|
Objective: Acute myocardial infarction as a common cardiovascular disease, a serious threat to human life. A large number of studies have shown that bone marrow stem cell transplantation in the treatment of myocardial infarction can significantly repair damaged myocardium induced neovascularization, improve heart function. But there are still a large number of transplanted cells early loss of transplanted timing and other issues, limiting its clinical application. After myocardial infarction, myocardial tissue ischemia and hypoxia, harmful cytokines and inflammatory mediators are released, the activity of inducible nitric oxide synthase (iNOS) is activated, may be the main reason for the massive death of transplanted cells. To this end, through animal experiments at different time points after myocardial infarction, male SD rat bone marrow mesenchymal stem cells transplanted to female rat model of myocardial infarction, transplanted cells was observed in the area of ??infarction survival and cardiac function change explore the best time of cell transplantation in the treatment of select suitable after myocardial infarction. And further by experimental observation after myocardial infarction iNOS expression variation observed early intervention iNOS inhibitor, on this basis, the impact on the survival of the transplanted cells and cardiac function, clear the survival of transplanted cells and cardiac repair, for stem cell transplantation in the treatment of myocardial infarction clinical applications to provide the necessary scientific basis. Method: 1, the model of acute myocardial infarction (AMI) Preparation: the body weight 150-180g, female SD rats with AMI model of permanent ligation of the left anterior descending artery was prepared according to the different time of cell transplantation randomized experimental observation . Bone marrow mesenchymal stem cells (MSCs) separation train: Take weighing 100 ~ 130g, male SD rats were isolated whole bone marrow adherent culture of MSCs using immunocytochemistry and flow cytometry culture MSCs were identified. Cell transplantation: According to different line in situ cell transplantation time point reoperation, the cultured MSCs under direct vision (about 1.2 × 106 unit) injectable implant to the middle of the infarcted area epicardium. 4, the location detection of the transplanted cells: Use Hoechst33342 and BrdU mark the transplanted cells, immunohistochemistry, and HE staining corresponding, observation of the location and distribution of the transplanted cells in the area of ??myocardial infarction. 5, quantitative detection of transplanted cells: the expression of the the sry gene sequences on real-time quantitative PCR female rat infarct zone determining region of the Y chromosome Gender, as the detection and quantitative comparison of the survival of the transplanted cells indicators. 6, the detection of cardiac function: 4 weeks after cell transplantation, the experimental rats cardiac ultrasound. 7, of myocardial infarcts iNOS expression pattern detection: 1h, 3d, 7d, 10d, 14d infarct zone after myocardial infarction iNOS expression was detected by Western-blot. Results: 1, MSCs are identified: the separation and purification of the fourth generation of MSCs by immunocytochemistry the expression of CD29 and CD44, CD34 expression by flow cytometry of CD44 / CD34 - to prove experimental cell of MSCs. 2 models of myocardial infarction: left anterior descending artery ligation, left ventricular anterior pale, heart enlargement, left atrial appendage congestive ventricular contraction weakened clear and not infarct zone boundaries, to show that the successful preparation of animal model of myocardial infarction. 3, transplanted cell survival and heart function observed: The rats were randomly divided into four groups, myocardial infarction (MI) group: the MSCs transplantation myocardial infarction, in four weeks undergoing cardiac function after myocardial infarction as a control; 1h transplant team: after myocardial infarction 1h line MSCs transplantation, transplant immediately taken as the Y chromosome sry gene detection control specimens; 3d transplant team: 3d line MSCs transplantation after myocardial infarction; 7d transplant group: the 7d line MSCs transplantation after myocardial infarction. 4 weeks of cardiac function after transplantation, 3d group and 7d group, the survival rate of the transplanted cells and histological detection. Real-time PCR detection of myocardial infarction after 7d MSCs survival of the transplanted group was significantly higher than the 3d transplantation group of MSCs survival rates were 8.1% and 2.5% (n = 10, p lt; 0.05). Cardiac function testing found that compared with the MI group, 4 weeks after transplantation of MSCs the 3d transplantation group, left ventricular end-diastolic diameter (LVDd) and end-systolic diameter (LVDs) no significant narrowing of left ventricular fractional shortening (FS) and significantly improved left ventricular ejection fraction (EF), there was no significant difference (n = 10, p gt; 0.05). 7d transplantation group four weeks after cell transplantation, LVDd and LVDs representing 3d transplant group and MI group were significantly reduced, FS and EF also improved significantly, compared with the 3d transplant group and MI group with a significant difference (n = 10 , p lt; 0.05). Histological detection the 1h transplant team: by Hoechst33342 markers and BrdU-positive cells are mainly distributed in the transplant area, no change in cell morphology; 3d transplantation group: cell transplantation around the infarct zone Hoechst33342 mark and BrdU-positive cells in the small number of; 7d transplantation group: with blue fluorescent transplanted cells in the infarcted area, some cells aggregate to form vessel-like structures. BrdU immunohistochemical staining and HE staining was also found that a large number of transplanted cells into a group gathered in the infarcted area, visible part of the vascular endothelial BrdU-positive cells, prompted the transplantation of MSCs in the host myocardium tissue survival, proliferation, and may be involved in the infarction District neovascularization, to promote myocardial tissue repair. Myocardial infarction, infarct zone iNOS expression pattern observed: the expression of iNOS in the infarcted myocardium after myocardial infarction immediately increase peaked three days after myocardial infarction, followed by a gradual decrease. At different time points after MI infarct zone iNOS expression intensity variation: 3d gt; 1h gt; 7d gt; 10d gt; 14d (n = 5, p lt; 0.05). INOS inhibitors on the survival of transplanted cells and cardiac function observed: cell transplantation, the treatment group before 12h given selective iNOS inhibitor 1400W 2mg/kg, intraperitoneal injection, 2 times / day for three days surrounded underwent heart function, the survival rate of the transplanted cells and histological detection, control group, replaced by normal saline. Real-time PCR detection 1400W treatment group and the control group Y chromosome the sry gene sequence of the expression level of 4.2%, 1.8%, respectively, results show that three days after myocardial infarction, cell transplantation, and to give early intervention iNOS inhibitor, transplant Cell viability may have significantly improve (n = 10, P LT; 0.01). Histological examination found that the control group of BrdU-positive cells scattered few in number; significantly increased the number of BrdU-positive cells in the treatment group, and the distribution is more concentrated. Corresponding to the HE staining showed a similar distribution of the transplanted cells. However, heart function tests between the treatment group and control group no significant difference (n = 10, p gt; 0.05). The experimental results suggest that iNOS inhibitors early intervention can improve to some extent the 3d transplantation group, the survival rate of transplanted cells after myocardial infarction, to improve heart function but no role. Conclusions: 1, seven days after myocardial infarction MSCs transplantation on the survival of the transplanted cells and improve heart function are better than three days after myocardial infarction cell transplantation. INOS expression peak, followed by a gradual decline in the first three days after myocardial infarction. INOS inhibitor of early intervention in 3d transplantation group, the survival rate of transplanted cells after myocardial infarction improved to a certain extent, but not enough to cause the change of heart function.
|