体外心肌细胞共培养诱导骨髓间充质干细胞向心肌样细胞的分化  

Differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells via co-culture with cardiac myocytes induction in vitro

作　　者：郭义威[1] 毛光兰[2] 范艳芬[3] 王庆志[1] 闫建国[1] 李娜娜[1] 王志勇[1] 

机构地区：[1]新乡医学院人体解剖学教研室 [2]新乡医学院第三附属医院康复科 [3]新乡医学院期刊社,河南新乡453003

出　　处：《解剖学报》2015年第3期336-341,共6页Acta Anatomica Sinica

基　　金：河南省卫生厅资助项目(200802008)

摘　　要：目的探讨体外心肌细胞间接接触共培养诱导骨髓间充质干细胞（BMSCs）向心肌样细胞分化的效果,以寻找最佳的诱导条件。方法 2-3周龄SD大鼠24只和新生1-3d SD大鼠96只。分别通过全骨髓贴壁筛选法和差速贴壁法获取BMSCs和心肌细胞（CMs）。根据诱导条件不同分成3组：A组：5-氮杂胞苷（5-Aza-CR）诱导组,取采用10μmol/L 5-Aza-CR避光诱导24h;B组：共培养CMs诱导组,实验开始时,CMs和BMSCs分开培养,待各自贴壁后进行共培养;C组：5-Aza-CR＋共培养CMs诱导组,CMs接种于Transwell小室上层,下层接种5-Aza-CR诱导的BMSCs。在相差显微镜下连续观察各组BMSCs的形态变化,诱导至第2、4周时收集细胞。应用免疫组织化学法和免疫荧光方法检测诱导后的BMSCsα-横纹肌肌动蛋白（α-actin）和心肌肌钙蛋白T（c Tn T）的表达情况。结果1.细胞形态的变化：B组细胞有聚集生长趋势,C组脱落或降解的细胞明显少于A组,但部分细胞内有脂肪空泡形成。2.免疫组织化学和免疫荧光检测结果均显示,诱导2周时,A、B、C组c Tn T均呈阴性或低表达,α-actin均呈弱表达;诱导4周时各组c Tn T和α-actin均呈阳性表达。A组c Tn T阳性表达率和α-actin阳性表达率分别为（20.22±2.30）%和（28.05±2.45）%、B组为（21.18±1.30）%和（29.06±1.86）%、C组为（26.28±2.89）%和（33.91±2.18）%,且C组与A、B组比较差异均有统计学意义（P〈0.05）,但A组与B组组间差异无统计学意义（P〉0.05）。结论体外心肌细胞间接接触共培养可以诱导BMSCs向心肌样细胞分化,和5-Aza-CR共同诱导可提高诱导率。Objective To investigate the effect of indirect contact co-cuhure with cardiac myocytes induction in the differentiation of bone mesenchymal stern cells （BMSCs） into cardiomyoeyte-like cells in order to find the optimal induction conditions. Methods 2-3 weeks old 24 SD rats and newborn 1-3 day old 96 SD rats were used. BMSCs were derived by adhesive screening, and cardiac myocytes （CMs）were isolated by differential adhesion. According to the induction conditions, the cells were divided into groups A, B, and C. In group A [ the 5-azacytidine（5-Aza-CR）induction group] , 10 μmol/L 5-Aza-CR was used to induce for 24 hours under a light-free environment. In group B （ the co-cultured CMs induction group） , at the beginning of the experiment, the CMs and BMSCs were cultured separately ; when each had attached, and they were co-cultured. In group C （the 5-Aza-CR and co-cuhured CMs group） , CMs were seeded in the upper side of the transwell chamber, and 5-Aza-CR-induced BMSCs were seeded in the lower side. The phase contrast microscope was used to continuously observe morphological changes of BMSCs in each group; the cells were collected in 2- 4 weeks. Expression of α-sarcomeric actin and cTnT in induced BMSCs was inspected by immunohistochemistry and immunofluorescence. Results 1. Cells of group B had a trend of aggregate growth. Shedding or degrading cells of group C were significantly less than in group A, but fatty vacuolization appeared in a part of the cells. 2. Results ofimmunohistochemistry and immunofluorescence ： The expression of cTnT in group A, B, and C was negative or at low levels at 2 weeks of induction, while the expression of α-actin was weak. The expression of cTnT and α-actin in each group was positive, respectively, at 4 weeks of induction. The positive expression rates of cTnT and α-actin were （20. 22 ± 2.30） % and （28.05 ±2.45）% in group A, （21.18±1.30）% and （29.06±1.86）% in group A, and （26.28±2.89）% and （33.91±2. 18）% in group C. The differe

关 键 词：骨髓间充质干细胞 心肌细胞 间接接触 共培养 5-氮杂胞苷 免疫组织化学 大鼠 

分 类 号：Q254[生物学—细胞生物学]