Nkx2-5基因诱导P19细胞分化中心肌标志物的表达  

Exogenous Nkx2-5 gene expression induces the expression of cardiac markers during P19 cell differentiation in vitro

作　　者：张金平[1] 王慧娟[1] 史玉兰[2] 王立轩[1] 赵昱[1] 郭艳红 赵春芳[1] 张雷[1] 

机构地区：[1]河北医科大学组胚教研室,河北石家庄050017 [2]白求恩军医学院生理教研室,河北石家庄050081 [3]石家庄妇产医院,河北石家庄050011

出　　处：《南方医科大学学报》2010年第12期2658-2662,共5页Journal of Southern Medical University

基　　金：河北省科技厅计划项目(07275555)

摘　　要：目的研究同源框基因Nkx2-5在心肌发生中的作用。方法实验分转染组和未转染组,转染组细胞为稳定表达Nkx2-5的P19细胞,未转染组细胞为P19细胞。两组细胞悬浮培养4d,形成聚集体后贴壁培养,于贴壁培养的4、8、12、16d,免疫细胞化学检测α横纹肌肌动蛋白(α-sarcomeric actin,α-SA)和心肌肌钙蛋白T(cardiac troponin T,cTnT)的表达;RT-PCR检测GATA-4、α-肌球蛋白重链(α-myosin heavy chain,α-MHC)、心房利钠因子(atrial natriuretic factor,ANF)基因的表达。结果转染组α-SA和cTnT两种蛋白在聚集体贴壁的8、12、16d出现表达,表达量有增高趋势;16d与8d比有显著差异(P<0.01);RT-PCR结果显示:转染组GATA-4在贴壁培养的4d有表达,8、12d表达逐渐增多,16d时表达有所下降。α-MHC和ANF在贴壁培养的8d有表达,12d和16d时表达逐渐增多。统计结果表明GATA-4在8、12、16d的表达量与4d比有显著差异(P<0.05或P<0.01),α-MHC在12、16d的表达量与8d比有显著差异(P<0.05,P<0.01),ANF在16d的表达量与8d比有显著差异(P<0.01),16d表达量与12d比也有显著差异(P<0.01);未转染组结果均为阴性。结论外源表达Nkx2-5基因可诱导P19细胞逐渐表达心肌标志物。Objective To investigate the role of homeobox gene Nkx2-5 in cardiac myogenesis. Methods Two P19 cell lines,namely cells transfected with exogenous expression of Nkx2-5 gene and non-transfected cells,were cultured in suspension for 4 days to induce cell aggregation,and the cell aggregates were transferred to the Petri dish for further adherent culture. On days 4,8,12 and 16 of adherent culture,the expressions of α-sarcomeric actin （α-SA） and cardiac troponin T （cTnT） protein were detected by immunocytochemistry,and the mRNA expressions of GATA-4,α-myosin heavy chain （α-MHC） and atrial natriuretic factor （ANF） genes by RT-PCR. Results In the transfected cells,α-SA and cTnT protein expressions were detected on days 8,12 and 16 of adhere culture,and their expressions increased gradually with time. α-SA and cTnT expression was significantly higher on day 16 than on day 8 of culture （P0.01）. RT-PCR analysis of the transfected cell showed the presence of GATA-4 expression on day 4 of adherent culture,and the expression increased on days 8 and 12 but decreased on day16. ANF and α-MHC expressions were found on days 8,12,and 16,increasing gradually over time and showing significant differences from those on day 4 （P0.05 or P0.01）. The expression of α-MHC was significantly higher on days 12 and 16 than on day 8 （P0.05 or P0.01）,and ANF expression was significantly higher on day 16 than on days 8 and 12 （P0.01）. The non-transfected cells were negative for the expressions of all these genes. Conclusion Exogenous expression of Nkx2-5 gene can induce P19 cells to express cardiac markers in vitro.

关 键 词：同源框基因 NKX2-5 P19细胞 心肌标志物 

分 类 号：R329.21[医药卫生—人体解剖和组织胚胎学]