Treatment Of Cardiomyopathy Heart Failure With Bone Marrow Derived Meshenchymal Stem Cells Overexpressing Hepatocyte Growth Factor

Part one: The expression of hHGF in the transfected MSCsObjective : To investigate the transfection efficiency of mesenchymal stem cells by adenovirus vector encoding GFP gene (Ad-GFP) and the expression of hHGF in MSCs infected with adenovirus vector encoding hHGF gene (Ad-hHGF).Methods: Rat,s bone marrow-derived mesenchymal stem cells (MSCs) were isolated using Percoll desity gradient method and according to the adherent property of MSCs . Then we infected rat,s MSC with adenovirus vector encoding green fluorescent protein (GFP )or hHGF. The efficiency of infection and expression of human hepatocyte growth factor (hHGF) were detected by flow cytometer (FCM) and enzyme-linked immunoadsorption assay (ELISA) respectively.Results: (1) MSCs can be successfully isolated and cultured by density gradient centrifugation followed by adherence-separation and be identificated by FCM. (2) It showed that transfection efficency was increasing along with the increase of multiplicity of infection (MOI). A nearly 100% cells were transfected when the 100 MOI was administrated. MSCs could be transfected efficiently as seed cells to accept target genes by Ad-hHGF, and a higher level of expression in vitro, which persisted 16 days at least.Conclusions:①MSC are ideal cells for cell-mediated HGF gene therapy or cell therapy.②MSC expression HGF at a high level when being transfected with the Ad-hHGF. The HGF experession could maintain for at least 16 days .Part two: Effects of vein transplantation of mesenchymal stem cells transfected with hepatocyte growth factor gene on improvement of heart function after cardiomyopathy heart failure with rats and the investigation of mechamismObjective: To establish a method to transfect Ad-HGF into MSCs and implant to cardiomyopathy heart failure model of rat, to investigate the effects of the gene transfected MSCs on heart function restoration biomarkers and fibrosis, angiopoiesis in rats with adriamycin (Adr)-induced heart failure, and to compare the difference among cell therapy and combined therapy .Methods: Thirty-six SD rats underwent injecting intraperitoneal injection with adriamycin so as to establish heart failure model. Forteen rats did not undergo any operation . twelty-four surviving rats that underwent injecting adriamycin were randomly divided into 3 equal groups : The MSCs-hHGF group to be injected via caudal vein with suspension of Ad-hHGF transfected MSCs after 2 weeks after the establishment of the model, the MSCs group to be injected with suspension of MSCs not transfected with Ad-hHGF, and the Normal group and Model group to be injected with PBS only to collect blood plasma from four groups rats in the different time to detect the expression of hHGF. Four weeks after the injectiong the surviving rats underwent examination of heart functions by a physiological recorder and collected blood plasma from four groups rats to detect the level of atrial natriuretic peptide( ANP). treponin T (cTnT) , tumor necrosis factor a (TNF-a), endothelin(ET ), interleukin 6 (IL-6).The rats were killed and their hearts were taken out. Myocardial cell morphology and interstitial collagen were observed by electron microscope and were stained by Sirus red in supersaturated picric acid solution . transforming growth factor-β1(TGF-β1)and factorⅧto measure the capillary density was detected by immunohistochemical method.Results: (1)Intravenous administration of Ad-hHGF into rats resulted in significantly elevation of serum hHGF level (n= 8) p <0.01 vs other groups in 7, 9 ,11,14 ,28 day. but in the 3d, the four groups have not statistical significance (p>0.05). (2) Four weeks after the cells were transplanted, among all groups but the normal group, cardiac pathological change of MSCs-hHGF group was lightly changed compared with those of the MSCs group and Model group. The heart function of the MSCs-hHGF group was better than those of other groups.(3) Four weeks after the cells were transplanted , Plasme ANP and serum ET, TNF-a, cTnT, IL-6 concentration in MSC group and MSC-hHGF group were significiently lower than that of model group (p <0.01), TNF-a ,IL-6 concentration had no statistical significance between MSCs and MSC-hHGF group, but TNF-a, cTnT concentration were higher in MSC group than in MSC-hHGF group(p <0.05). (4) Myocardial collagen in model group increased obviously compared with normal group and MSC group and MSC-HGF group from the results of electron microscope, HE, Sirus red in supersaturated picric acid solution Myocardial collagen (p <0.01). The level of TGFβ1was significantly decreased in the MSC-HGF group and MSC group compaired with the model group. There was significant difference in the level of TGFβ1 in the MSCs compaired with MSC-HGF group (p <0.01) (5)The capillary density of the MSCs-hHGF group was 11.85±1.34 /visual field, significantly greater than those of both the MSCs group (9.68±0.96/visual field , p <0.05) and that of the model group (6.55±1.50 /visual field , p <0.01).Conclusions Transplantation of HGF gene by means of transfected MSCs brings better improvement in myocardial perfusion and in restoration of heart function than cellurlar therapy alone .