amount of neovascularity in the muscular tissue that the dyes section, and the sign of oriented large artery trunks blood vessel development; And matched group is not seen the formation (as Fig. 8, Fig. 9) of tangible new vessels.
11. after rat limb ischemia model shifts recombiant plasmid, do not observe the regression or the fibrosis of partial musculature in the section of H.E. dyeing muscular tissue; And matched group has been observed tangible regression of muscular tissue or fibrosis phenomenon (as Figure 10).
12. after observing new zealand rabbit limb ischemia model injection recombiant plasmid, can be observed the formation of a large amount of neovascularity in the section of H.E. dyeing muscular tissue, do not observe the regression or the fibrosis of partial musculature; And matched group is not seen the formation of tangible new vessels, observes tangible muscle fiber and attenuates, ruptures, muscle congestion (as Figure 11, Figure 12).
13. set up rabbit ear cicatrix model by operation method with new zealand rabbit, this recombiant plasmid is applied in wound surface (prevention group), the result shows that the prevention group is fast than the matched group wound healing, and inflammatory reaction is light, and the formation of cicatrix is little; H.E. dyeing shows that the prevention group is light than the matched group keratinization of epidermis, skin corium thickness is little, and fibrous tissue is thin.Thereby confirm that this recombiant plasmid has certain preventive effect (as Figure 13) to cicatrization
14. observed the dose-effect relationship that recombiant plasmid excessively forms rabbit ear healing speed and prevention of scar (see Table 1, table 2).
The invention provides a kind of recombiant plasmid of carrier's liver cell growth factor gene, can be preserved by lyophilizing after this recombiant plasmid is purified, can be made into injection or be wrapped in gold grain surface or baste etc. and be used for the treatment of ischemic diseases and promote wound healing, prevention of scar to form.Ischemic diseases can be a periphery infraction property angiopathy, also can be coronary artery infraction and cerebral infarction disease, also can be the ischemia of ischemic optic atrophy and other organs.Wound can be a skin trauma, also can be skin burn or operative incision etc.Embodiment will block the gene therapy of angiopathy (limb ischemia) and the preventive effect that skin scar excessively forms is further set forth the present invention periphery by the recombinant vector of describing carrier's hepatocyte growth factor.
Use the formation of recombinant vector treatment ischemic diseases provided by the invention and prevention of scar, the one, start with from the purpose that promotes vascularization, sets up collateral circulation and improve local blood circulation, the HGF gene of angiogenesis is urged in importing to patient's diseased region tissue in.The 2nd, from promoting vascularization, accelerating wound healing and inhibition fibrous tissue excessively form mechanism and start with, smear the recombiant plasmid of carrier's liver cell growth factor gene to the wound surface, by promoting vascularization, suppress the generation of TGF β and the activity of enhancing extracellular matrix degrading enzyme such as collagenase, reduce level and the degrade collagen fiber of TGF β in the tissue, reach the purpose that promotes that wound healing, prevention of scar excessively form.Therefore, use recombiant plasmid provided by the invention, use simple and convenient, cost is lower.If the present invention is implemented, will provide a kind of alternative new treatment means for the clinical treatment of present ischemic diseases; To provide a kind of effective means for the prevention of still not having at present the pathologic cicatrization of effective means of prevention.
