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目的研究在单层培养条件下,5-氮胞苷(5-azacytidine,5-aza)诱导表达Nkx2-5的P19细胞向心肌分化的可能性和效率。方法实验分为实验组和对照组,实验组细胞为稳定表达Nkx2-5的P19细胞,对照组为P19细胞。2组细胞均在单层培养条件下,以5-aza(1μmol/L)诱导。倒置显微镜观察细胞的生长状态;诱导4、8、12、16d,Western blot检测α-sarcomeric actin和cTnT的表达;并用免疫荧光双标检测α-sarcomeric actin和cTnT的共表达。结果实验组和对照组细胞在5-aza诱导下,生长缓慢,死亡细胞数目增多。随贴壁细胞密度增加,形成类似于聚集体贴壁生长的状态,中心细胞小、密集,周边细胞呈放射状排列,胞体长梭形,伸出突起。实验组诱导8、12、16d检测到α-sarcomeric actin和cTnT2种蛋白的表达和共表达,表达量呈增多趋势;诱导12、16d时α-sarcomerica ctin的表达量与8d比差异有统计学意义(P<0.01),诱导16d的表达量与12d比差异有统计学意义(P<0.01);cTnT在诱导12、16d的表达量与8d比差异有统计学意义(P<0.01)。对照组没有2种蛋白的表达和共表达。结论5-aza可诱导单层生长的表达Nkx2-5的P19细胞向心肌分化。
Objective To investigate the possibility and efficiency of 5-azacytidine (5-aza) -induced P19 cells expressing Nkx2-5 to differentiate into cardiomyocytes under monolayer culture conditions. Methods The experiment was divided into experimental group and control group. The experimental group was P19 cells stably expressing Nkx2-5 and the control group was P19 cells. Cells in both groups were induced by 5-aza (1μmol / L) under monolayer culture conditions. The growth of cells was observed by inverted microscope. The expression of α-sarcomeric actin and cTnT was detected by Western blot at 4, 8, 12 and 16 days after induction. The co-expression of α-sarcomeric actin and cTnT was detected by double immunofluorescence staining. Results The cells in experimental group and control group grew slowly and the number of dead cells increased under the induction of 5-aza. With the adherent cells increased in density, the formation of adherent growth similar to the aggregation state, the central cells small, dense, peripheral cells were radially arranged, cell body fusiform, protruding. The expression and co-expression of α-sarcomeric actin and cTnT2 protein were detected in the experimental group at 8, 12 and 16 days after induction, and there was an increasing trend in the expression of α-sarcomerica ctin and 8d after induction (P <0.01). The difference between the expression of cTnT on the 16th day and that on the 12th day was statistically significant (P <0.01). The control group did not express and co-express the two proteins. Conclusions 5-aza induces monolayer growth of P19 cells expressing Nkx2-5 into cardiomyocytes.