miR-20b过表达对P19细胞增殖和细胞分化的影响  

Effect of miR-20b over-expression on the proliferation of P19 cells and the differentiation of P19 cells into cardiomyocytes

作　　者：朱莎莎[1] 刘雪华[1] 余章斌[1] 朱春[1] 李萌萌[1] 韩树萍[1] 胡晓山[1] 朱金改[1] 彭宇竹[1] 

机构地区：[1]南京医科大学附属南京妇幼保健院儿科,南京210004

出　　处：《发育医学电子杂志》2014年第2期87-92,共6页Journal of Developmental Medicine (Electronic Version)

基　　金：国家自然科学基金项目资助(81200126);江苏省医学重点人才基金项目资助(RC2011021)

摘　　要：目的探讨miR-20b过表达对P19细胞增殖和细胞分化的影响。方法将包含鼠miR-20b成熟序列的慢病毒载体及空载体分别感染P19细胞,通过嘌呤霉素筛选2周,建立稳定过表达鼠mR-20b的P19细胞系。用Real-time PCR的方法验证稳定细胞表达株。用CCK-8方法检测稳定感染miR-20b过表达慢病毒和空载体的P19细胞间细胞增殖的差别。将稳定感染的两组细胞以无血清培养液37℃孵育24小时使细胞停留于G0期,而后恢复血清培养,取不同时间点的细胞,使用流式细胞仪检测两组间细胞周期的差别,从而间接验证CCK-8的结果。以1%二甲基亚砜(DMSO)将两组稳定感染的P19细胞诱导分化为心肌细胞,Real-time PCR检测心肌分化相关标志基因的表达,并用光镜观察分化过程中的形态变化。结果建立了稳定表达miR-20b的P19细胞系,与空载体组相比,miR-20b过表达可抑制P19细胞的增殖,促进心肌分化相关标志基因的表达(P<0.05,P<0.01或P<0.001)。结论 miR-20b过表达可显著抑制P19细胞的增殖,并具有促进P19细胞向心肌细胞分化的作用。Objectives To explore the effect of miR-20 b over-expression on the proliferation of P19 cells and the differentiation of P19 cells into myocardiocyte. Methods P19 cells were infected with the lentiviral vector containing mmu- miR- 20 b and the empty vector,respectively. Stably miR- 20 b over- expressed P19 cell line was successfully constructed by puromycin screening for two weeks,which was validated by fluorescent observation and real- time quantitative PCR. The proliferation of P19 cells was analyzed by CCK- 8 method. Two cell groups were cultured in FBS- free- α- MEM for 24 h to make them both at G0 phase,which were assayed cell cycle by flow cytometry. Then we detected the relative expression of some heart related marker genes by real- time quantitative PCR and observed the cell morphology under light microscope after inducing P19 cells into cardiomyocytes under 1% DMSO. Results We successfully established stably infected P19 cell line and miR- 20 b was significantly over- expressed. Compared with the empty vector,miR- 20 b over- expressed could suppress the proliferation of P19 cells and promote the relative expression of related marker genes( P < 0. 05,P < 0. 01 or P < 0. 001). Conclusion MiR- 20 b could inhibit the proliferation of P19 cells and promote the cardiomyocytes differentiation.

关 键 词：P19细胞 miR-20b 细胞增殖 心肌分化 

分 类 号：R363[医药卫生—病理学]