5-氮胞苷诱导外源表达Nkx2-5的P19细胞向心肌分化的研究.docVIP

5-氮胞苷诱导外源表达Nkx2-5的P19细胞向心肌分化的研究 张金平，赵 静，王慧娟，赵秀军，赵春芳，张 雷 （河北医科大学组胚教研室，石家庄 050017） 摘 要：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cTnT 2种蛋白的表达和共表达，表达量呈增多趋势；诱导12、16d时α-sarcomeric actin的表达量与8d比差异有统计学意义（P＜0.01），16d的表达量与12d比差异有统计学意义（P＜0.01）。cTnT在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。 关键词：5-aza；Nkx2-5；P19细胞；心肌分化 中图法分类号： 文献标识码：A The study of 5-azacytidine induced P19 cells with exogenous expression of Nkx2-5 gene to differentiate toward cardiomyocytes ZHANG Jin-ping, ZHAO Jing, WANG Hui-juan, ZHAO Xiu-jun, ZHAO Chun-fang, ZHANG Lei (Department of Histology and Embryology , Hebei Medical University , Shijiazhuang 050017, China) Abstract: Objective To research the possibility and efficiency of the myocardial differentiation of P19 cells that exogenous expressing Nkx2-5 gene under monolayer culture condition with 5-aza induction. Methods The experiment contains two groups, experimental group and control group. The cells of experimental group were P19 cells which expressed Nkx2-5 gene constantly. While control group were P19 cells. Under the monolayer culture condition, the cells of two groups were induced by 5-aza with a concentration of 1μmol/L.The growth state of cells were observed by inverted microscope. At 4th day, 8th day, 12th day and 16th day after induction, Western blot was used to detect the expression of α-sarcomeric actin and cTnT. The co-expression of α-sarcomeric actin and cTnT was detected with immunofluorescence methods. Results With 5-aza inducing monolayer culture, the cells of experimental group and control group all grew slowly. The