CAJ | 학술논문

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。
목적：통과침묵 microRNA（miRNA）-30c 연구 miRNA-30c 대 P19세포증식급분화공능적영향。방법장성공구건적 miRNA-30c 침묵질립（miRNA-30c 침묵조）여공재질립（음성대조조）통과 lipo2000법분별전염 P19세포，형광표체관찰질립전염효솔；통과살도온균소 Blasticidin 사선출 miRNA-30c 침묵은정표체적P19세포주；사용쌍형광소매보고기인간접험증성공침묵은정표체적 P19세포주；세포증식（CCK-8）법검측세포증식활성；사용이갑기아풍（DMSO）유도 P19세포분화；도치현미경관찰세포분화형태변화；사용취합매련반응검측심기분화표지기인（cTnT、NKX2.5、GATA4）적핵산표체수평。결과통과형광표체관찰도질립성공전염입 P19세포；쌍형광소매보고기인제시 miRNA-30c 침묵현저해제료 miRNA-30c 대파기인 Gli2적억제작용，세포주성공건립（P ﹤0.001）；P19세포분화과정중，도치현미경관찰도 miRNA-30c 침묵조도동심기세포적수량급빈솔균저우음성대조조；검측심기표지물발현 miRNA-30c 침묵조심기세포분화수량、표지기인표체균현저저우음성대조조（P 균﹤0.05）。결론 miRNA-30c 침묵가이현저억제 P19세포증식공능，병억제기향심기세포적분화，위기용우심장발육적진일보연구전정기출。
Objective 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.