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目的:探讨同源盒基因Nkx2-5基因在5-氮胞苷(5-aza)诱导单层生长的P19细胞向心肌细胞分化中的作用。方法:以稳定表达Nkx2-5的P19细胞为实验组,P19细胞为对照组。两组细胞均单层培养并以5-aza(1μmol/L)诱导,倒置显微镜下观察细胞的生长状态;在诱导后4、8、12、16 d,以RT-PCR检测转录因子GATA-4、α-肌球蛋白重链(α-MHC)和心房利钠多肽(ANP)基因的表达。结果:对照组经5-aza诱导后ANP没有表达;GATA-4在8、12、16 d有表达;α-MHC在12、16 d有表达。实验组5-aza诱导后,GATA-4在诱导4、8、12、16 d有表达;α-MHC和ANP都是在5-aza诱导后的8、12和16 d表达。两组结果比较显示实验组GATA-4和α-MHC基因的表达时间提前;两组除α-MHC在12 d的表达无差异外,其他取材时间点实验组两个基因的表达量与对照组相比均增高,差异有统计学意义(P<0.05,P<0.01)。结论:Nkx2-5促进5-aza诱导的单层生长的P19细胞向心肌分化。
Objective: To investigate the role of homeobox gene Nkx2-5 in cardiomyocyte differentiation induced by 5-azacytidine (5-aza) -induced monolayer growth in P19 cells. Methods: P19 cells stably expressing Nkx2-5 were used as experimental group and P19 cells as control group. Both groups were cultured in monolayer and induced by 5-aza (1μmol / L). The growth of the cells was observed under inverted microscope. The expression of GATA-4 was detected by RT-PCR at 4, 8, 12 and 16 days after induction , Α-myosin heavy chain (α-MHC) and atrial natriuretic polypeptide (ANP) gene expression. Results: The ANP was not expressed in the control group after 5-aza induction; GATA-4 was expressed at 8, 12 and 16 days; α-MHC was expressed at 12 and 16 days. After the induction of 5-aza in experimental group, GATA-4 was expressed on the 4th, 8th, 12th and 16th day after induction. Both α-MHC and ANP were expressed at 8, 12 and 16 days after 5-aza induction. The results of two groups showed that the expression of GATA-4 and α-MHC genes in the experimental group was earlier than that in the control group. The expressions of two genes in the experimental group were significantly higher than those in the control group Compared with the control group, the difference was statistically significant (P <0.05, P <0.01). CONCLUSIONS: Nkx2-5 promotes 5-aza-induced monolayer growth of P19 cells toward cardiomyogenic differentiation.