摘要
目的观察乳鼠心肌细胞条件培养液、SalB、5-氮胞苷(5-aza)及SalB联合5-aza体外诱导骨髓间充质干细胞(MSCs)分化为心肌样细胞的作用及其机制。方法分离培养及鉴定大鼠MSCs,培养乳鼠心肌细胞,收集条件培养液对其进行分组诱导:①SalB(终浓度为250μg/L),②5-aza组(终浓度为10μg/L),③SalB联合5-aza组(终浓度分别为250μg/L与10μmol/L),④乳鼠心肌细胞条件培养液,⑤未加诱导剂的阴性对照组。选取MSCs进行诱导,诱导周期为4w。倒置相差显微镜下观察细胞的生长情况及形态变化,免疫细胞化学法鉴定心肌特异性肌钙蛋白T(cTnT)表达,实时荧光定量RT-PCR法检测心肌早期基因NKX2.5、GATA-4 mRNA的相对表达量。结果细胞形态学显示,诱导后的MSCs体积增大,增殖减慢,并出现肌管样结构;免疫细胞化学结果显示,诱导后的MSCs表达心肌特异性蛋白cTnT,与5-aza组相比,SalB组和条件培养液两组蛋白表达较低,SalB联合5-aza组蛋白表达升高;实时荧光定量RT-PCR结果显示除阴性对照组外,其他四组表达心肌分化早期基因,SalB联合5-aza组诱导后的MSCs的心肌早期分化基因表达明显高于其他三组。结论SalB联合5-aza诱导MSCs表达心肌特异性肌钙蛋白cTnT;上调心肌早期基因NKX2.5、GATA-4mRNA的表达,证实其能够向心肌细胞分化能力;条件培养液不能上调心肌细胞的早期基因和蛋白,不能诱导其分化。
Objective To explore the role and mechanism of bone marrow mesenchymal stem cells （MSCs） differentiated into myocardial cells induced by neonatal rat myocardial cells conditioned medium, salvianolic acid B （SalB）, and SalB combined with 5-azacytidine. Methods MSCs of rats were separated, cultured and indentified. Neonatal rat myocardial cells were cultured and condition culture fluid was collected. MSCs were induced by SalB （terminal concentration 250 μg/L）, 5-azacytidine （5-aza） （250 μg/L）, SalB combined with 5-aza （250 μg/L and 10 μmol/L）, Neonatal rat myocardial cells condition culture fluid, and wihout any inducer respectively for 4 weeks. Cells growth condition and morphologic change were observed by inverted phase contrast microscope, cTnT expression was indentified by immnocytohistochemical assay, and myocardial early gene NKX2.5, and GATA -4 mRNA expression were detected by RT-PCR. Results ①Cell morphologic observation： after inducing, MSCs volume enlarged, proliferation speed slowed down and appearred structure of myotube. ②Expression of cTnT： cTnT was detected in induced MSCs. Compared with 5-aza group, the cTnT expressions in SalB group and condition culture fluid group were low in SalB group and condition culture fluid group and high in SalB combined with 5-aza group. ③Expressions of myocardial early gene： myocardial early gene was measured in other 4 groups except negative control group, and was high in SalB combined with 5-aza group than in other 3 groups.Conclusions SalB combined with 5-aza can induce MSCs to express myocardial specific troponin （cTnT）, upregulate early gene NKX2.5 and GATA- 4 mRNA, which confirm MSCs can differentiate into myocardial cells and condition culture fluid can′t upregulate the expressions of myocardial early gene and protein, and induce MSCs differentiation.
出处
《中国老年学杂志》
CAS
CSCD
北大核心
2009年第9期1069-1071,共3页
Chinese Journal of Gerontology
基金
国家自然基金资助项目(No.co3050402)
关键词
骨髓间充质干细胞
诱导分化
丹酚酸B
5-氮胞苷
Marrow mesenchymal stem cells
Induced and differentiated
Salvianolic acid B
5-azacytidine
作者简介
郭茂娟（1980-），女，硕士，主要从事干细胞诱导分化的基础研究工作。
通讯作者：范英昌（1951-），男，教授，博士生导师，主要从事干细胞诱导分化的基础研究工作。
