Study Of Guanxin Ⅱ Combined With Transplantation Of Bone Marrow Mesenchymal Stem Cells For Treatment Of Myocardial Infarction

Part 1 Culturing,Identification and Labelling of Rat Marrow Mesenchymal Stem CellsExperiment 1The Isolating,Culturing and Identification of Marrow Mesenchymal stem cellsObjective To culture rat marrow mesenchymal stem cells(BMSCs)fitting the requirement of experimentation for further study.Methods Whole bone marrow adherent cultivation or density gradient centrifugate cultivation were adopted to culture rat BMSCs.The morphology and characteristics of cultured cells were observed and the surface marker identification of CD34,CD44,CD45,CD29,CD90 and CD11b of cells identified by flow cytometry.Results Cells cultured by whole bone marrow adherent cultivation showed CD34-(95.4%),CD44+(94.2%),CD45-(91.6%),CD29+(93.8%), CD90+(98%)and CD11b-(87.6%);Cells cultured by density gradient centrifugate cultivation showed CD34-(96.9%),CD44+(97.3%), CD45-(97.5%),CD29+(99.4%),CD90+(99.8%)and CD11b-(96%)Conclusions The purified BMSCs are isolated and cultured by both methods. The whole bone marrow adherent cultivation are simple and easy to operate and Its primary culture have shorter cycle.But the cells cultured by density gradient centrifugate cultivation are more purify.Experiment 2 A Survey of CM-DiI Labelling and Tracing of Rat Marrow Mesenchymal Stem CellsObjective A method of labeling and tracing bone marrow-derived mesenchymal stem cells(BMSCs)is established for further study.Methods CM-DiI cell-labeling solution was used to label rat BMSCs(P3)and trace labeled BMSCs.CM-DiI-labeled BMSCs were transplanted into the cardiac muscle of acute myocardial infarction(AMI)rat models by intramyocardial injection.In 1week,2weeks,3weeks,4weeks after cells were injected,The labeled cells were traced and expression of connexin43 was surveyed(only in 4weeks after cells were injected)in the cardiac muscle tissue sections by fluorescence microscope.Results Efficiency of CM-DiI labeling BMSCs was 100%,The growth and proliferation of BMSCs were not changed by CM-DiI.Their fluorescence were stayed in 14 days after labeled.The CM-DiI -labeled BMSCs can been found in the cardiac muscle tissue sections in 1week,2weeks,3weeks,4weeks after cells were injected.The labeled cells expressed a small quantity connexin43. Conclusion The method of CM-DiI labeling BMSCs is simple to perform and effective and can be use in the further study.The BMSCs can be survival in infarct cardiac sites in 4 weeks after cells are injected.Part 2Effects of GuanXinⅡCombined with Transplantation of Bone Marrow Mesenchymal Stem Cells on Acute Myocardial Infarction in RatsObjective To observe the effects of GuanXinⅡcombined with BMSCs transplantation on cardiac function,neovascularization and ventricle remodeling of acute myocardial infarction(AMI)rat models,compared with only BMSCs transplantation,and investigate the synergetically therapeutical effects of GuanXinⅡand BMSCs transplantation on AMI and their mechanism of action. To provid the evidences of the traditional Chinese medicine's application in enhancing curative effect of stem cells transplantation on AMI.Methods SD rats were randomedly divided into 5 groups:(1)Control group (without AMI model):intragastric administration with saline;(2)model group (with AMI model):L-DMEM medium was injected into the infracting myocardia sites and saline was fed for 1 week by intragastric administration;(3) GuanXinⅡgroup(with AMI model):L-DMEM medium was injected into the infracting myocardia sites and GuanXinⅡbroth(equivalent to rude drug 10g/kg)was fed for 1 week by intragastric administration;(4)BMSCs group (with AMI model):2×10~6 BMSCs labelled CM-DiI were injected into 5 different points in the infracting myocardia sites and saline was fed for 1 week by intragastric administration;(5)GuanXinⅡ+BMSCs group(with AMI model): 2×10~6 BMSCs labelled CM-DiI were injected into 5 different points in the infracting myocardia sites and GuanXinⅡbroth(equivalent to rude drug 10g/kg) was fed for 1 week by intragastric administration.4 weeks after cells transplantation,These data were detected and collected:(1)Doppler echocardiography was used to detect cardiac function and record LVIDd,LVIDs, FS and EF.