pEGFP-N2-BMP2转染诱导P19细胞分化为心肌样细胞

目的 探讨真核表达质粒pEGFP-N2-BMP2转染P19细胞后能否诱导其分化为心肌样细胞.方法 实验分转染组和对照组,转染组以真核表达质粒pEGFP-N2-BMP2转染P19细胞并使骨形态发生蛋白2(BMP2)基因稳定表达,然后结合细胞聚集培养,对照组为培养的P19细胞.两组细胞聚集培养4d,形成聚集体后贴壁培养,于贴壁培养的4d、8d、12d、16d,免疫细胞化学和Western blotting方法检测心肌标志物心肌肌钙蛋白T (cTnT)和心肌肌动蛋白α;半定量RT-PCR检测心脏早期转录因子GATA-4和NKx2.5基因的表达.结果 转染组心肌肌钙蛋白T和心肌肌动蛋白α两种蛋白形成聚集体贴壁4d时无表达,8d、12d、16d出现表达,其表达随时间延长逐渐增强.取材各时间点之间有显著差异(P＜0.01).在转染诱导后4d时GATA-4、NKx2.5两个基因即出现弱的表达,随着时间延长,8d、12d、16d两个基因的表达逐渐增强.取材各时间点之间有显著差异(P＜0.01).对照组未发现心肌肌钙蛋白T和心肌肌动蛋白α两种蛋白的表达,亦未发现GATA-4、NKx2.5两个基因的表达.结论稳定表达BMP2基因的P19细胞可以分化为心肌样细胞,BMP2可能通过调控转录因子GATA-4、NKx2.5进而影响心肌肌钙蛋白T和心肌肌动蛋白α的表达.

Cardiomyocyte differentiation from P19 cells transfected with pEGFP-N2-BMP2

Objective To identify occurrence of cardiomyocyte differentiation from P19 cells transfected with pEGFP-N2-BMP2 eukaryotic expression plasmid. Methods P19 cells were divided into transfected and non-transfected groups.In the transfected group,the cells with stably expressed of BMP2 as being transfected with pEGFP-N2-BMP2 eukaryotic expression plasmid and then were cultured in suspension. For 4 days, and the formed aggregates were transferred to Petri dish for adherent culture.On days 4, 8, 12, and 16 of the adherent culture, the expressions of cardiac troponin T（cTnT）and α-cardiac actin were detected with immunocytochemistry and Western blotting,while the gene expression of GATA-4 and NKx2-5 was detected with semiquantitative RT-PCR. Results In the transfected group, no expression of cTnT and α-cardiac actin was found on day 4, and the expression of these 2 proteins existed on days 8, 12, and 16 by both immunocytochemistry and Western blotting. The expression of two proteins was gradually increased with the prolong of time. There was a obvious difference between every two different drawing materials time point(EM>P/EM>0.01). The expression of GATA-4 and NKx2-5 was appeared with a weak expression at 4 days after transfection, as time went by, at 8 days, 12 days and 16 days after transfection, the expression of two gene intensified gradually. Also,the expression of two genes at every two different drawing materials time point had a significantly difference (EM>P/EM>0.01). In the non-transfected group, neither the protein expression of cTnT and α-cardiac actin nor the gene expression of GATA-4 and NKx2-5 was found. Conclusion Stable expression of BMP2 gene can induce cardiomyocyte differentiation from P19 cells. Possibly, BMP2 regulates the transcrip