【作者】 王静；

【导师】 王爱玲；

【作者基本信息】 安徽医科大学 ， 内科学， 2009， 硕士

【摘要】 目的:研究单独及联合应用MSCs移植及EPO注射治疗心肌梗死的疗效,观察围移植期腹腔注射EPO后对MSCs移植的影响,探讨其可行性及可能机制。方法:①实验动物:60只Sprague-Dawley(SD)大鼠,永久结扎左冠状动脉前降支制备心肌梗死模型,造模成功后随机分成MI组(14只)、MSCs组(13只)、EPO组(13只)、MSCs-EPO组(14只),实验过程中对动物的处置符合动物伦理学标准。②干细胞分离、培养、标记及鉴定:采用贴壁法结合消化控制法体外培养骨髓间充质干细胞,流式细胞仪检测细胞表面抗原的表达,5-溴脱氧尿苷(BrdU)标记备用。③实验方案:冠脉结扎后30min,MSC组和MSC-EPO组将经BrdU标记的MSCs200uL分4点注射到缺血周边区域;EPO组与MSC-EPO组围手术期每天腹腔注射EPO 5000 U/kg,共7天(即术前1天、手术当天、手术后5天)。MI组注射相同剂量的生理盐水。④指标检测:术后4周行血流动力学检测,采用原位末端标记法(TUNEL)检测心肌细胞凋亡情况、免疫荧光染色检测移植细胞存活及发布情况,免疫组织化学染色法检测缺血区毛细血管密度及Bcl-2、Bax蛋白表达情况。结果:①原代细胞接种24h后可见散在分布的贴壁细胞,为短梭形、椭圆形、三角形、长梭形;换液传代后,细胞形态趋于一致,呈长梭形。骨髓间充质干细胞体外培养分裂增殖能力强,细胞呈集落式生长,集落外观呈‘漩涡’状。②传代后的骨髓间充质干细胞CD34和CD45阳性率小于5%,而CD44和CD90阳性率高达90%以上。③4周后移植部位的标本切片经抗BrdU免疫荧光染色后呈阳性,说明移植的骨髓间充质干细胞已在心肌梗死部位定值和存活。④MI后4周,MSCs组、EP0组、MSCs-EP0组血流动力学指标较MI组均显著改善,其中以MSCs-EP0组最明显(P<0.05)。⑤经TUNEL染色后凋亡细胞核呈棕黄色,与MI组相比,MSCs组、EP0组、MSCs-EP0组心肌细胞凋亡指数显著降低(P<0.05)。⑥干细胞移植后4周,MSCs组、EP0组、MSCs-EP0组毛细血管密度明显高于MI组(P<0.05);其中,MSCs-EP0组毛细血管密度明显高于MSCs组、EP0组,MSCs组与EP0组比较差异无统计学意义。⑦MSC-EPO组、MSC组、EPO组Bcl-2 A值均高于MI组(P<0.05),MSC-EPO组高于MSC组和EPO组(P<0.05),但MSC组与EPO组比较差异无统计学意义;MSC-EPO组、MSC组、EPO组Bax A值低于MI组(P<0.05) , MSC-EPO组低于MSC组及EPO组(P<0.05),MSC组Bax A值与EPO组比较,差异无统计学意义。结论:①MSCs移植和EPO注射均可减少心肌细胞凋亡,增加缺血区毛细血管密度,显著改善心梗后心功能。②EPO注射可提高MSCs移植治疗心梗的疗效,其机制可能与EPO改善局部心肌微环境,提高MSCs的存活分化比例,以及EPO增加MSCs的促血管生成作用有关。更多还原

【Abstract】 AIMS To investigate the effect of intramyocardial-transplantion mesenchymal stem cell(MSCs) and erythropoietin(EPO) for the treatment of myocardial infarction. We investigated whether EPO enhances the therapeutic potency of MSCs transplantation in rats with experimental myocardial infarction(MI).METHODS The experiment was performed at the animal laboratory and the central laboratory, The First Affiliated Hospital of Anhui Mdical University between December 2007 and March 2009.①animal: Sixty Sprague-Dawley(SD) rats were randomly divided into 4 groups:MI group,MSCs group,EPO group and MSCs-EPO group after MI was made by ligating the anterior descending coronary artery successfully. What to do with animals in the experiment is consistent with animal ethical standard.②MSCs culture、inducing and labeling in vitro:MSCs were isolated and purified with adherence plasticity method. The shape was observed and CD34, CD44, CD45, CD90 were detected with Flow cytometry analysis. MSCs were labeled by 5-bromodeoxyuridine (BrdU) and were to be applied.③MSCs transplantation and EPO infusion: Transplantation of MSC and/or 7-day infusion of EPO was performed immediately after coronary ligation. MSC(2×106 cells in 200ul saline solution) was injected into four sites, one within the infarct area and three in the myocardium bordering the ischemic area. EPO (5000U/kg body weight) was subcutaneously administered for 7 days, whereas control animals were injected saline solution for the same time duration.④experiment evaluation: Hemodynamics was performed four weeks afer operation. Then the rats were sacrificed. Cardiocyte apoptosis was determined by terminal deoxynucleotidyl transfease-mediated dUTP nick-end labeling(TUNEL) methods. Fluorescence microscope was used to identify the BrdU-labeled cells.Vascular density and the level of Bcl-2,Bax were evaluated by immunohistochemical technique.RESULTS①Cells cultured in vitro adhered obviously after 24 h, cells were many kinds of shape such as round、fusiform、and irregularity; after passages, the form of cells was becoming to be conformity, being fusiform. The reproductive activity of bMSCs cultured in vitro was strong, MSCs grew colony and the shape of cell colony was on“swirl”.②After passages of bMSCs, the positive rate of CD34 and CD31 on bMSCs cell surface is below 5% while the positive rate of CD44 and CD90 is above 90%.③Four weeks after transplantation, some cells were observed in the MSCs group and MSCs-EPO group, which were positive for BrdU. The immunofluorescence staining demonstrated that transplanted cells can survive in the peri-infarction region.④Compared with those in MI group, hemodynamic indexes of rats in MSCs group,EPO group and MSCs-EPO group were significantly improved.Hemodynamic indexes of rats in MSCs-EPO group were the most significantly improved(P<0.05) .⑤the cell nucleus of apoptotic cells were buffy by TUNEL staining, and compared with MI group, cardiocyte apoptotic index is much lower than that of MSCs,EPO and MSCs-EPO group(P<0.05).⑥Capillary density was markedly higher in the MSC-EPO group, followed by MSC and EPO groups, when compared with control group (P<0.05).⑦Compared to other groups,Bcl-2 was significantly up-regulated and Bax down-regulated in MSCs-EPO group.CONCLUSIONS:①MSCs transplantation and EPO infusion could reduce cardiocyte apoptosis ,stimulate angiogenesis and improve cardiac function.②EPO enhances the therapeutic potency of MSCs transplantation. The mechanisms may be associated with the effect of EPO, which EPO can improve the cardiac micro-environments, facilitate the survival and differentiation of implanted MSCs in vivo and drive a pro-angiogenesis program within the MSCs.更多还原