外环境对骨髓间充质干细胞分化成心肌样细胞的影响

Effect of microenvironment in vitro on the differentiation of bone marrow mesenchymal stem cells into cardiomyogenic cells

目的:研究外环境对骨髓间充质干细胞(mesenchymalstemcells,MSCs)定向分化成心肌样细胞的影响。方法:取SD大鼠骨髓,通过贴壁法进行MSCs培养,经过多次传代后获得较纯的MSCs。用5-氮胞苷进行诱导分化,显微镜下观察其形态变化,通过RT-PCR鉴定心肌特异基因心房利尿肽和脑钠肽表达。实验分Ⅰ组为在玻片上的5-氮胞苷干预的MSCs移入到心肌细胞培养环境中;Ⅱ组为5-氮胞苷干预的MSCs;Ⅲ组为玻片上未用5-氮胞苷干预的MSCs移入到心肌细胞培养环境中;Ⅳ组为未用5-氮胞苷干预的MSCs。显微镜下观察4组形态变化,并通过免疫组化鉴别结蛋白和心肌肌钙蛋白I(cardiactroponinI,cTnI)表达及其出现时间的早晚。结果:RT-PCR显示诱导6周后有心房利钠肽,脑钠肽的表达。Ⅱ组MSCs诱导后10d可见细胞由原来扁平多角形变成长梭状,15d后部分细胞胞体明显增粗,可见到有些细胞间有融合样情况发生,20d左右类似肌管样细胞出现;而Ⅰ组在17d左右可以看到类似肌管样细胞;Ⅲ,Ⅳ组未见上述形态改变。免疫组化表明Ⅲ,Ⅳ组未见阳性细胞出现;Ⅰ,Ⅱ组可见阳性结果,且Ⅰ组阳性结果出现的比Ⅱ组更早。结论:MSCs在体外的化学诱导物条件下可以诱导分化成心肌样细胞,心肌细胞培养环境对其定向分化成心肌样细胞有促进作用。

AIM:To study the effect of microenvironment in vitro on bone marrow mesenchymal stem cells(MSCs) in differentiating into cardiomyogenic cells. METHODS:Bone marrow cells, derived from adult SD rats,were cultured by the method of adherence to plastic in vitro,and the purified MSCs were obtained after several passages.5 azacytidine (5 aza) was used for induction and differentiation, and morphological changes were observed under microscope.Cardiomyocyte specific genes,including atrial natriuretic polypeptide(ANP) and brain natriuretic peptide(BNP) were evaluated by reverse transcription polymerase chain reaction(RT PCR). In group Ⅰ,MSCs interfered by 5 aza,were shifted to the culture dishes seeded with cardiomyocytes. In group Ⅱ,there were MSCs cultured by 5 aza.In group Ⅲ, MSCs without 5 aza were shifted to the culture dishes seeded with cardiomyocytes.In group Ⅳ,there were MSCs without 5 aza cultured only. The morphological changes of MSCs in the 4 groups were observed under microscope,and immunohistochemical staining was used to identify the expression of desmin and cardiac troponin I(cTnI), and the emergency time. RESULTS: RT PCR showed that there were expressions of ANP and BNP 6 weeks after the induction. In group Ⅱ, the cells turned from thin and flat with multi angles to fibroblast like after treated with 5 aza for 10 days, the cells became thicker and connected with adjoining cells after 15 days, and myotube like cells were observed at about 20 days. and in group Ⅰ, the myotube like cells were observed at about 17 days, while in group Ⅲand Ⅳ, there were no above mentioned morphological changes. The results of immunohistochemical staining indicated that no positive findings were observed in group Ⅲand Ⅳ, while in group Ⅰand Ⅱ, those in group Ⅰappeared earlier than those in group Ⅱ.MSCs can be induced to differentiate into cardiomyogenic cells in vitro by profit chemical inductor, and the cardiomyocyte microenvironment can accelerate the differentiation.