摘要目的：通过沉默 microRNA（miRNA）-30c 研究 miRNA-30c 对 P19细胞增殖及分化功能的影响。方法将成功构建的 miRNA-30c 沉默质粒（miRNA-30c 沉默组）与空载质粒（阴性对照组）通过 lipo2000法分别转染 P19细胞，荧光表达观察质粒转染效率；通过杀稻瘟菌素 Blasticidin 筛选出 miRNA-30c 沉默稳定表达的P19细胞株；使用双荧光素酶报告基因间接验证成功沉默稳定表达的 P19细胞株；细胞增殖（CCK-8）法检测细胞增殖活性；使用二甲基亚砜（DMSO）诱导 P19细胞分化；倒置显微镜观察细胞分化形态变化；使用聚合酶链反应检测心肌分化标志基因（cTnT、NKX2.5、GATA4）的核酸表达水平。结果通过荧光表达观察到质粒成功转染入 P19细胞；双荧光素酶报告基因提示 miRNA-30c 沉默显著解除了 miRNA-30c 对靶基因 Gli2的抑制作用，细胞株成功建立（P ﹤0.001）；P19细胞分化过程中，倒置显微镜观察到 miRNA-30c 沉默组跳动心肌细胞的数量及频率均低于阴性对照组；检测心肌标志物发现 miRNA-30c 沉默组心肌细胞分化数量、标志基因表达均显著低于阴性对照组（P 均﹤0.05）。结论 miRNA-30c 沉默可以显著抑制 P19细胞增殖功能，并抑制其向心肌细胞的分化，为其用于心脏发育的进一步研究奠定基础。

abstractsObjective To explore the effects of microRNA(miRNA)- 30c knockdown on proliferation,diffe-rentiation of P19 cells. Methods miRNA - 30c knockdown plasmid(miRNA - 30c knockdown group)or no - load vector(negative control group)was transfected into P19 cells by lipo2000 and stable cell lines were selected by Blastici-din;Dual luciferase reporter gene system was used to confirm miRNA - 30c knockdown. Cell counting kit - 8(CCK - 8) assay was adopted to detect cell proliferation activity. An inverted microscope was used to observe morphological chan-ges of P19 cell differentiation. Cells were induced to differentiated to myocardiocyte with dimethyl sulfoxide(DMSO). Differentiation marker genes including cTnT,NKX2. 5,GATA4 relative mRNA expression levels were detected with real - time quantitative polymerase chain reaction,respectively. Results Observation of green fluorescent protein ex-pression under a fluorescence microscope indicated similar transfection efficiencies,and miRNA - 30c knockdown re-leased the activity of target gene Gli2. As a result,miRNA - 30c knockdown vector was constructed successfully(P ﹤0. 001). During differentiation of mouse P19 cells into myocardial cells,the beating cell clusters in miRNA - 30c knockdown cells were much lower than those in the control cells,and cTnT,NKX2. 5,GATA4 in miRNA - 30c knock-down cells showed significantly lower expression than those in the control cells( all P ﹤ 0. 05). Conclusions miRNA - 30c inhibits the P19 cell proliferation and differentiation. This study gives us a new insight of heart develop-ment and we need more efforts on exploring the deep function of heart diseases.