Description of drawings
Further specify the present invention in conjunction with the accompanying drawings:
The design of graphics of Fig. 1 pUDKH
Former primary cultures of rat Skeletal Muscle Cell of Fig. 2 pUDK-lacZ transfection and transfection efficiency thereof
(a) (b) transfection efficiency of x-gal dyeing (* 100)
The expression of HGF behind the former primary cultures of rat Skeletal Muscle Cell of Fig. 3 transfection recombiant plasmid
Fig. 4 pUDK-lacZ rotaring copolymering NIH 3 T 3 cell and transfection efficiency thereof
(a) (b) transfection efficiency of x-gal dyeing (* 100)
The expression of HGF behind Fig. 5 NIH3T3 cell transfecting recombiant plasmid
Fig. 6 liver cell growth factor gene expression product is to the stimulating activity of Human umbilical vein endothelial cells propagation
(a) to the stimulating activity of former generation Human umbilical vein endothelial cells
(b) to Human umbilical vein endothelial cells be the stimulating activity of (HUV-EC)
(tasty and refreshing transfection plasmid supernatant on the ■ untransfected plasmid)
Fig. 7 hepatocyte growth factor is to the function analysis of NIH3T3 cell
Fig. 8 rat hindlimb acute ischemia model is local shift or do not shift recombiant plasmid after, vessel density is observed in gross anatomy
(a) shift side (b) and do not shift side
Fig. 9 rat hindlimb acute ischemia model is local shift or do not shift recombiant plasmid after, the microphotograph (the transfer group has a large amount of little neovascularization) of local skeletal muscle tissue section (H.E. dyeing * 00)
Transfer group: (a) intramuscular injection (b) particle gun
Not transfer group: (c) intramuscular injection (d) particle gun
Figure 10 rat hindlimb acute ischemia model is local shift or do not shift recombiant plasmid after, the microphotograph (the obvious regression of not transfer group muscle fiber) of local skeletal muscle tissue section (H.E. dyeing * 100)
(a) not transfer group of transfer group (b)
Figure 11 rabbit hind leg acute ischemia model is local shift or do not shift recombiant plasmid after, the microphotograph (the transfer group has more neovascularization) of local skeletal muscle tissue section (H.E. dyeing * 100)
(a) not transfer group of transfer group (b)
Figure 12 rabbit hind leg acute ischemia model is local shift or do not shift recombiant plasmid after, the microphotograph (not transfer group muscle congestion, muscle fiber attenuates, rupture) of local skeletal muscle tissue section (H.E. dyes * 100)
(a) not transfer group of transfer group (b)
The microphotograph of the tissue slice that Figure 13 recombiant plasmid prevention of scar forms (H.E. dyeing * 100)
(a) not transfer group of transfer group (b)
The specific embodiment
The experimentation of embodiment 1 using recombinant plasmid pUDKH gene therapy rat limb ischemia
One, construction of recombinant plasmid and preparation
(1) structure of plasmid pUDKH
Building process such as Fig. 1:, reclaim the dna fragmentation of 2.3kb with pUC18 cloning vehicle PvuII enzyme action.With NruI and PvuII double digestion pcDNA3, reclaim the dna fragmentation of 1kb, this fragment contains cytomegalovirus promoter, multiple clone site and growth hormone terminator.Get these two fragments continuously plasmid pUD (3.3kb) mutually.With AvaII and SspI double digestion pUD, reclaim the dna fragmentation of 2.7kb, in addition with AvrII enzyme action pEGFP-N1, reclaim the dna fragmentation of 1.1kb, this fragment contains complete kanamycin gene; Connect two recovery dna fragmentations and get plasmid pUDK (3.8kb).Then human hepatocyte growth factor coding region cDNA is inserted into the multiple clone site of pUDK carrier, obtains the recombinant vector pUDKH of carrier's liver cell growth factor gene.Identify with restricted enzyme whether insertion is correct.
(3) preparation of plasmid
The plasmid purification preparation flow comprises: fermentation, centrifugal collecting cell, alkaline denaturation cracking, separation and purification.The centrifugal gained antibacterial in fermentation back adds the resuspended liquid of cell, fully mixing.Slowly add lysate immediately, mixing was put ice bath 10 minutes.Slowly add neutralizer, form white depositions, put ice bath 60 minutes.Centrifugal, obtain cleared lysate.Behind 350 milliliters of buffer equilibrium separation post Ultrapure100 (QIAGEN company), with sample on the cleared lysate, wash impurity with 3.0 liters of buffer then, with 400 milliliters of another kind of buffer solution elution recombiant plasmid.The isopropanol precipitating plasmid that in eluent, adds 0.7 volume.Institute obtains precipitation with a small amount of 70% washing with alcohol once, adds the source water dissolution precipitation of reducing phlegm and internal heat on a small quantity, promptly gets the pure product of recombiant plasmid after the dialysis.Agarose gel electrophoresis and ultraviolet spectrophotometry are determined the purity and the content of plasmid.The result shows that the super spirial plasmid ratio is greater than 85%, and agarose gel electrophoresis is not seen RNA, spectrophotometric analysis A
260nm/ A
280nmBetween 1.75-1.85.The quality control standard that meets plasmid.
