| Objective:To observe the effects of Brain marrow derived marrow mesenchymal stem cells(BMSCs)induced by tissue inhibitor of kallikrein 1 combined with Astragalus membranaceus on myocardium ultrastructure,Ion channel protein and tissue kallikreceptor B2.Method:Twenty randomly selected male rats(180±20g)from SPF grade Wistar were randomly divided into sham operation group and normal group(10 rats in ea-ch group).A model of myocardial infarction was established by coronary artery lig-ation.70 rats were randomly divided into two groups:model group,negative control group,BMSCs group,BMSCs+huangqi group,BMSCs+KLK1group,BMSCs+5-AZA group,BMSCs+KLK1+huangqi group,a total of 11 groups.Cell transplantation was to induce rat BMSCs into adenovirus or 5-aza Cytidine(5-AZA),respectively.14 days a-fter the establishment of the model,4 sites were selected around the infarcted myocardium and injected into each group of KLK1 cells.Astragalus injection(dose 0.66 m L/0.2 kg d)was injected intraperitoneally.After cell transplantation,third day were performed,BMSCs group,BMSCs+5-AZ A group,BMSCs+KLK1 group,after operation 3 days,were injected with normal saline,such as the volume o-f Astragalus Injection.In the negative control group,Negative control group,Fourteenthd after molding,two thoracotomy,the cell culture medium was injected only in the infarcted area.Each group was taken 4 weeks after the intervention.The ultrastructure of the pathological section of the heart was observed by electron microscopy,The expression of KLK1 and kallikrein type B2 receptor in the heart were detected by RT-PCR.The expression of KLK1,sustained release peptide B2 receptor and potassium channel Kv1.2 and Kv1.5 were detected by Western Blotting.protein expression levels were observed by electron microscopy.Results:1.Electron microscopy results: normal group,sham operation group: myocardial myofibers arranged neatly,continuous,A band,I band,H band,Z line,M lineis clearly visible,intercalated disk was ladder-like,With many mitochondria.In the model group,the negative control group: the myocardium of the infarcted myotomy was shortened,the I band disappeared,the myofascicular myofibus was irregular,the H band and the M line disappeared and the mitochondria swelled.BMSCs transplantation group,BMSCs + Astragalus group: pathological changes were mild,and can see the naive cells,large nuclei,chromatin particles fine and uniform,mainly to the main chromatin,nucleus mitochondria,ribosomes,Silk just formed a small myofibrate and the degree of development of different myofibrils,considered for the differentiation of transplanted BMSCs cells,surrounded by more capillaries,some visible and the surrounding normal myocardial formation gap junction.BMSCs+KLK1 group: In the electron microscopy also found that foreign cells and myocardial cells to form a close gap junction,similar to the intercalated disk structure.In the myocardium,usually only between the cardiomyocytes canform a close gap junction.BMSCs +5-AZA group: some foreign cell morphology between fibroblasts and cardiomyocytes,there are also silk-like structure exists BMSCs+KLK1+ Astragalus group: observed in the MI scars in the presence offoreign transplant cells: some cells longer,large nuclei,both sides blunt round,similar to myocardial cells oval nucleus(unlike fibroblasts,the nucleus on both sides of the shuttle Shape),the internal chromatin and heterochromatin coexist ence,and often a higher proportion of chromatin,cytoplasm is not rich,with mitochondria and more just developed myofilament,irregular arrangement.2.The expression of KLK1 mRNA content: BMSCs group compared with the control group,the difference was not statistically significant;Compared with BMSCs group,BMSCs+KLK1 group and BMSCs+Huangqi group content of KLK1 mRNA expression level was increased;Comparison with the BMSCs+Huangqi group,BMSCs+KLK1 group KLK1 m RNA in the expression level increased;Further com-parison,compared with BMSCs+KLK1+Huangqi group,BMSCs+KLK1groupm RNA content were up-regulated,BMSCs+Huangqi group m RNA in The expressionlevels