(2)Myocardic fibrosis was investigated by Masson's trichrome staining.(3)The transplanted labeled BMSCs were detected in the cardiac muscle tissue sections by fluorescence microscope.(4)immunohistochemical method was used to detect the expression of VWF and VEGF and calculate microvessel density(MVD).(5)Expression of TGFβ1mRNA and VEGFmRNA in the marginal zones of infracting myocardia sites was measured by fluorescent quantitation RT-PCR.(6)Enzyme-linked inmunosorbent assay was used to detect the concentrations of PⅠCP and PⅢNP in blood serum.Results(1)dead cases in each group:4 in model group,3 in GuanXinⅡgroup,4 in BMSCs group,3 in GuanXinⅡ+BMSCs group.There were not significant different among the 4 groups.A total of 60 animals(10 in the control group,12 in model control group,13 in GuanXinⅡgroup,12 in BMSCs group, 13 in GuanXinⅡ+BMSCs group),were involved in the detection of cardial function;(2)cardiac function comparison:4 weeks after AMI,except the control group,the argument of ventricular wall contraction decreased and the chambers heart extended at different extents;besides,ejection fraction of left ventricle and shortening fraction decreased and the arterior wall of left ventricle did not move or move more slowly than before.Cardiac function of the model group was most severely damaged in comparison with the other the three groups. Compared with the control group,LVIDd,LVIDs,FS and EF in model group, BMSCs group,GuanXinⅡgroup and GuanXinⅡ+MSCs group declined remarkably(P＜0.01).LVIDs in GuanXinⅡ+MSCs group markedly shortened versus BMSCs group;besides,FS and EF of GuanXinⅡ+BMSCs group elevated significantly in comparison with the BMSCs group(P＜0.05,P＜0.01); (3)Masson trichrome staining showed that AMI sites were taken the place of collagen fiber;ventricular wall thinned;collagen content and fibrotic extent increased(model group＞BMSCs group＞GuanXinⅡ+BMSCs group).There was no fiber formation and the cardiac muscles lined up in order in the control group.(4)Transplanted BMSCs,erupting red fluorescence,distributed diffusedly in the frozen section of AMI marginal zone of the left ventricle.(5)In the three curative groups,the expression of VWF and VEGF and MVD increased(P＜0.01).The expression of VWF and VEGF of the three curative groups increased versus the model group(P＜0.01).The expression of VWF and VEGF and MVD of GuanXinⅡ+BMSCs group also increased in comparison with the BMSCs group.(6)The expression of TGFβ1mRNA and VEGFmRNA of the three curative groups were higher than the control group;the expression of VEGFmRNA was lower in the three curative groups than in the model control group;The expression of TGFβ1mRNA is lower,but the expression of VEGFmRNA is higher in the GuanXinⅡ+BMSCs group than in the BMSCs group.(7)After AMI,typeⅠand typeⅢcollagen synthesis increased at different extents.Compared with the model group,the serum level of PICP and PⅢNP in GuanXinⅡ+BMSCs group,GuanXinⅡgroup and MSCs group significantly decreased(P＜0.01);the serum level of PICP and PⅢNP in GuanXinⅡ+BMSCs group declined remarkably in comparison with the GuanXinⅡgroup and MSCs group.The results indicated that GuanXinⅡcombined with transplantation of BMSCs may effectively decreased the formation of collagen.Conclusions(1)The three curative therapies(GuanXinⅡbroth,BMSCs transplantation and GuanXinⅡcombined with transplantation of BMSCs) may improve the cardiac function after AMI and decrease the extent of ventricular dilation.GuanXinⅡcombined with transplantation of BMSCs may most effectively improve the cardiac function after AMI.GuanXinⅡand transplantation of BMSCs have synergetically therapeutical effects on AMI.(2)GuanXinlI both,BMSCs transplantation and GuanXinⅡcombined with BMSCs transplantation may enhance neovascularization after AMI. GuanXinⅡbroth can enhance neovascularization after BMSCs transplantation, which may correlate with the secretion of VEGF.(3)The three curative group can decrease collagen formation,heart muscle fibrosis and cardial remodeling. GuanXinⅡCombined with BMSCs transplantation may most effectively improve the cardiac remodeling via decreasing the secretion of TGFβ1.