摘要
目的:观察5-氮胞苷在体外诱导人骨髓间充质干细胞转化为心肌样细胞的情况及GATA-4和Nkx2.5基因的表达,从而确定人骨髓间充质干细胞体外诱导为心肌样细胞的条件、规律及转化细胞的特点。方法:实验于2005-08/2006-05在解放军沈阳军区总医院心血管外科和中国医科大学实验技术中心一部完成。取非血液疾病的5例胸科手术患者术中已切除掉的肋骨,抽取骨髓。利用淋巴细胞分离液行密度梯度离心法和差异贴壁法进行分离、提纯骨髓间充质干细胞,并进行培养扩增。用流式细胞仪对第2代的骨髓间充质干细胞表面抗原进行测定。应用10μmol/L5-氮胞苷对第2代的骨髓间充质干细胞诱导24h,于诱导后1,2,3,4周,用反转录-聚合酶链反应检测GATA-4和Nkx2.5基因表达。结果:①3份第2代骨髓间充质干细胞样本重复测定,表达CD29,CD44,不表达CD34,CD45。②以5-氮胞苷诱导培养24h后,骨髓间充质干细胞形态和排列方式发生明显变化,诱导1周后,细胞体积增大,多呈长梭行,平行排列;诱导2周后,细胞变为短柱状,突起位于两端,相邻细胞的突起紧密接触;诱导3周后,短柱状细胞的突起相互连接,部分细胞可见类肌管样结构;诱导4周后,细胞体积变小,可见由几个细胞连接形成的多核肌管样结构。③诱导组细胞GATA-4和Nkx2.5的聚合酶链反应产物凝胶电泳呈阳性,对照组为阴性,诱导组基因表达量均随着时间延长逐渐增加。结论:5-氮胞苷可诱导骨髓间充质干细胞向心肌样细胞转化,其处于祖心肌细胞和分化的心肌细胞之间。
AIM： To study the differentiation of bone marrow derived from human mensenchymal cells （MSCs） induced into cardiomyocytes by 5-azacytidine in vitro and the expressions of GATA-4 and Nkx2.5, so as to confirm the requirements, rules and characteristics of MSCs induced into cardiomyocytes. METHODS： The experiment was conducted in the Department of Cardiovascular Surgery, General Hospital of Shenyang Military Area Command of Chinese PLA and Department of No. 1 Experiment Technology Center, China Medical University between August 2005 and May 2006. Ribs cut in the thoracic operation from 5 patients without hematologic disease were selected to draw the bone marrow. The methods of density gradient centrifugation and adherent filtration were used to isolate and purify MSCs. The MSCs were cultured and amplified. MSCs surface antigen profiles were obtained by flow cytometry. The MSCs of the second passage were induced with 5-azacytidine at 10 μmol/L for 24 hours, and the expressions of GATA-4 and Nkx2.5 were tested by RT-PCR at the 1^st, 2^nd, 3^rd and 4^th week, respectively after the MSCs were treated with 5-azacytidine. RESULTS： ①Three samples of MSCs of second passage were tested repetitively. The MSCs were positive for CD29, CD44 and negative for CD34, CD45.②After the MSCs treated with 5-azacytidine for 24 hours, their morphology and arrangement changed obviously. After one week, the cells became larger, and most of them became long shuttle-shape and parallel. After two weeks, most of the cells became rod-shape, and there were umbos on the two sides of the cells, and the umbos of near cells touched tightly. After three weeks, the umbos of near cells touched each other to form myotube-shape. After four weeks, the cells became small, and the multinuclear myotube-shape constructions were formed by a few cells. ③The GATA-4 and Nkx2.5 were positive in the MSCs treated group, and negative in control group. With the culture time passed, the levels of GATA-4 and Nkx2.5 in MSCs were increased. CONCLUSION： MSCs can be induced into cardiomyocytes with 5- azacytindine. The stage of differentiation of the MSCs is between cardiomyocyte-progenitor and differentiated cardiomyocytes.
出处
《中国临床康复》
CSCD
北大核心
2006年第45期8-11,共4页
Chinese Journal of Clinical Rehabilitation
基金
辽宁省自然科学基金资助项目(20032052)~~