were down re-gulated;Compared with the BMSCs+5-AZA group,BMSCs+KLK1 group KLK1 m RNA in the expression level increased;BMSCs Huangqi group was no significant difference.The expression of KLK1 protein: compared with the negative control group,the relative expression of BMSCs protein decreased;Compared with BMSCs group,BMSCs+KLK1 group and BMSCs+Huangqi group protein expression level was increased;Compared with BMSCs+KLK1+Huangqi group,protein content BMSCs+KLK1 group expression of water increases;BMSCs Huangqi group expression level is lower;Compared with the BMSC+5-AZA group,the expression levels of KLK1 protein in BMSCs+KLK1 group were up-regulated;The expression level of KLK1 protein in BMSCs+ Astragalus membranaceus group was down regulated.3.The expression of release peptide B2 mRNA receptor: Compared with the BMSCs group,BMSCs+KLK1 group and BMSCs+huangqi group the levels of B2 mRNA in the were up-regulated;Compared with BMSCs Huangqi group,BMSCs+KLK1 group the levels of B2 mRNA in the were up-regulated;Com-pared with BMSCs+KLK1+Huangqi group,BMSCs+KLK1 group the levels of B2 m RNA in the were up-regulated,BMSCs+ Huangqi group had no statistical significance difference.Compared with the BMSCs+5-AZA group,BMSCs+KLK1 group the levels of B2 mRNA in the were up-regulated;BMSCs+ Huangqi group he levels of B2 mRNA was down regulated.Expression of sustained release peptide B2 protein:Compared with the BMSCs group,BMSCs+KLK1 group and BMSCs+huangqi group the relative expression levels of B2 protein in were all up-regulated;Compared with BMSCs +Huangqi group,BMSCs+KLK1 group the relative expression levels of B2 protein in were all up-regulated;Compared with BMSCs+ KLK1+huangqi group,the expression level of B2 proptein in BMSCs+huagqi group was significantly lower than that in group,BMSCs+KLK1 group The difference was not statis-tically significant;Compared with the BMSCs+5-AZA group,BMSCs +Huangqi group The expression level of B2 protein in the sustained release peptide was significantly lower,BMSCs+KLK1 group The difference was not statistically significant.4.Expression of potassium channel proteins:The expression of Kv1.2 protein: Compared with BMSCs group,the relative expression of BMSCs+KLK1 group and BMSCs+Huangqi group Kv1.2 protein level was increased;Compared with the BMSCs+huangqi group,the expression Level of Kv1.2 protein in BMSCs+KLK1 group was significantly increased;Compared with BMSCs+KLK1+Huangqi group,in BMSCs+Huangqi group the expression of Kv1.2 protein was down significantly.The expression of Kv1.2protein in BMSCs+KLK1 group was significantly decreased;Compared with the BMSCs+5-AZA group,BMSCs Huangqi group Kv1.2 protein expression was significantly decreased,BMSCs+KLK1 group,there was no statistically significant difference.The expression of Kv1.5 protein: Compared with BMSCs group,the relative expression of BMSCs+KLK1 group and BMSCs+Huangqi group Kv1.5 protein level was increased;Compared with BMSCs+Huangqi group,BMSCs+KLK1 group Kv1.5 protein expression increased significantly;Further compared with BMSCs+KLK1+Huangqi group,BMSCs+KLK1 protein expression of the groups increased significantly,BMSCs+Huangqi group protein expression level was significantly decreased;Compared with the BMSCs+5-AZA group,BMSCs+KLK1 protein expression level increased si-gnificantly,BMSCs Huangqi group the level of protein expression was down regulated.Conclusion:1.KLK1 has an active therapeutic effect on the recovery of heart tissue after myocardial infarction;2.KLK1 transfection of BMSCs plays a very important role in the recovery of cardiac function after myocardial infarction,but there is no synergistic effect between the two doses of Astragalus membranaceus injection.3.5-AZA is effective in the treatment of myocardial infarction,but its own toxic effects can not be ignored,and even toxic effects are one of the factors that hinder the recovery of myocardial cells;4.KLK1 is superior to 5-AZA.in the protection of cardiac tissue in myocardial infarction model... |
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