摘要
目的 研究二甲亚砜 (dimethylsulfoxide ,DMSO)诱导人骨髓基质细胞 (humanbonemarrowstromalcells ,hBMSC)心肌分化过程中心肌特异转录因子和心肌基因的表达变化。方法 取第 8代hBMSC ,以DMSO诱导 2 4h。在诱导后 2 1天 ,通过免疫荧光染色检测干细胞标志CD90和肌性标志Desmin表达变化 ,并在诱导后1、2、3周提取RNA。以RT PCR方法检测细胞心肌特异转录因子 (Nkx2 .5和GATA4 )和特异基因 (connexin4 3、ANP等 )的表达变化。结果 经DMSO诱导后 ,MSC体积变大 ,细胞排列趋于一致 ,连接紧密 ,并逐渐形成肌节样结构 ,CD90阳性细胞减少 ,Desmin阳性细胞增加。在DMSO诱导hBMSC后 1周 ,开始可检测到细胞发生转录水平变化 (Nkx2 .5、GATA - 4表达和connexin4 3、ANP表达 ) ,并均可持续至诱导后 3周 ;前三者的表达量有随诱导后时间延长而增加的趋势。结论 DMSO在体外可诱导hBMSC向心肌前体细胞分化 。
Purpose: To investigate the potential and temporal transcription regulation of human bone marrow stromal cells (hBMSC) being induced to the cardiovascular precursors in vitro with the treatment of dimethyl sulfoxide (DMSO). Methods: Human bone marrow was obtained from healthy adults. hBMSC were isolated using Percoll gradient centrifugation method. Cells were cultured in L-DMEM with FGF (0.5 μg/L) at 37°C in humid air with 5% CO2. The specific surface antigens of the mesenchymal stem cell were assayed by FACS after the 2nd to 8th passage. Cardiomyocyte differentiation was induced by the treatment of 0.8% of DMSO. Three weeks after induction of DMSO, immunofluorescence was used to detect the CD90 and desmin in hBMSC. A series of RT-PCR of cardiomyocyte-specific transcription factors and cardiomyocyte-specific genes were performed on different differentiation days to investigate the temporal regulation of cardiomyogenesis at a transcription level. Results: hBMSC showed a fibroblast - like morphology before differentiation. After the treatment of DMSO, the former fibroblast-like cells increased in size and length in one week. The link between adjacent cells became apparent. The morphology of cell culture inclined to be unidirectioned and formed a sarcomere-like structure. The CD90-positive cells were decreased and desmin-positive cells were increased after treatment of DMSO. RT-PCR assessment showed that the differentiated cells began to express Nkx2.5 and GATA4, two important cardiac transcription factors, from week 1 to week 3 of differentiation. Meanwhile, the cells began to express atrial natriuretic protein and connexin43, the specific cardiac genes, which could last at least 3 weeks. Conclusions: These findings show that hBMSC possesses the differentiation potential of cardiomyocyte. We demonstrated that DMSO showed the power in inducing the cardiomyocyte differentiation in hBMSC. The reagent functioned at the transcription level to promote cardiomyogenesis of hBMSC.
出处
《复旦学报（医学版）》
EI
CAS
CSCD
北大核心
2004年第5期454-457,F002,共5页
Fudan University Journal of Medical Sciences
基金
上海市医学发展基金 -重点研究课题基金 ( 2 0 0 0I-2D0 0 2 )
上海市科委优秀学科带头人计划 ( 0 3XD 14 0 10 )资助项目
