| Background:With the development of molecular biological and modern biotechnological techniques, the research of biological treatment for the ischemic heart disease has made significant progress.Existing research results have proved that angiogenic growth factor gene therapy and autologous bone marrow mesenchymal stem cell transplantation have significant therapeutic effects on both acute and chronic ischemic heart disease.The angiogenic growth factor recombinant adenovirus can effectly transfected cells,and have large quantity of protein expression and the advantages of moderate duration,so it is thought to be good in similar studies.Animal and clinical study results show that the angiogenic growth factor can effectively promote angiogenesis in ischemic myocardium and improve the blood supply.But further research found that the improvement of cardiac function can is not significant,the reason may be associated with that the number of the heart homing stem cells is too small to replenish necrotic myocardial cells.The study on myocardial cell transplantation in the region of myocardial infarction is popular noe.The experts fully affirmed the effect of biological treatment for ischemic heart disease,but they also pointed out that the methods still have disadvantages.The core is micro-environment of ischemic affects the survival rate of transplanted cells and increase of the tendency of local fibrosis after regional cell transplantation.The survivorship and growth of stem cells,as well as their offspring cells,need appropriate micro-environment. When the bone marrow stem cells are implanted after myocardial injury,infarction marginal have been well improved,but the most of the transplanted stem cells in the core region of infarction can not survive effectively,as a result of serious local tissue hypoxia-ischemia,micro-poor environment,and eventually affect the therapeutic effect. Therefore,how to improve the survival rate of transplanted stem cells has become a new hotspot the of treatment for ischemic heart disease.In addition,the differentiation of transplanted cells to fibroblasts in the treatment should not be overlooked as the potential constraints.Because of the ability of multi-directional differentiation,stem cells have a clear tendency differentiation to fibroblasts after transplantation to the scar tissue in which the micro-environmental impacts.In a certain sense,implanted cells can even further increase the possibility of fiber scar formation.Existing research results show that hepatocyte growth factor(HGF) can not only promote the restoration of endothelial cell and the promotion of angiogenesis functions, but can also significantly decrease the apoptosis of the cell in the infarctial region. Moreover,HGF can also significantly inhibite fibrosis.As a multi functional cytokine which both can promote angiogenic growth,inhibite of apoptosis and fibrosis,HGF is a kind of can ideal cytokines which can potential improve the micro-environment of ischemic tissue.Methods:1.MSCs isolation,culture and adenovirus transfectedMSCs were harvested from pig ilium and isolated by combination of gradient centrifugation of Percoll and preplating treatment,then were cultured in EMDM medium with 10%fetal bovine serum.Cell growth curve was obtained by the continual cell counting and the ultra-structure of cells were observed by electron microscope.The cells were identified by the immunohistology staining and flow cytometer of CD71,CD34,CD44 and desmin.MSCs were incubated with HGF and other growth factors to observe their pluripotential differentiation.Transfection efficiency of adenovirus vector to MSCs were evaluated by infection with various titrations of Ad.LacZ.MSCs were transfected by Ad.HGF.HGF expression were measured by immunohistochemical staining and western blot.The concentration of secretive protein in the medium were measured by ELISA.The cells proliferation effect of secretive proteins were evaluated by proliferation of the endothelial cells.2.Bionomics of MSCs transfected by Ad.HGFMSCs was transfected by AdHGF,AdVEGF respectly,and detected the change of MSCs through MTT method.Intensity of fluorescence and cell-distribution were measured by flow cytometer.Using Annexin V-FITC and PI to mark apoptosis of MSCs. Composition and externalization of collagen fiberâ…¢were detected by ELISA in order to compare diversity of them.3.Establish of pig myocardium ischemic model and transplantation of MSCsThe distal left anterior descending artery(LAD) was occluded with the balloon of an OpenSail dilatation catheter for 90 minutes to create myocardial infarction. Electrocardiography was used during the procedure to monitor the induction of ischemia, and to prevent premature death from excessive ventricular arrhythmia,guiding catheter was introduced into the ostium of the coronary sinus through the jugular vein under fluoroscopy.Next,the guiding catheter was inserted into the GCV,and advanced into the distal AIV.The cells were rotated inside the vial to achieve re-suspension before delivery, and loaded into a 10cc angiography syringe.The balloon was re-inflated,and an image was recorded(cine and still) to show the location of occlusion.To deliver the cells(all 10ml in about 10-15 sec),the injection syringe containing the cell suspension was attached to the infusion port of the infusion catheter,and the injection was performed. Saline(2ml) was injected to flush the infusion lumen,and the balloon was left inflated for 3 minutes before deflation and