心肌微环境与5-氮杂胞苷诱导脂肪间充质干细胞心肌分化能力评价

发布时间：2018-07-09 11:26

本文选题：cTnT + 5-氮杂胞苷　；参考：《新乡医学院》2017年硕士论文

【摘要】：背景当心肌组织受损,成纤维细胞可以很快形成瘢痕组织,而心肌细胞再生能力有限,严重影响心肌组织的结构稳定性及心脏的正常功能。心肌组织工程的理想种子细胞脂肪间充质干细胞(Adipose-derived Mesenchymal Stem Cells,ADMSCs)有多向分化潜能,可向成脂、成骨、成心肌等分化,并且具有取材方便,伦理限制较少,增殖速率高等优点。目前将ADMSCs诱导分化为心肌细胞的方法多为化学试剂诱导。由于化学试剂具有一定的细胞毒性,在一定程度上制约了其在基础研究和临床中的应用,寻找安全有效的诱导方法是心肌细胞治疗和心肌组织工程的重要条件。目的探讨体内、外心肌微环境与体外5-氮杂胞苷(5-aza)诱导ADMSCs分化为心肌样细胞的作用差异,为进一步开展细胞治疗心肌疾患提供实验参考。方法1.取小鼠腹股沟的皮下脂肪组织,分离培养ADMSCs,流式细胞术对其表面标记检测;分别用成骨、成脂诱导剂诱导,并使用茜素红与油红O鉴定ADMSCs多向分化能力。2.取小鼠乳鼠的心脏组织,分离培养心肌细胞。3.选取生长状态良好的第3代ADMSCs分为:5-aza体外诱导组、体外与心肌细胞共培养组和ADMSCs心肌内移植组,体外诱导以及体内移植1~3周后,检测ADMSCs的心肌特异性肌钙蛋白(cTnT)的免疫荧光表达。结果1.小鼠ADMSCs表达间充质干细胞的表面标记CD29,不表达造血干细胞标记CD45;且具有成脂、成骨的分化能力。2.三种诱导方法ADMSCs均表达cTnT:5-aza诱导组3周表达率(32.65±3.79)%,心肌细胞共培养组3周表达率(36.31±5.12)%,心肌内移植组1周表达率(42.93±4.04)%,3周表达率(78.43±1.32)%;心肌内移植组1周及3周较5-aza诱导组和心肌细胞共培养组3周均具有更高的分化效率(P0.05)。结论ADMSCs在体外经5-aza化学诱导可分化为心肌样细胞,但分化效率明显低于ADMSCs体外与心肌细胞共培养及体内移植两种心肌微环境诱导的分化效率。
[Abstract]:Background when myocardial tissue is damaged, fibroblasts can quickly form scar tissue, but the ability of myocardial regeneration is limited, which seriously affects the structural stability of myocardial tissue and the normal function of heart. Adipose-derived mesenchymal stem cells (Adipose-derived mesenchymal stem cells), which are ideal seed cells for myocardial tissue engineering, have the potential to differentiate into adipogenic, osteogenic and myocardial cells, and have the advantages of convenient selection, less ethical limitation and high proliferation rate. At present, the methods of inducing ADMSCs to differentiate into cardiomyocytes are chemical reagents. Because of the cytotoxicity of chemical reagent, its application in basic research and clinic is restricted to a certain extent. It is an important condition for cardiomyocyte therapy and myocardial tissue engineering to find a safe and effective induction method. Objective to investigate the differentiation of 5-azacytidine (5-aza) into cardiomyocytes in vitro and in vivo, and to provide an experimental reference for the further development of cell therapy for myocardial diseases. Method 1. The subcutaneous adipose tissue of mouse groin was isolated and cultured, and the surface markers were detected by flow cytometry. ADMSCs were induced by osteogenesis and lipogenic inducer, and ADMSCs were identified by alizarin red and oil red O. Cardiomyocytes were isolated and cultured from the heart tissue of neonatal mice. The third generation of ADMSCs in good growth state was divided into three groups: 1: 5-aza in vitro, co-cultured with cardiomyocytes in vitro, and in vivo transplanted with ADMSCs, then induced in vitro and transplanted in vivo for 1 to 3 weeks. The expression of cardiac troponin (cTnT) in ADMSCs was detected. Result 1. Mouse ADMSCs expressed CD29 on the surface of mesenchymal stem cells, but not CD45 on the surface of hematopoietic stem cells, and had the ability of adipogenic and osteogenic differentiation. The expression rate of cTnT: 5-aza was (32.65 卤3.79) in three weeks, 36.31 卤5.12 in co-cultured cardiomyocytes, (42.93 卤4.04) in intramyocardial transplantation in 1 week and (78.43 卤1.32) in intramyocardial transplantation. The differentiation efficiency was higher in the co-culture group for 3 weeks (P0.05). Conclusion AdMSCs can differentiate into cardiomyocyte-like cells induced by 5-aza chemically in vitro, but the differentiation efficiency is significantly lower than that induced by co-culture with cardiomyocytes and transplantation in vivo.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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