microRNA-1诱导大鼠骨髓间充质干细胞向心肌样细胞分化过程中Notch信号分子的表达变化  

Expression of Notch signaling molecules in the process of microRNA-1 inducing rat bone marrow mesenchymal stem cells into cardiomyocyte-like cells

作　　者：邓海燕[1] 曾俊义[2] 魏云峰[1,2] 王梦洪[1,2] 郑泽琪[1,2] 张婉[2] 文通[1] 

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

出　　处：《基础医学与临床》2014年第9期1204-1210,共7页Basic and Clinical Medicine

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：目的探讨微小RNA-1对大鼠骨髓间充质干细胞向心肌样细胞分化的影响及分化过程中Notch信号分子的表达变化。方法全骨髓贴壁培养法分离培养大鼠MSCs并进行流式细胞学鉴定,miR-1慢病毒载体感染大鼠MSCs(MSCsmiR-1)按培养时间将细胞分成4组:对照组、培养4、6和15 d组;光镜下观察细胞形态变化,q PCR检测miR-1及心肌细胞特异性基因GATA-4、c Tn I、α-actin表达,免疫荧光检测c Tn I表达,Western blot检测α-actin表达;q PCR检测Notch信号通路相关基因的表达变化。结果原代大鼠MSCs呈长梭形、漩涡状生长,98%以上细胞表达CD44和CD29,不足1%的细胞表达CD45。MSCsmiR-1中miR-1表达水平持续上升,同时伴随心肌特异性基因GATA-4、c Tn I和α-actin的表达逐渐增强,感染4 d后可见c Tn I在部分MSCsmiR-1中表达,同时可检测到α-actin在MSCsmiR-1中表达;MSCsmiR-1向心肌样细胞分化过程中,Notch信号分子Jagged1、Notch1、Notch3和Hey2表达水平逐渐下调,于15 d时下调幅度最大。结论传导miR-1至大鼠MSCs中可促使其向心肌样细胞的分化;且分化过程中伴随着Notch信号分子Jagged1-Notch1/Notch3-Hey2表达水平下调。Objective To investgate the effect of miR-1 on MSCs differentiation pression changes of Notch signaling molecules in this process. Methods MSCs into cardiac phenotypes and the ex- were isolated from rat bone marrow by the whole bone marrow adherence method; MSCs were introduced by the lentiviral vectors expressing miR-1 （MSCsmin1 ） ,which were then divided into four groups： control group, 4-day culture group, 6-day culture group, 15-day culture group; The cell morphology was examined by light microscopy, miR-1 and cardiomyocyte-specific genes including GATA-4, cTnI and ct-actin were examined by real-time qPCR, and the expression of eTnI and ct-actin was detected by immunofluorescence and Western blot respectively;Meanwhile, nSCsmiR-1 cells were detected for the expression of genes related to notch signaling pathway by qPCR. Results Isolated MSCs displayed a sta- ble spindle-phenotype and showed characteristic swirling growth. More than 98% of the MSCs population expressed CIM4 and CD29 for MSCs phenotype; Meanwhile, less than 1% cells were CD45 positive. MSCsmIR-1 highly ex- pressed miR-1 and showed a higher expression of cardiomyocyte-specific genes, including GATA-4, cTnI and α-actin, cTnI was detected by immunofluorescence in MSCsmiR-1 after miR-1 transduction for 4 days, and gradually in- creased afterwards. Western blot further confirmed the expression of α-actin in MSCsmiR-1. The mRNA expression of Jaggedl, Notchl, Notch3 and Hey2 reduced significantly in MSCsmiR-1 during its differentiation into cardiomyocyte- like cells, and reached the minimum on day 15. Conclusions Transduction of miR-1 into rat MSCs induce cell differentiation into cardiomyocyte-like cells, which is in company Notchl/Notch3-Hey2 in the Notch pathway. with down-regulation mRNA expression of Jaggedl-

关 键 词：MIR-1 骨髓间充质干细胞 心肌样细胞 Notch 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]