Two, the cultivation of Skeletal Muscle Cell and plasmid transfection
(1) former being commissioned to train of neonate rat Skeletal Muscle Cell supported and evaluation
The rat that was born two, peel off a rear flank limb skin after the whole body sterilization, take off this hind leg and put in the aseptic ware, peel off its muscle in a bottle, fully wash with PBS, and shred, add 0.125% trypsinization 3-4 time, 1500 rev/mins are centrifugal 8 minutes; as far as possible collect discrete cell, behind Skeletal Muscle Cell feeding liquid suspension cell, inoculate in the sub-culture bottle, hatch for 37 ℃.After 75 minutes suspension cell is moved in another culture bottle, to remove easily adherent non-myocyte's composition.Identify whether be Skeletal Muscle Cell with the SABC method, one anti-is mouse-anti desmin monoclonal antibody (ZEMED), and two anti-ly are biotin labeled anti-Mus IgG, and three anti-ly are that the avidin of horseradish peroxidase-labeled, Color Appearance System are DAB.The result shows more than 90% to be Skeletal Muscle Cell.
(2) plasmid DNA transfection Skeletal Muscle Cell of former generation
The Lipofectin method is adopted in transfection, and each transfection is with 5 micrograms of DNA+20 microlitre liposomees.Behind the transfection pUDK-lacZ, recording transfection efficiency is 0.057%; (cell number is 4 * 10 behind the transfection pUDKH
5), 24 hours, 48 hours and 72 hours sampling after liquid is changed in transfection detects the expression of HGF with the ELISA method, and one anti-ly is mouse-anti people HGF monoclonal antibody (R﹠amp; D).The result shows, detects the expression of HGF thereon in can be clear behind the former generation Skeletal Muscle Cell transfection pUDKH, and expression is about 16-20ng (as Fig. 2, Fig. 3).
Three, the HGF expression product is to the stimulating activity of Human umbilical vein endothelial cells propagation
Former being commissioned to train of people's umbilical cord endotheliocyte supported and the proliferation activity analysis: fully wash people's umbilical cord chamber with PBS under the aseptic condition, with an end ligation, inject 20 milliliter of 0.125% trypsin and the sealing of 37 ℃ of preheatings from the other end, umbilical cord is inserted among 37 ℃ of preheating PBS, digested 15 minutes, Digestive system moves in the centrifuge tube, and 1200 rev/mins centrifugal 10 minutes, DMEM re-suspended cell with containing 80% new-born calf serum is inoculated in the culture bottle.
HGF expresses supernatant to former generation Human umbilical vein endothelial cells and the function analysis of Human umbilical vein endothelial cells system (HUV-EC): 96 orifice plate cell number that every hole adds are 2500, the expression supernatant added amount of HGF in former generation Skeletal Muscle Cell accounts for 5% (80 pik) of cultivating cumulative volume respectively, 10% (160 pik), 15% (240 pik), 25% (400 pik) and 50% (800 pik).Mtt assay is measured cell-proliferation activity.The result shows that supernatant has the activity of tangible stimulation Human umbilical vein endothelial cells propagation, has confirmed that fully HGF is the strong mitogenesis former (as Fig. 6 a, Fig. 6 b) of vascular endothelial cell.
