Experimental Study On Therapy For Ischemia Of Rabbit Hindlimb By Mesenchymal Stem Cells Transduced With Ad-HGF

BackgroundTherapeutic angiogenesis promotes the formation of both new blood vessels and collateral circulation by means of transferring heterogeneous angiogenesis inducing factors into ischemic tissue. It mainly contains two strategies. One is using cytokines or cytokine gene carriers, the other is transplanting multi-directional differentiation potent stem cells to ischemic tissue, where they would differentiate into vascular endothelium and produce vascular growth factors to achieve angiogenesis.Combined gene therapy and stem cell therapy in genetic engineering, the application of genetically modified stem cells to promote angiogenesis is a promising treatment. HGF gene-modified bone marrow mesenchymal stem cells not only transport therapeutic protein HGF to the ischemia organizations but play a important role as therapeutic materials to promote angiogenesis.This study including three parts investigates those questions:(1) To research the separation, in vitro culture and expanding method of mesenchymal stem cells (MSCs) and identifying the MSCs cultured by this mothod.(2) Rabbit bone marrow mesenchymal stem cells (BMSCs) were transduced with hepatocyte growth factor (HGF) by adenovirus vector. Transduction efficiency was detected by flow cytometry, transgene expression of HGF in vitro were evaluated by ELLISA and immunohistochemical methods.(3) To investigate whether a new strategy that combines MSCs transplantation and exvivo hepatocyte growth factor (HGF) gene transferring with adenovirus vector was more therapeutically efficient than MSCs cell therapy alone in a rabbit hindlimb ischemia model.Part Ⅰ. In vitro culture and identification of bone marrow mesenchymal stem cellsObjective:To research the separation, in vitro culture and expanding method of mesenchymal stem cells (MSCs) and identifying the MSCs cultured by this mothod. Study the biological characteristics of MSCs.Method:MSCs were isolated and purified from rabbits using density centrifugation and anchoring culture, then cultured in a-MEM supplement with10%fetal bovine serum (FBS) for expanding. Observed the morphous and growth velocity of MSCs, identify the phenotype of MSCs, draw the growth curve of MSCs and determine the mitotic index and cloning efficiency of MSCs. Observed the survival rate and growth state of MSCs underwent freeze thawing. Flow cytometric was used to dentify BMSC.Result:The cultured MSCs were typical spindle-shaped, grew in whirlpool anchoring the culture plates, arranged gyrately. Through detection by flow cytometry, the expressions of CD44and CD29were positive; the exprssions of CD11b and CD45were negative, which was in accordance with the phenotype of MSCs. The passage MSCs proliferated fast. MSCs were purified at3rd generation. As the passage increases, the cloning efficiency and proliferative ability of MSCs decrease. The survival rates of MSCs thawed after frozen1month was90%.Conclusion:BMSCs can be harvested using density centrifugation and anchoring culture. In vitro expanding of BMSCs is feasible. Freeze thawing has no obvious influence on BMSCs. Part Ⅱ. Preparation of BMSCs transduced with Ad-HGF and invitro experimentObjective:Rabbit bone marrow mesenchymal stem cells (BMSCs) were transduced with hepatocyte growth factor (HGF) by adenovirus vector.Transduction efficiency was detected by flow cytometry, transgene expression of HGF invitro were evaluated by ELISA and immunohistochemical methods.Method:After digested with pancreatic enzyme, BMSCs were plated and cultivated in6holes plate with a density of1.5×105/per hole.50ul Ad-GFP with6different MOI(multiple of infection) was added into each hole for BMSCs transduction, with3duplicated holes for each MOI. BMSCs were transduced with Ad-hgf at MOI=150.After digested with pancreatic enzyme, BMSCs were added to silicificated slice, and cultivated. Then BMSCs were detected with ELISA and immunohistochemical methods.Also, transwell was used to detect the efficiency of BMSC migration mediated by HGF. Result:48h after BMSCs transduction with Ad-GFP at different MOI, percentage of GFP-positive cells showed significant difference(P<0.05). Percentage of GFP-positive cells increased as MOI increased, however there was no significant difference (P>0.05) when MOI was bigger than150. When MOI=150, GFP-positive cells were over98%, so MOI=150was optimal for BMSCs transduction. When BMSCs were transduced with Ad-HGF at MOI=150, the results of ELISA and immunohistochemistry showed that Ad-HGF might effectively transducer BMSCs, and that the transduced BMSCs revealed high efficiency of HGF expression.The result also demonstrated HGF promoted the migration of BMSCs.Conclusion:Ad-GFP successfully transduced BMSCs. As MOI=150, GFP-positive cells were over98%.There were GFP-positive cells just24h after transduction, with peak percentage from2d to7d. These results indicated that adenovirus vector possessed high effieiency transduction and stable gene expression. Results of ELISA and immunohistochemitry demonstrated that Ad-HGF was capable of effectively transducing BMSCs, and that transduced BMSCs possessed high efficiency of HGF gene expression in vitro.Part Ⅲ. The research of bone marrow-derived mesenchymal stem cells transduced with HGF gene transplantation for treatment of rabbit limb ischemiaObjective:To investigate whether a new strategy that bone marrow-derived mesenchymal stem cells transduced with HGF gene transplantation was more therapeutically efficient than BMSCs transplantation or HGF gene therapy in a rabbit hindlimb ischemia model.Methods:New Zealand white rabbits hindlimb ischemia model were randomly divided into four groups:(a) Ad-GFP injection (Ad-GFP group,n=10),(b)BMSCs transplantation (BMSC group, n=10),(c) Ad-HGF injection (Ad-HGF group, n=10),(d)BMSCs transduced with HGF (HGF-BMSCs) transplantation (HGF-BMSC group,n=10). The ischemic thigh muscle were analyzed for arteriography,capillary density, transplanted cell differentiation, the expression of HGF,c-met and VEGF protein after treatment. Serum levels of HGF in HGF-BMSC group after transplantation were determined by ELISA. Results:Three weeks after transplantation, angiogenesis was significantly enhanced by b,c and d group, while capillary density was highest in the HGF-BMSC group (P<0.05). The number of transplanted cell-derived endothelia cells was higher in the HGF-BMSC group than in the BMSC group since one week after treatment (P<0.05). In treated ischemic hindlimb muscles, allogeneic HGF-BMSC and BMSC injection were associated with comparatively few T lymphocyte infiltration in Ad-HGF group(P>0.05). The expression of angiogenic growth factors like HGF and VEGF in local ischemic muscles undergoing intramuscular cell transplantation was more abundant in HGF-BMSC group than the other three groups (P<0.05).Serum levels of HGF in HGF-MSC group one week after transplantation have no significant difference(P>0.05).Conclusions:The present study shows that BMSC transduced with HGF gene transplantation induced more potent angiogenesis and collateral vessel formation than BMSC transplantation or HGF gene therapy. Stem cell-based angiogenic gene therapy may be a new therapeutic strategy for the treatment of severe ischemic cardiovascular disease.