微小RNA-1慢病毒载体介导大鼠骨髓间充质干细胞分化为心肌样细胞的研究  

MicroRNA-1 lentiviral vector mediates differentiation of rat mesenchymal stem cells into cardiomyocyte-like cells

作　　者：邓海燕[1] 曾俊义 魏云峰[1] 王梦洪[1] 郑泽琪[1] 彭景添[1] 张婉 文通[1] 张玉崚 

机构地区：[1]南昌大学第一附属医院心血管内科,330006 [2]江西省高血压病研究所

出　　处：《中华老年心脑血管病杂志》2013年第5期514-520,共7页Chinese Journal of Geriatric Heart,Brain and Vessel Diseases

基　　金：江西省自然科学基金(2010GZY0321);江西省科技支撑计划(2010BSA11700);江西省教育厅青年科学基金(GJJ12153);江西省卫生厅科技计划(20111021)

摘　　要：目的通过微小RNA-1(microRNA-1,miR-1)慢病毒载体转染大鼠骨髓间充质干细胞(MSC),探讨miR-1对MSC向心肌细胞分化的影响。方法采用全骨髓贴壁培养法分离培养大鼠MSC并进行流式细胞学鉴定;构建表达miR-1慢病毒载体;将miR-1慢病毒载体转染大鼠MSC(MSCmiR-1)连续培养15d,光镜下观察转染后细胞形态,分别于0、4、6、15dqRT-PCR检测miR-1、心肌相关基因锌指结构蛋白(GATA-4)、肌钙蛋白I(cTnI)、α肌动蛋白(α-actin)的表达;免疫荧光观察cTnI的表达;Western blot检测α-actin的表达。结果原代大鼠MSC呈长梭形、漩涡状生长,流式细胞学检测显示,98%以上细胞表达CD44和CD29,不足1%细胞表达CD45;成功构建miR-1重组慢病毒载体,病毒滴度为3×108 TU/μl;转导miR-1后,MSCmiR-1高表达心肌特异性相关基因GATA-4、α-actin、cTnI,并随时间逐渐增强,转染4d免疫荧光检测可见cTnI在部分MSCmiR-1中表达,于15d时表达最强,Westernblot进一步证实了α-actin在MSCmiR-1中表达。结论转导miR-1至大鼠MSC中可促使其向心肌样细胞的分化。Objective To study the effect of mieroRNA-1 lentiviral vector on differentiation of mes- enchymal stem cells （MSC） into cardiomyocyte-like cells by tranfecting rat MSC. Methods Rat MSC were isolated from rat bone marrow by whole bone marrow adherent culture and identified by flow cytometry. A microRNA-1 lentiviral vector was constructed. Morphology of rat MSC （MSC^miR-1） was observed under light microscope with immunofluorescence staining after they were transfected with microRNA-1 lentiviral vector and cultured for 15 days. Expressions of microRNA-1,myoeardium-related genes GATA-4, cTnI and α-actin were detected by RT-PCR and Western blot, respectively, with immunofluoreswcence staining. Results The primary rat MSC were long spindle-like in shape and showed a characteristic swirling growth. Flow cytometry dem- onstrated that over 98 % rat MSC expressed CD44 and CD29 while less than 1%rat MSC expressed CD45. The microRNA-1 lentiviral vector was successfully constructed with a titer of 3 × 10^8 TU/μl. The MSC^miR-1 highly expressed the GATA-4,cTnI, α-actin,and cTnI after transfection,which in- creased with the prolongation of time and reached their peak on day 15. Western blot further con- firmed that the α-actin was expressed in MSC^miR-1. Conclusion Transfection of microRNA-1 into rat MSC promotes their differentiation into cardiomyocyte-like cells.

关 键 词：微RNAs 慢病毒属 间质干细胞 转染 肌钙蛋白I 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]