摘要
目的:研究Nkx2-5基因在5-氮胞苷(5-aza)诱导单层P19细胞向心肌分化中的作用.方法:实验分实验组和对照组,实验组细胞为稳定表达Nkx2-5的P19细胞,对照组为P19细胞.两组细胞均单层培养,以5-aza(1μmol/L)诱导,倒置显微镜观察细胞的生长状态;5-aza诱导的4,8,12,16d,免疫细胞化学检测横纹肌α-actin(α-SA)和心肌肌钙蛋白T(cTnT)的表达;取5-aza诱导16d的细胞透射电镜观察超微结构的变化.结果:实验组5-aza诱导的8,12,16d检测到α-SA和cTnT的阳性表达,表达量逐渐增多.对照组没有两种抗体的阳性表达;实验组5-aza诱导16d,电镜观察发现部分相邻细胞分化出细胞连接和心肌发育早期的闰盘结构,胞质中出现密集平行排列肌丝样结构.对照组没有发现分化的细胞.结论:外源表达Nkx2-5基因促进P19细胞单层生长时向心肌分化.
AIM： To research the effect of Nkx2-5 gene on the myocardial differentiation of monolayer-cultured P19 cells induced by 5-azacytidine（5-aza）. 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 the cells of control group were normal P19 cells. Under the monolayer-euhure condition, the cells of both two groups were induced by 5-aza with a concentration of 1 μmol/L. The growth state of cells were observed by inverted microscope. At 4th, 8th, 12th and 16th day, the expression of α-sarcomeric actin（α-SA） and cardiac troponin T（cTnT） was detected with immunocytochemical methods. The ultrastructural changes of cells were observed at 16th day after induced by 5-aza. RESULTS： In experimental group, α-SA and cTnT expression was detected at 8th, 12th and 16th day after induced by 5-aza and the expression of the two proteins was gradually increased. In control group, the expression of the two proteins was not detected. In experimental group, observation of the uhrastructure showed that cell junction appeared between adjacent cells and some of the adjacent cells extended mutual chimeric processes which were similar to the early myocardial intercalated disk-like structure. In the cytoplasm of some cells, some dense, myofilament-like structure arranged in parallel way was found. No differentiated cell was found in control group. CONCLUSION： Myocardial differentiation from monolayercultured P19 cells can be promoted by exogenous expression of Nkx2-5 gene.
出处
《第四军医大学学报》
北大核心
2009年第20期2086-2089,共4页
Journal of the Fourth Military Medical University
基金
河北省科技厅资助项目(07275555)
