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lip02000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Gf口的抑制作用，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。
Objective To explore the effectof microRN（miRNA） -30e knockdown on proliferation, diffe- rentiation of P19 cells. MethodmiRN- 30knockdown plasmid （ miRN- 30knockdown group） ono - load vector（ negative control group） watransfected into P19 cellby lipo2000 and stable cell linewere selected by Blastici- din ;Dual luciferase reportegene system waused to confirm miRNA- 30knockdown. Cell counting kit- 8 （ CCK- 8） assay waadopted to deteccell proliferation activity. An inverted microscope waused to observe morphological changeof P19 cell differentiation. Cellwere induced to differentiated to myocardioeyte with dimethyl sulfoxide（DMSO）. Differentiation markegeneincluding cTnT, NKX2. 5, GATA4 relative mRNexpression levelwere detected with real -time quantitative polymerase chain reaction, respectively. ResultObservation of green fluorescenprotein ex- pression undefluorescence microscope indicated similatransfection efficiencies, and miRN- 30knockdown re- leased the activity of targegene Cdi2. Aresult, miRN- 30knockdown vectowaconstructed successfully （ P 〈 0. 001 ）. During differentiation of mouse P19 ceilinto myocardial cells, the beating cell clusterin miRN- 30knockdown cellwere much lowethan those in the control cells, and cTnT, NKX2. 5, GATA4 in miRN-30knockdown cellshowed significantly loweexpression than those in the control cell（ all P 〈 0. 05 ）. ConclusionmiRN-30inhibitthe P19 cell proliferation and differentiation. Thistudy giveunew insighof heardevelop- menand we need more efforton exploring the deep function of heardiseases.
出处
《中华实用儿科临床杂志》
CAS
CSCD
北大核心
2015年第13期992-995,共4页
Chinese Journal of Applied Clinical Pediatrics
基金
国家自然科学基金（81370200）
江苏省医学重点人才基金（RC2011021）
