| Objective: Studies have shown that immunogenicity may be one of the reasons for the low survival rate of BMSCs after allogeneic transplantation.In this study,bone marrow mesenchymal stem cells(BMSCs)with low immunogenicity were designed to observe whether it could improve the survival rate of BMSCs after transplantation and improve myocardial infarction Posterior state,in order to provide reference for rational clinical stem cells use.Methods: 1,recovery the rat bone marrow mesenchymal stem cells,culture in vitro,when the cell fusion reached 70%-80%,with 0.25% trypsin digestion,passaged,adjust the cell status,to maintain good cell activity,for the late Of the lentivirus infection.2,take 5 generations of cells,add different concentrations of puromycin,72 hours later,using CCk-8 measured absorbance OD,select the cells just all the death of the smallest purine,which is the best screening concentration.3,BMSCs were infected with MOI = 5,20,50,100.After 72 hours of infection,the fluorescence expression of the cells was observed by fluorescence microscopy.The infection efficiency was detected and the MOI value was obtained.4,The BMSCs were infected with MOI at the best infection efficiency.After 72 hours,the puromycin was selected and the puromycin concentration was selected.The optimal concentration of puromycin was selected and the cells were screened for one week to obtain stable cell lines.IFN-? was added to the obtained stable cell line and normal BMSCs.After 24 hours of extraction,RT-QPCR and western blotting were used to confirm the silencing of MHC? gene.5,the myocardial infarction model of rat,legating the anterior descending coronary artery branch.The cells were transplanted in the marginal area of myocardial infarction after 1 week.The patients were divided into four groups: sham operation group,AMI group,BMSCs transplanted group,MHC? gene silenced.6,At the end of 4 weeks,the echocardiography was performed and the cardiac tissue was taken.The RT-QPCR of the SRY gene was used to estimate the survival of the cells.HE,Masson staining,immunohistochemistry was performed to determine the cardiac function,Macrophage infiltration,Fibrosis and neovascularization.7,data analysis using SPSS 16.0 statistical software statistics,the data with the mean ± standard deviation(x± s)that the two groups were compared between the two independent samples of the t test,multiple groups using single factor analysis of variance,P <0.05 suggest differences Has a statistically significant.Results: 1,bone marrow mesenchymal stem cells are mostly long spindle shape,a few were polygonal.Bone marrow mesenchymal stem cells were successfully obtained2,lentivirus infection of bone marrow mesenchymal stem cells,24 h no obvious fluorescent protein expression,48 h can be seen the expression of fluorescent protein,72 h fluorescence expression reached its peak.MOI = 5,20 when the expression of fluorescent protein,but the positive rate of transfection is low,MOI = 50,100,showing significant fluorescent protein expression,lentivirus transfection,the cell state slightly worse,taking the positive rate of infection and the influence of the virus to cells into consideration,MOI = 50 was chosen as the best infection virus titer.3,We acquire the stabilized expressed cell lines through puromycin selection.when the puromycin concentration of 0,2,2.5ug / ml,the stem cells will not completely die,with the puromycin concentration increased,puromycin 3,3.5,4,4.5,5ug / ml when the cells were completely dead,and there was no statistically significant difference between the groups,3.0 ug / ml for the cells just the exact minimum concentration of death,so choose 3.0ug / ml as the best Screening concentration.4,The m RNA expression of MHC? gene was significantly lower in the silencing group than in the untreated group(p <0.05).The results of RT-QPCR and MHC? gene silencing group were significantly higher than those in the untreated group(P <0.05)(P <0.05).5,through the rat anterior descending coronary artery ligation,establish a rat myocardial infarction model,the following myocardial became White after ligated,the boundary between non-infarcted and infracted area are clear,wall motion becaming week,heart rate increased.6,myocardial infarction after 4 weeks,the results of Echocardiography showed that the stem cell transplantation group has some improvement than the simple myocardial infarction group in cardiac function,and MHC? gene silencing group improved heart function is more obvious.7,RT-QPCR results of SRY gene showed that SRY gene expression in MHC? gene silencing group has a high survival rate of bone marrow mesenchymal stem cells(BMSCs)than in simple BMSCS transplantation group.8,The results of cardiac and histopathological findings showed that,fibrosis,connective tissue hyperplasia,tissue thinning,softening,depression,HE and Masson staining showed that myocardial fibrosis and myocardial normal tissue destruction were observed in myocardium.The results of VEGF and F4 /80 staining showed that angiogenesis occurred in the myocardial infarction area,and the proliferation of the stem cell transplantation group was more obvious.The expression of VEGF was increased in the MHC? gene silencing group.Conclusions: 1,reduce the immunogenicity of BMSCs can improve the survival rate.2,low immunogenicity of BMSCs improved better at the situation after myocardial infarction,the mechanism may be related to macrophage. |
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