心肌样微环境诱导脂肪干细胞分化的研究  

Adipose Tissue-derived Stem Cells Differentiated into Cardiomyocytes in Cardiac Microenvironment

作　　者：朱艳霞[1] 刘天庆[1] 宋克东[1] 马学虎[1] 崔占峰 

机构地区：[1]大连理工大学干细胞与组织工程研发中心,大连116024 [2]Oxford Centre for Tissue Engineering and Bioprocessing,Department of Engineering Science,Oxford University,Oxford OX1 3PJ,UK

出　　处：《生物化学与生物物理进展》2009年第5期624-632,共9页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目(30670525);大连理工大学青年教师培养基金资助项目(893228)~~

摘　　要：微环境在促进干细胞分化过程中起着重要的作用,研究心肌样微环境介导脂肪干细胞向心肌细胞分化有重要意义.将脂肪干细胞与心肌细胞直接或通过细胞培养小室间接共培养,检测脂肪干细胞的分化情况.对于直接共培养体系,采用绿色荧光蛋白CFSE对脂肪干细胞进行标记,然后与心肌细胞以1:5混合后进行直接共培养,2周后,通过流式细胞仪分选分化的脂肪干细胞,并检测其分化情况.检测方法包括:扫描电镜和透射电镜观察细胞的超微结构;免疫细胞化学检测心肌特异性肌球蛋白重链(MHC)、肌钙蛋白(TnⅠ)和连接蛋白(Cx43);Western blot定量分析;RT-PCR检测心脏特异性转录因子mRNA的表达.结果表明,分化的脂肪干细胞呈现心肌样超微结构,并表达心肌特异性蛋白和转录因子,并且直接共培养体系中分化的脂肪干细胞其表达率明显高于间接共培养体系中的表达率.因此,在心肌样微环境中,除了可溶性细胞因子对分化起作用以外,心肌细胞产生的机械牵拉对脂肪干细胞向心肌细胞分化也起着重要的作用.Microenvironment plays a critical role in directing the progression of stem cells into differentiated cells. It is necessary to investigate the role of cardiac microenvironment in directing the differentiation of adipose tissue-derived stem cells （ADSCs）. Human ADSCs were cocultured with rat cardiomyocytes directly or indirectly by cell culture inserts. For direct coculture, hADSCs were labeled with carboxyfluorescein succinimidyl ester （CFSE）, then mixed with cardiomyocytes at a 1∶5 ratio in complete media and seeded at a cell density of 50 000 cells/ml. Fluorescence-activated cell sorting was used to extract and examine the directly cocultured differentiated ADSCs. For indirect coculture, differentiated ADSCs were collected directly. The assays used include scanning electron microscope（SEM） and transmission electron microscope（TEM） for the ultrastructure of differentiated cells, immunostaining against myosin heavy chain, troponinⅠ and connecxin43, Western blotting and RT-PCR for the expression of cardiac specific proteins and genes respectively. Results showed that the differentiated ADSCs experienced the coculture presented cardiac ultrastructure and expressed cardiac specific genes and proteins, and the fractions of ADSCs expressing these markers by direct coculture were higher than those of indirect coculture. These data indicate that in addition to soluble signaling molecules, the direct cell-to-cell contact is obligatory in relaying the external cues of the microenvironment controlling the differentiation of ADSCs to cardiomyocytes.

关 键 词：脂肪干细胞 心肌细胞 共培养 肌钙蛋白 连接蛋白 

分 类 号：R318[医药卫生—生物医学工程]