withdrawal of all catheters.Blood samples were collected at 0,90,and 150 minutes after ischemia,and were centrifuged to obtain the serum for measurement of pig cardiac Troponin I(cTnI) levels.One week after cell implantation, under general anesthesia,animals were euthanized by injection of 20ml of potassium chloride into the right atrium.The hearts were excised and stainied for analysis.4.Transplantation of autologous MSCs transfected by AdHGF gene in a porcine ischemic heart model through coronary sinusOne weeks later,20 pigs were randomized(10 pigs for each group) to treatment with the MSCs transfected ex vivo by HGF group(Groupâ… ),or MSCs transfected ex vivo by VEGF group(Groupâ…¡),The amount of total implated cells was 108 and the transfected virus titrations was 1010pfu,.All cells were labeled by CM-DiI before injection.Four weeks after initiation of therapy,the animals were evaluated by regional perfusion,Rentrop score,ejection factor(EF),fraction shortening(FS),regional systolic wall thickening (RSWT) using DSA,echocardiography,contrast echocardiography or SPECT.After sacrificing the animals,the percentage of infarct area,vessels counting,TUNEL staining of the implanting sites were performed.The CM-DiI labeled cells were observed under fluorescence microscope and the target proteins were detected by western blot.Results:1.MSCs isolation culture and adenovirus transfected The MSCs demonstrated typical MSC fibroblastic morphology with the ability to proliferate in culture.They are positive for CD71 and CD44,while negative for CD34 and desmin by immunohistology staining. FACS analysis demonstrated that 73.9%cells expressed CD34 and 85.4%for CD 71.The cells displayed the MSCs characters under transmission electron microscope and possessed the capabilities of rapid proliferation and pluripotential differentiation.The cells showed CD34 positive when induced by HGF and other growth factors.The transduction efficiency of Adenovirus to MSCs was proved to be over 95%at MOI 50 using Ad.LacZ transfection in vitro.The MSCs were detected to express of target protein after Ad.HGF transfection by immunohistochemical staining and western blot.2.Bionomics of MSCs transfected by Ad.HGF MSCs was transfected by AdHGF and detected the change of MSCs through MTT method and found HGF may induce increment of MSCs,and antagonism apoptosis of MSCs.Intensity of fluorescence and cell-distribution were expression by flow cytometer.Using Annexin V-FITC and PI. Composition and externalization of collagen fiberâ…¢were degrade compare to AdVEGEThis show that HGF was one better biotherapy to IHD than VEGF.3.Evaluation of pig myocardium ischemic model and transplantation of MSCs Of the total 40 animals,12 animals died from excessive ventricular arrhythrnia.yocardial infarction were obviously in animal mode,Di-I labeled cells seen at the site of implantation on fluorescence microscopy.TroponinI levels changed obviously(P＜0.01).4.The improvement of perfusion and heart function after the transplantation of Ad.HGF-MSCs.4 weeks after treatment,compared with those before treatment,the perfusion were greatly enhanced in the Groupâ… ;partial enhanced in Groupâ…¡(P＜0.05).EF, FS,RSWT showed greatly improved in Groupâ… ,and the same results were reached by SPECT.The lateral infarctions were observed in the gross left ventricular(LV) cross-sections,consistent with the echocardiography results.The infarction areas in Groupâ… (3.12%±1.10%) were much smaller than the other groups(P＜0.05)(Groupâ…¡8.45%±3.73%).Groupâ… showed the apparent normal gross L V geometry and maintenance of wall thickness.There was successful engraftment of MSCs in all MSCs treated animals as shown by the Di-I labeled cells seen at the site of implantation on fluorescence microscopy.A large number of labeled MSCs could be identified in Groupâ… ,while only sporadic labeled MSCs were seen in Groupâ…¡.Cells appeared to preferentially engraft in regions of necrotic tissue and adhere to the collagen rich matrix.The vessels counting of the ischemic area for each group were:30.3±3.5/HPF,34.5±3.7/HPF respectively.The vascular density in Groupâ… have increased more diffusely than the other groups(P＞0.05). For TUNEL staining,the positive rate in each group were:3.3±0.8%,9.5±1.3% respectively(P＜0.01).Conclusions:1.High purified MSCs can be obtained by the combination of gradient centrifugation of Percoll and preplating treatment.The obtained cells showed MSCs surface markers and possessed the capabilities of pluripotential differentiation.The MSCs can be expanded rapidly in vitro;Transduction HGF gene to MSCs by adenovirus is safe and high efficient, and the target protein can be expressed in cell and secreted to surrounding at a high level in vitro or in vivo.2.MSCs was transfected by AdHGF may induce increment of MSCs,and antagonism apoptosis of MSCs.Intensity of fluorescence and cell-distribution were expression by flow cytometer.Collagen fiberâ…¢were degrade compare to AdVEGF.This show that HGF was one better biotherapy to IHD than VEGF.3.The distal left anterior descending artery(LAD) was occluded with the balloon and implantation of MSCs through vena coronaria was feasible.This method consistent with clinical.4.The implantation of AdHGF-MSCs developed the collateral vessel,improved the local reperfusion,attenuate contractile dysfunctons and pathologic remodeling of the ventricular wall after infarction and significantly augmented left ventricular function.
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