Four, revascularization evaluation
(1) rat acute limb ischemia animal model
20 of male rats, body weight 200-250 gram.Pentobarbital sodium intraperitoneal anesthesia (50 mg/ml), left side hind leg femoral artery section far away and main branch thereof cause acute posterior-limb ischemia angiopathy model all to cut off after the ligation of thin operation silk thread, and ligation is refused on the right side.The acute ischemia rat model is divided into 2 groups at random, intramuscular injection group and particle gun transfer group.
(2) gene transfer
Intramuscular injection group rat when undergoing surgery in each 200 microgram of ligation position muscle direct injection plasmid: 5 rat models injection pUDKH plasmids, 5 rat models injection pUDK plasmids (matched group).About 0.8 milliliter of the total amount of liquid of every animal injection.
Particle gun transfer group rat postoperative next day with portable particle gun (ST-500, Ningbo Xin Zhike device institute) metastatic gene in the ischemia intramuscular.Acute lower limb ischemia rat model is fixed in operating-table after the anesthesia in postoperative next day, and art side limbs are lost hair or feathers.Get a certain amount of carrier suspension (the bronze carrier of parcel plasmid) and place a stainless (steel) wire center membrane, as bullet, on the rearmounted particle gun to be dried, with the high-pressure helium is power, particle gun is close to the skin emission, and (each bullet carries about 2.5 μ micrograms of DNA, 25 kPas of helium pressures, target area can reach 2 square centimeters), launch twice continuously for every.Experimental group shifts and carries pUDKH (5 s') bronze carrier, and matched group shifts and carries pUDK (5 s') bronze carrier.
(3) expression of HGF in skeletal muscle tissue
Behind transferring plasmid, got the muscle samples at gene transfer position on the 10th day, do paraffin section after 10% formalin fixed, carry out SABC and detect: an anti-mouse-anti people HGF monoclonal antibody (R﹠amp of being; D), two anti-are biotin labeled sheep anti-mouse igg, and three anti-ly are that the Avidin of horseradish peroxidase-labeled, chromogenic substrate are the DAB system.Shift pUDKH plasmid group in the time of .10 days, comprise particle gun group and intramuscular injection group, muscle specimen all has stronger HGF to express.And two groups of shifting the pUDK plasmid there is no tangible HGF and express.
(4) revascularization analysis
Behind transferring plasmid the 10th day, gross anatomy was seen: shift the visible significantly blood vessel of the rat limbs that carry the HGF gene plasmid and increase, and offside and the extremity vascular that shifts empty plasmid are not seen significant change (as Fig. 8).Get local muscle simultaneously and do the transverse section paraffin section, H.E. dyeing, observe under the optical microscope and shift pUDKH plasmid group, comprise particle gun group and intramuscular injection group, little vascularization is obviously shifted pUDK plasmid group and is increased, during to 30 days, lumen of vessels has increase tendency, and the logical again sign (as Fig. 9) of arteries is arranged.In addition, in the section of matched group muscular tissue, observed the regression or the fibrosis of muscular tissue, and do not observed (as Figure 10) in transfer recombiant plasmid group.
The animal model of the rat hindlimb acute ischemia that adopts in this experiment, the ability that forms little blood vessel in surgery alone or after shifting empty plasmid 30 days the time is still very weak.By comparison, the acute ischemia hind leg that shifts HGF genetic animal group had a large amount of neovascularity to generate (packed red blood cell in the tube chamber is blood vessel) in 10 days after surgery, had fully confirmed the short vascularization effect of HGF and the effectiveness of gene transfer.
The recombiant plasmid pUDKH of embodiment 2 carrier's liver cell growth factor genes is pre-to cicatrization
Anti-effect
One, the structure of recombiant plasmid pUDKH and large scale purification preparation
With embodiment 1
Two, recombinant plasmid dna rotaring copolymering NIH 3 T 3 cell
Plasmid DNA transfection NIH3T3 cell adopts the lipofectin method, and each transfection is with 5 micrograms of DNA+20 microlitre liposomees.Behind the transfection pUDK-lacZ, getting its transfection efficiency is 7.96%; (cell number is 1.0 * 10 behind the transfection pUDKH
6), 24 hours, 48 hours and 72 hours sampling after liquid is changed in transfection detects the expression of HGF with the ELISA method, and one anti-ly is mouse-anti people HGF monoclonal antibody (R﹠amp; D).The result shows, detects the expression (as Fig. 4, Fig. 5) of HGF thereon in can be clear behind the NIH3T3 cell transfecting pUDKH.
