论文部分内容阅读
目的研究Islet-1诱导C3H10T1/2细胞定向分化为心肌样细胞过程中表达增加的心肌特异蛋白基因GATA4、NKX2.5、MEF2c上组蛋白乙酰化水平的变化情况。方法 Islet-1基因慢病毒感染小鼠骨髓间充质干细胞C3H10,Western blot检测转染后乙酰化的组蛋白H3表达情况,逆转录及实时荧光定量PCR时序性检测出心肌特异性蛋白基因GATA4、NKX2.5、MEF2c的mRNA表达达高峰的时间点,采用染色质免疫共沉淀技术(chromatin immunoprecipitation,CHIP),用CHIP级乙酰化H3抗体免疫沉淀与其所结合的DNA,采用实时荧光定量PCR扩增抗体所富集的心肌特异性蛋白基因GATA4、NKX2.5、MEF2c启动子区域的DNA量,比较感染组和未感染组与抗体所结合的上述3种心肌特异蛋白基因的表达情况。结果感染高表达Islet-1的C3H10T1/2细胞组蛋白H3的乙酰化水平较未感染组升高3.04倍(P<0.05),在心肌特异性蛋白基因GATA4、NKX2.5、MEF2C的mRNA表达达高峰的2周时间点,乙酰化的H3抗体所结合的心肌特异性蛋白基因GATA4、NKX2.5、MEF2c与未转染Islet-1的细胞组相比较,表达增加(P<0.05)。结论高表达Is-let-1之后C3H10T1/2细胞定向分化为心肌样细胞过程中表达增加的心肌特异蛋白基因上组蛋白乙酰化水平增高。
Objective To investigate the changes of histone acetylation levels of myocardial specific protein GATA4, NKX2.5 and MEF2c in Islet-1-induced C3H10T1 / 2 cells towards cardiomyocyte-like differentiation. Methods Mouse bone marrow mesenchymal stem cells C3H10 were infected with Islet-1 lentivirus and the expression of acetylated histone H3 was detected by Western blot. The expression of cardiac specific protein GATA4 was detected by reverse transcription and real-time PCR. NKX2.5 and MEF2c at the peak time point, the chromatin immunoprecipitation (CHIP) was used to immunoprecipitate the bound DNA with the CHIP acetylated H3 antibody. The real-time fluorescent quantitative PCR amplification The amount of DNA of the GATA4, NKX2.5, and MEF2c promoter regions of the myocardium-specific protein gene enriched by the antibody was compared with that of the three kinds of cardiac-specific protein genes that were combined with the antibody-infected and uninfected groups. Results The acetylation level of histone H3 in C3H10T1 / 2 cells infected with Islet-1 was 3.04-fold higher than that in non-infected cells (P <0.05). The mRNA expression of cardiac specific protein GATA4, NKX2.5 and MEF2C At 2 weeks of peak, the expression of cardiac specific protein GATA4, NKX2.5 and MEF2c bound by acetylated H3 antibody increased compared with that of untransfected Islet-1 cells (P <0.05). Conclusion The histone acetylation level of myocardial specific protein gene increased during the directional differentiation of C3H10T1 / 2 cells into cardiomyocytes was observed after Is-let-1 was overexpressed.