大鼠骨髓间充质干细胞体外诱导分化心肌样细胞膜电流的研究

发布时间：2018-08-26 10:16

【摘要】： 背景与目的既往研究认为,心肌坏死只能通过形成疤痕组织进行修复,使用药物可以通过延缓心肌重构来降低心衰的发生和临床死亡率。近年来,人们一直在寻求用一种细胞取代坏死的心肌细胞以期维持正常的心脏功能,而干细胞移植的研究进展为心肌坏死患者的治疗带来了新的希望。骨髓间充质干细胞(Mesenchymal stem cells ,MSCs)是一种具有自我复制和增殖潜能的细胞,并可在适当的环境条件下分化为具有不同生理功能的细胞,且不会涉及伦理问题,是目前认为最有希望成为坏死心肌细胞替代治疗的干细胞。1999年Makino等将MSCs连续传代4个月以上,得到永生细胞(immortalized cells),其在5-氮胞苷(5-aza)的作用下诱导分化,筛选出部分细胞类似胎儿的心肌细胞,这为MSCs的心脏细胞移植提供了理论基础。近年来,国内外虽对MSCs在各种条件下诱导分化为心肌样细胞进行了诸多研究,但对诱导后MSCs的鉴定主要集中在形态学和免疫组化方面,有关MSCs体外诱导分化心肌样细胞的电生理特性研究极少,而诱导后的心肌样细胞是否具有与心肌细胞相似的电生理特性,直接关系到细胞移植到体内后是否能与心肌细胞在功能上融合,具备可兴奋性及传导性特征。本实验拟在体外将大鼠MSCs经5-aza诱导心肌化,采用全细胞膜片钳技术,对细胞膜电流进行研究。方法 1.取健康SD大鼠骨髓,经密度梯度离心和贴壁培养获得MSCs,传两代后,以10μmol/L的5-aza孵育诱导24小时。培养4周,用免疫细胞化学染色方法鉴定心肌样细胞;并与未经诱导分化的MSCs进行比较。 2.取诱导后4周的MSCs为实验组,原代培养一周的未诱导MSCs和急性分离的大鼠心肌细胞为对照组,采用全细胞膜片钳技术对各组细胞进行膜电位检测。使用各种离子通道阻断剂对记录到的各种膜电流进行鉴定。并对各组细胞记录到的膜电流进行比较和分析。结果 1.免疫组织化学染色结果显示,经5-aza诱导后约40-50%的MSCs表达心肌特异
[Abstract]:Background & AIM previous studies suggest that myocardial necrosis can only be repaired by scar formation and that drug use can reduce the incidence of heart failure and clinical mortality by delaying myocardial remodeling. In recent years, people have been seeking to replace the necrotic cardiomyocytes with a kind of cells in order to maintain normal cardiac function, and the research progress of stem cell transplantation has brought new hope for the treatment of myocardial necrosis patients. Bone marrow mesenchymal stem cells (BMSCs) are self-replicating and proliferative cells that can differentiate into cells with different physiological functions under appropriate conditions, and have no ethical implications. In 1999, Makino et al subcultured MSCs for more than 4 months, and obtained immortalized cells (immortalized cells), which induced differentiation under the action of 5-azacytidine (5-aza). Some fetal cardiomyocytes were screened, which provides a theoretical basis for cardiac cell transplantation of MSCs. In recent years, although many studies have been carried out on the differentiation of MSCs into cardiomyoid cells under various conditions, the identification of induced MSCs is mainly focused on morphology and immunohistochemistry. There are few studies on the electrophysiological properties of cardiomyoid cells induced by MSCs in vitro, and whether the induced cardiomyocytes have similar electrophysiological properties to cardiomyocytes. It is directly related to whether the cells can fuse with cardiomyocytes after transplantation in vivo and possess the characteristics of excitability and conductivity. In this experiment, rat MSCs was induced by 5-aza in vitro and the cell membrane current was studied by whole-cell patch clamp technique. Method 1. The bone marrow of healthy SD rats was obtained by density gradient centrifugation and adherent culture. After two generations, MSCs, was incubated with 10 渭 mol/L 5-aza for 24 hours. Cultured for 4 weeks, cardiomyoid cells were identified by immunocytochemical staining and compared with undifferentiated MSCs. 2. Four weeks after induction, MSCs was used as experimental group, uninduced MSCs and acute isolated rat cardiomyocytes were cultured for one week as control group. The membrane potential of each group was detected by whole-cell patch clamp technique. Various ion channel blockers were used to identify the recorded membrane currents. The membrane currents recorded in each group were compared and analyzed. Result 1. Immunohistochemical staining showed that about 40-50% of MSCs expressed myocardial specificity after induction by 5-aza.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

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