经去分化预处理脂肪间充质干细胞移植治疗大鼠急性心肌梗死的实验研究

发布时间：2018-02-15 19:03

本文关键词： 脂肪间充质干细胞 bFGF 心肌梗死细胞移植　出处：《新乡医学院》2013年硕士论文　论文类型：学位论文

【摘要】：背景干细胞移植是治疗心肌梗死的一种非常有前景的新方法。在特定的微环境下移植间充质于细胞可分化为血管内皮细胞或者心肌细胞,并分泌血管生长因子,促进心肌血管生成,改善心脏功能。然而,急性心肌梗死经干细胞移植治疗后,移植干细胞在梗死区会因为缺血/再灌注损伤、炎性因子等恶劣环境的作用而大量死亡,将极大的影响干细胞移植的疗效。因此,人们试图通过采取干预措施即利用细胞因子、药物、化合物等对移植干细胞进行体外预处理,或采用有利于干细胞存活、黏附或促进血管形成的基因对干细胞进行修饰,直接和间接地提高干细胞在缺血心肌微环境中的存活和分化能力,从而有助于促进移植干细胞修复坏死心肌和改善心功能。目的研究经去分化预处理诱导后的脂肪间充质干细胞应用于细胞移植治疗是否能有效地提高干细胞在缺血心肌微环境中的生存率,为今后研究脂肪源性干细胞治疗急性心肌梗死提供实验依据。方法 1分离雌性小鼠(30-50g体重)腹股沟皮下脂肪组织用于脂肪问充质干细胞分离与培养；取第3代脂肪间充质干细胞用于本实验。 2结扎雌性SD大鼠的左冠状动脉前降支,建立急性心肌梗死模型；在心肌梗死模型建立后1小时,于梗死区边缘分别移植单纯ADMSCs、5-aza预处理的ADMSCs和碱性成纤维细胞生长因子(bFGF)预处理的ADMSCs;根据细胞预处理方式不同随机分组进行实验观察；在细胞移植后1、2、3和4周,用M型超声心动图检测心功能变化。在动脉结扎后不同时间取梗死区心肌组织,作HE染色和Masson染色,观察梗死区心肌组织的病理学变化；荧光显微镜下验证移植后的ADMSCs存活情况。结果 1体外培养的脂肪源性干细胞表达CD29、CD90,不表达CD45；证明实验所使用细胞为脂肪间充质干细胞。 2心肌梗死模型：冠状动脉前降支结扎后,左室前壁变苍白,心脏膨大,左心耳充血,心室收缩减弱,与未梗死区界线清楚,表明心肌梗死动物模型制备成功。 3移植细胞存活情况和心功能观察：心功能检测发现,在ADMSCs移植后1周,与模型对照组相比,bFGF预处理ADMSCs移植组左室舒张末期内径(LVDd)左室收缩期内径(LVDs)无明显缩小,左室射血分数(LVEF)及左室短轴缩短率(LVFS)也无明显提高,两组无显著性差异(n=10,p0.05)。而在细胞移植后4周,bFGF预处理ADMSCs移植组LVDd和LVDs较1周移植组和模型对照组组均有明显缩小,EF及FS也明显提高,与一周移植组和模型对照组以及单纯ADMSCs移植组相比具有显著性差异(n=10, p0.05)。HE染色可看到心肌梗死区域瘢痕增生紊乱,新生血管形成。结论经去分化预处理诱导后的脂肪干细胞移植治疗心肌梗死,有利于移植细胞在受损心肌内的存活,并促进心功能恢复,其疗效优于单纯干细胞移植。
[Abstract]:Background. Stem cell transplantation is a promising new way to treat myocardial infarction. Transplantation of mesenchymal cells in specific microenvironments can differentiate into vascular endothelial cells or cardiomyocytes and secrete vascular growth factors. Promote myocardial angiogenesis and improve cardiac function. However, after transplantation of stem cells in acute myocardial infarction, stem cells in the infarct area will die in large numbers because of the effects of ischemia / reperfusion injury, inflammatory factors and other adverse circumstances. It's going to have a huge impact on the effectiveness of stem cell transplantation. So people are trying to pretreat the transplanted stem cells in vitro by using cytokines, drugs, compounds and so on, or by using them that are good for the survival of stem cells. The adhesion or promotion of angiogenic genes modifies the stem cells, directly and indirectly improves the viability and differentiation of stem cells in the ischemic myocardial microenvironment, thus facilitating the transplantation of stem cells to repair the necrotic myocardium and improve the cardiac function. Purpose. To study whether the application of adipose mesenchymal stem cells induced by dedifferentiation pretreatment in cell transplantation can effectively improve the survival rate of stem cells in ischemic myocardial microenvironment. To provide experimental evidence for the treatment of acute myocardial infarction by adipose-derived stem cells. Method. 1 the inguinal subcutaneous adipose tissue was used for the isolation and culture of adipose mesenchymal stem cells, and the third generation adipose mesenchymal stem cells were used in this experiment. 2ligation of left anterior descending coronary artery of female SD rats to establish acute myocardial infarction model, 1 hour after myocardial infarction model was established, ADMSCs pretreated by ADMSCs5-aza and ADMSCs pretreated by basic fibroblast growth factor (bFGF) were transplanted at the edge of infarct area respectively. Cardiac function was detected by M-mode echocardiography. Myocardial tissue of infarcted area was taken at different time after artery ligation and stained with HE and Masson to observe the pathological changes of myocardial tissue in infarcted area. The survival of ADMSCs after transplantation was verified by fluorescence microscope. Results. 1 the adipose-derived stem cells expressed CD29 CD90, but not CD45, which proved that the cells used in the experiment were adipose mesenchymal stem cells. 2Myocardial infarction model: after ligation of anterior descending coronary artery, left ventricular anterior wall became pale, heart dilated, left atrial appendage congested, ventricular contraction weakened, and the boundary between left atrial appendage and uninfarcted area was clear, which indicated that myocardial infarction animal model was successfully prepared. 3The survival of transplanted cells and cardiac function: at 1 week after ADMSCs transplantation, the left ventricular end-diastolic diameter (LVDD) and left ventricular systolic diameter (LVDss) were not significantly decreased in the ADMSCs group pretreated with bFGF compared with the model control group. Left ventricular ejection fraction (LVEF) and left ventricular shortening rate (LVFS) were not significantly increased. There was no significant difference between the two groups, but the LVDd and LVDs in the ADMSCs transplantation group pretreated with bFGF 4 weeks after transplantation were significantly smaller than those in the 1-week transplantation group and the model control group. Compared with the one-week transplantation group, the model control group and the simple ADMSCs group, there was a significant difference between the two groups. The hypertrophic scar and neovascularization in the myocardial infarction area could be seen by HE staining. Conclusion. The transplantation of adipose stem cells induced by dedifferentiation preconditioning is beneficial to the survival of transplanted cells in the injured myocardium and to the recovery of cardiac function, and the effect is better than that of stem cell transplantation alone.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R542.22

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