HGF is to the function analysis of NIH3T3 cell: 2500 cells of the every hole inoculation of 96 porocyte culture plates, with the pure product of HGF albumen (5 nanograms, 2.5 nanograms, 1.25 nanograms, 0.5 nanogram, 0.25 the nanogram) (R﹠amp of variable concentrations; D) stimulate the NIH3T3 cell, mtt assay detects.The result does not observe the stimulation (see figure 7) of HGF to the NIH3T3 cell.
Three, the effect assessment of prevention of scar
1. the foundation of cicatrix animal model
The female White Rabbit of New Zealand, body weight 2.5-3.0 kilogram.The ear veutro does not have the blood vessel place shave hair sterilization after, until cartilage surface, the diameter of otch is about 0.7 centimetre, 5 of every sides with operation method excision holostrome skin.
2. effect assessment
Recombiant plasmid pUDKH with various dose in the time of excision skin is applied in the wound surface, and each wound respectively is coated with 10 micrograms, 8 micrograms, and 4 micrograms, 2 micrograms, 0 microgram only is coated with once.2 rabbits of each dosage group, 20 wounds.Experimental result shows that the 8th day healing rate promptly has significant difference after smearing, and is coated with 10 micrograms and 8 microgram groups and all is coated with 4 micrograms, 2 microgram groups and matched group healing fast (seeing Table 1), and significant difference (p＜0.01) is arranged on the statistics; Though and 4 micrograms, 2 microgram groups and matched group comparison wound area dwindle but not statistically significant (p＞0.05).To smearing the back the 30th day, wound surface and contiguous no site of injury skin are downcut in the lump, do paraffin section after 10% formalin fixed, row H.E. dyeing.Observation by light microscope shows that it is light to be coated with pUDKH group tissue slice keratinization of epidermis, and skin corium thickness is little, and the fibrous tissue amount is few; And the matched group fibrous tissue is many, corium bed thickness (seeing Figure 13).Calculate simultaneously each scar loose index (Hypertrophic Index, HI) (HI=scar holostrome thick/the wound proximate skin is thick), as an index of healing quality, loose index and cicatrix big or small proportional.The result shows that each group of smearing pUDKH is all little than the loose index of matched group, i.e. the formation of cicatrix is little, and outward appearance is more smooth, but only is coated with 10 micrograms, and 8 microgram groups and matched group relatively have significant difference (p＜0.01 sees Table 2).
Simultaneously we also to smear pUDKH (8 microgram) and smear Bfgf-ESSEX (bFGF) (east, Zhuhai mcroorganism pharmaceutical Co. Ltd), recombinant human epidermal growth factor (rhEGF) (Shanghai Great River Stocks Trading Co. company limited biopharmaceutical company) compares.The result tentatively shows, smears the back and promptly can be observed in the 8th day and be coated with pUDKH group healing rate fast (seeing Table 3) slightly.Smeared the back the 25th day, loose Index for Calculation is coated with pUDKH class value minimum, and relatively there were significant differences (p＜0.01 sees Table 4) with other groups.
