摘要
目的:体外分离培养成人脂肪组织来源的间充质干细胞,探索其基本生物学特性及向心肌细胞诱导分化的潜能,为心肌再生提供理想的自体干细胞来源。方法:实验于2005-01/12在解放军兰州军区兰州总医院医学实验中心完成。取本院微创外科中心一急性单纯性阑尾炎患者大网膜脂肪组织(取得家属同意并告知仅用作实验室研究)。体外分离成人脂肪组织来源的间充质干细胞并传代扩增培养。采用相差显微镜及透射电镜观察细胞形态学特点,四氮唑蓝比色法绘制细胞生长曲线,流式细胞仪测定细胞周期及CD44、CD34的表达,第4代细胞用5-氮胞苷诱导使其向心肌细胞分化,免疫细胞化学鉴定心肌样细胞。结果:①分离培养的脂肪间充质干细胞经传代纯化后细胞形态呈均一梭形生长,电镜显示细胞较为幼稚,核大,核仁明显,常染色质多,异染色质少,细胞器结构及种类简单,以粗面内质网和线粒体为主。②细胞生长曲线呈“S”形,第1和2天为细胞生长潜伏期,第3天开始进入对数生长期,第7天达顶点,群体细胞倍增时间为18～20h。③流式细胞仪检测91.83%细胞CD44表达阳性、CD34阴性。④细胞周期分析显示76.2%细胞处于G0/G1期,S期细胞占8.7%,G2/M细胞占15.1%。⑤5-氮胞苷诱导后4周免疫细胞化学显示部分细胞缝隙连接蛋白-43表达阳性,表达率约为(29.0±1.2)%。结论:成人脂肪组织存在细胞活力旺盛的间充质干细胞且易于体外分离扩增,并在体外一定条件下具有向心肌细胞分化的潜能,可望成为心肌再生医学理想的自体干细胞种子来源。
AIM： To study the basic biological characteristics of adult adipose-derived mesenchymal stem cells （ADMSCs）, which are isolated and cultured from adult adipose in vitro, and the potential of differentiating into cardiomyocytes by chemical induction to provide the ideal autoallergic stem cells source for myoeardium regeneration.
METHODS： The experiment was carried out in the Medical Experimental Center, Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA from January to Decemhcr 2005. One patient with acute simple appendicitis treated in the Microtraumatic Surgery Center, Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA was informed of the experimental application and adopted in this study with the consent of his relatives. ADMSCs were isolated and cultured from adult greater omentum adipose tissue in vitro. The morphological characteristics of cells were observed by the phase-contrast microscope and the transmission electron microscope （TEM）, MTY colorimetry method was used to investigate cell growth curve; The cell cycle and expressions of CD44 and CD34 were detected by flow cytometry; The fourth passage cells were induced with 5-azacytidine to differentiate into cardiomyocytes. Then the immunocytochemistry method was used to identify eardiomyocytes. RESULTS： ①The isolated and cultured ADMSCs presented even fusiform shape after being purified. The cells were relatively immature with obvious nucleolus, many euchromatins but few heterochromatins, and simple organelle structure and type under the TEM and contained plenty of rough endoplasmic reticulum and bioblasts②The cell growth curve showed ＂S＂ shape. The cell growth was latent on the 1^st and 2^nd days, entered the logarithmic phase on the 3^rd day, and peaked on the 7^th day. The doubling time of group cells was 18-20 hours.③ADMSCs expressed CD44 （91.83%） but not CD34 by flow cytometry.④The cell cycle studies revealed that there were 76,2% of the cells in G0/G1, 8.7% in S and 15.1% in G2/M.⑤After 4-week induction by 5-azacytidine, the cells expressed gap connecxin-43 by immunocytochemistry, with the translation rate to cardiomyocyte of （29± 1.2）%.
CONCLUSION： Adult adipose tissues contain plenty of productive mesenchymal stem cells that can be easily in vitro isolated, cultured and differentiate into cardiomyocytes. ADMSCs might be a new ideal source of cell transplantation for myocardium regeneration.
出处
《中国临床康复》
CSCD
北大核心
2006年第37期14-17,i0002,共5页
Chinese Journal of Clinical Rehabilitation
