电磁场对大鼠骨髓间充质干细胞体外诱导分化为心肌样细胞影响的研究

发布时间：2018-05-01 11:14

本文选题：大鼠骨髓间充质干细胞 + 5-氮胞苷　；参考：《第三军医大学》2005年硕士论文

【摘要】：背景与目的各种病因造成心肌组织不可逆损伤均可导致心肌细胞数量的减少、心肌丢失。成年心肌再生能力差,坏死心肌无法通过自身的增殖、分化进行修复,丢失的心肌被瘢痕组织替代,造成心脏重构、心功能下降,最终导致慢性心力衰竭的发生。成体心肌细胞的再生是医学界急待解决的难题之一。骨髓间充质干细胞(mensenchymal stem cell,MSCs)所具有的多能分化特性为心肌细胞再生的研究提供了新的方向和思路。骨髓中MSCs 的含量非常少,必需在体外分离纯化、培养扩增才能满足要求,而如何获得大量纯化的MSCs 则成为临床利用这些细胞治疗疾病的前提。1999 年Makino 等首次体外给予3μmol/ L 的5-氮胞苷(5-Azacytidine, 5-aza)诱导MSCs 定向分化心肌样细胞获得成功,开创了MSCs 体外诱导分化心肌样细胞研究的先河。但仍然存在着诱导出功能性心肌样细胞的重复性差、诱导比率低、诱导条件不成熟等问题。已有研究证实,脉冲电磁场(pulsed electromagnetic fields, PEMFs)可以促进MSCs 向成骨方向分化。但其对MSCs 向心肌样细胞分化有无影响尚不清楚。因此本研究拟在用5-aza 体外诱导MSCs 分化为心肌样细胞的基础上,探讨电磁场对MSCs的增殖及向心肌样细胞分化的影响。方法 1. 通过对比不同鼠龄、不同纯化方法对大鼠MSCs 集落形成和生长特性的影响,筛选大鼠MSCs 分离、培养的条件,选取CD44、CD105 细胞免疫组化方法对大鼠MSCs进行初步鉴定。 2. 取第三代大鼠MSCs 随机分A、B、C 为三组,分别加入5μmol/L、10μmol/L 和20μmol/L 的5-aza;每组分别孵育12h、24h 和48h,继续培养28d。相差显微镜观察细胞形态变化以及电境观察诱导前后超微结构变化,细胞免疫化学染色检测α-肌动蛋白(α-actin)和心肌肌钙蛋白T(cardiac troponinT ,cTnT) 表达,并通过Western 印迹法对细胞cTnT 进行半定量分析。 3. 50Hz 的PEMFs 干预5-aza 诱导7d 后的大鼠MSCs,0.5mT、1 mT 和5 mT 三种感应强度分别为A、B、C 三组,各组根据暴露时间10min/d、20min/d、30min/d 及60min/d 又分1、2、3、4 亚组,作用4 d 后采用MTT 法测定细胞增殖变化,作用14d
[Abstract]:Background & objective the irreversible injury of myocardial tissue caused by various causes can lead to the decrease of myocardial cell number and myocardial loss. The regeneration ability of adult myocardium is poor, the necrotic myocardium can not be repaired through its own proliferation, differentiation and repair, the lost myocardium is replaced by scar tissue, resulting in cardiac remodeling, cardiac function decline, and eventually lead to the occurrence of chronic heart failure. The regeneration of adult cardiomyocytes is one of the urgent problems in medical field. The pluripotent differentiation of bone marrow mesenchymal stem cells provides a new direction and thought for the study of cardiomyocyte regeneration. The content of MSCs in bone marrow is very small. It is necessary to isolate and purify in vitro, culture and amplify to meet the requirements. However, how to obtain a large number of purified MSCs has become the precondition for the clinical treatment of these cells. In 1999, Makino et al were given 3 渭 mol/ L 5-Azacytidine (5-aza) for the first time in vitro to induce MSCs directionally differentiated cardiomyocytes. The study of MSCs induced differentiation of cardiomyoid cells in vitro was initiated. However, there are still some problems such as poor reproducibility, low induction rate and immature induction conditions. It has been proved that pulsed electromagnetic fields (PEMFs) can promote the differentiation of MSCs into osteogenesis. However, it is not clear whether it has any effect on the differentiation of MSCs into cardiomyocytes. Therefore, the purpose of this study was to investigate the effects of electromagnetic fields on the proliferation and differentiation of MSCs into cardiomyocyte-like cells on the basis of the induction of 5-aza into cardiomyocyte-like cells in vitro. Method 1. By comparing the effects of different ages and purification methods on the colony formation and growth characteristics of rat MSCs, the conditions for isolation and culture of rat MSCs were screened, and CD44-CD105 cells were selected to identify rat MSCs by immunohistochemical method. 2. The third generation MSCs rats were randomly divided into three groups, 5 渭 mol / L (10 渭 mol/L) and 20 渭 mol/L (5-aza), each group was incubated for 12 h for 24 h and 48 h for 28 days. Phase contrast microscopy was used to observe the changes of cell morphology and ultrastructure before and after induction. The expression of 伪 -actin (伪 -actin) and cardiac troponin (T(cardiac troponinT) was detected by immunocytochemical staining. The cTnT of cells was analyzed by Western blotting. 3. The three induction intensities of 5-aza induced by PEMFs of 50Hz for 7 days were as follows: 1 Mt and 5 Mt, respectively. According to the exposure time of 10 min / d, 20 min / d and 30 min / d respectively, each group was divided into two groups: 1 / 2 / 30 min / d and 1 / 2 / 3 / 4 subgroup respectively. After 4 days of treatment, the cell proliferation was measured by MTT method, and the effect was 14 days.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R35

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