Table one various dose pUDKH is to the influence of wound healing
Wound area (mm
2)
Time (my god) 10 μ g, 8 μ g, 4 μ g, 2 μ g, 0 μ g n
1 40.84±5.32 42.33±2.52 40.13±2.52 41.23±2.75 40.68±2.69 20
8 6.96±7.64 7.67±8.68 15.62±4.96 19.32±4.93 20.02±16.34 20
12 1.00±3.14 4.06±8.23 4.22±5.12 5.26±8.40 5.88±10.80 20
Table two is respectively organized loose index (HI) after smearing various dose pUDKH
Dosage (μ g) 10 8420
HI 1.456±0.386 1.236±0.232 1.689±0.358 1.592±0.448 1.896±0.390
n 20 20 20 20 20
Table three reorganization plasmid pUDKH and Bfgf-ESSEX, rbEGF are to the influence of wound healing
Wound area (mm
2)
Time (my god)
pUDKH bFGF rhEGF control
1 41.23±2.75 41.78±2.69 40.68±2.69 41.23±2.75
8 19.68±11.6 27.96±13.5 28.90±9.96 28.51±9.74
12 1.276±1.84 3.278±4.59 6.283±9.76 7.504±13.56
n 10 10 10 10
Table four is smeared loose index (HI) of each group behind pUDKH and Bfgf-ESSEX (bFGF), the rhEGF
Liniment pUDKH bFGF rhEGF control
HI 1.861±0.53 2.156±0.32 2.255±0.64 2.794±0.58
n 10 10 10 10
Claims (6)
1, a kind of eukaryotic expression recombinant plasmid that is used for the treatment of ischemic diseases and control cicatrix is characterized in that this recombiant plasmid carrier liver cell growth factor gene, contains the kalamycin resistance gene sequence; Liver cell growth factor gene is subjected to the polyadenylic acid signals-modulating of cytomegalovirus promoter, bovine growth hormone gene, and the structure of this recombiant plasmid is:
2, the purposes of the described recombiant plasmid of claim 1 in the medicine of preparation disease preventing and treating, wherein said medicine can or be wrapped in gold (tungsten) granule, balloon surface for injection, baste.
3, purposes according to claim 2, wherein said disease are limbs infraction property angiopathy, coronary artery infraction property angiopathy or cerebral arteries infraction property angiopathy, and route of administration is local intramuscular injection or particle gun or conduit intervention transfer.
4, purposes according to claim 2, wherein said disease is the ischemic optic atrophy.
5, purposes according to claim 2, wherein said disease is amyotrophy or myofibrosis.
6, the described recombiant plasmid of claim 1 promotes wound healing in preparation, prevention of scar excessively forms or treat purposes in the medicine of cicatrix.
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|---|---|---|---|---|
| EP1547618B1 (en) * | 2002-10-02 | 2010-05-26 | AnGes MG, Inc. | Drug for auditory dysfunction |
| CN101189023B (en) * | 2005-03-31 | 2013-01-30 | 通用医疗公司 | Monitoring and modulation of HGF/HGFR activity |
| CN102046792B (en) | 2008-04-09 | 2014-12-10 | 百疗医株式会社 | Lyophilized DNA formulations for enhanced expression of plasmid DNA |
| CN101701219B (en) * | 2009-11-06 | 2013-02-20 | 哈尔滨医科大学 | cDNA for encoding rabbit hepatocyte growth factor and expression vector and application thereof |
| KR101779775B1 (en) | 2013-10-22 | 2017-09-21 | 주식회사 바이로메드 | Composition for preventing or treating amyotrophic lateral sclerosis using two or more isoforms of hepatocyte growth factor |
| CN106497975A (en) * | 2016-10-21 | 2017-03-15 | 浙江生创精准医疗科技有限公司 | The preparation method of genetic recombination stem cell medicine and its application in skin injury reparation, scar suppress |
| CN108611367B (en) * | 2018-04-24 | 2019-11-19 | 北京诺思兰德生物技术股份有限公司 | The gene therapy recombinant vector that one kind is mediated by plasmid vector |
| SG11202100371RA (en) | 2018-07-19 | 2021-02-25 | Helixmith Co Ltd | Lyophilized pharmaceutical compositions for naked dna gene therapy |
-
2000
- 2000-12-21 CN CNB00132196XA patent/CN1150035C/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100441226C (en) * | 2002-04-26 | 2008-12-10 | 中国人民解放军军事医学科学院放射医学研究所 | Method of speeding wound repair and preventing complications |
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| Publication number | Publication date |
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| CN1358543A (en) | 2002-07-17 |
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