体外培养成人脂肪间充质干细胞生物学特性及诱导分化为心肌细胞的研究  

Biological characteristics of adult adipose-derived mesenchymal stem cells and differentiation into cardiomyocytes in vitro

作　　者：任刚[1] 边云飞[1] 武晓佳[1] 武卫东[1] 肖传实[1] 

机构地区：[1]山西医科大学第二医院心内科,干细胞研究中心,太原030001

出　　处：《中国药物与临床》2010年第5期519-522,I0001,共5页Chinese Remedies & Clinics

摘　　要：目的通过分离、培养脂肪间充质干细胞观察其生物学特性及诱导分化为心肌细胞,为心肌再生提供良好的干细胞来源。方法胶原酶消化分离成人脂肪来源的间充质干细胞并进行传代培养,倒置相差显微镜观察细胞形态,流式细胞仪测定CD29、CD31、CD34、CD44及细胞周期,MTT绘制细胞生长曲线。用第3代细胞进行诱导分化,观察不同浓度5-氮杂胞苷(5-Aza,1,3,5,10,15,20μmol/L)及不同作用时间(12,24,48,72h)诱导其向心肌细胞分化的差别,采用最佳浓度10μmol/L,最佳作用时间24h进行实验,分别在第7,14,21,28天用免疫细胞荧光染色鉴定心肌细胞α-横纹肌、肌球蛋白重链(MHC)、心肌肌钙蛋白I(cTnI)表达,第14天反转录-聚合酶链反应(RT-PCR)法检测心肌发育相关基因NKX2.5的表达。结果倒置相差显微镜下观察原代细胞,可见细胞呈梭型、核圆形或椭圆形,偶见双核。传代细胞核原代细胞形态相似,排列有了一定的方向性。流式细胞仪检测结果显示,第1、3、5代细胞均高表达CD29和CD44;而CD31始终表达很弱,可认为呈阴性表达;CD34在第1、3代细胞弱表达,在第5代细胞表达逐渐减弱为阴性。细胞生长曲线显示前3d处于细胞潜伏状态,第4天进入对数生长期,第10天达到顶峰。细胞周期检测结果显示G1期细胞为85.93%,S期为7.24%,G2期为6.83%。10μmol/L5-Aza诱导后7d进行免疫细胞荧光染色,未见有α-横纹肌、MHC、cTnI表达。14d少量细胞α-横纹肌和MHC阳性表达,cTnI阴性表达。21d表达α-横纹肌和MHC的细胞数量增多,并可见少量cTnI阳性表达。28dα-横纹肌、MHC、cTnT阳性表达数目均增多,RT-PCR结果显示NKX2.5呈阳性表达。结论成人脂肪中可以分离出脂肪间充质干细胞并且可以在体外培养传代,经过5-Aza的诱导可以向心肌细胞分化,为干细胞移植治疗和组织工程学种子细胞提供了更多的选择。Objective To isolate and culture adult adipose-derived mesenchymal stem cells（ADMSCs） in vitro,observe their biological characteristics,and to induce them differentiate into cardiomyocytes,so as to provide a good source of stem cells for myocardial regeneration.Methods ADMSCs were isolated with collagenase digestion and cultured from adult adipose tissue in vitro.The morphological characteristics of cells were observed by inverted phase contrast microscope.The expressions of CD29,CD31,CD34,CD44 and cell cycles were detected by flow cytometry.MTT colorimetry was used to investigate cell growth curve.The third-generation AMDSCs were induced and examined for outcomes of differentiation with various concentrations of 5-azacytidine（5-Aza,1,3,5,10,15 and 20 μmol/L） and various duration（12,24,48 and 72 h）.The optimal concentration（10 μmol/L） and duration（24 h） were used in another experiment.At 7,14,21 and 28 days after induction,expressions of α-Sarcomeric Actin,myosin heavy chain（MHC） and Troponin I（cTn1） in rabbit cardiomyocytes were detected by immunocytochemistry.The expression of ANP was detected by RT-PCR.Results The ADMSCs were fibroblast-like spindle cells,with round or oval nuclei and occasional dual-nuclei.The morphology of passage cells were similar to that of primary cells,arranged a certain of direction.Expression of cell surface antigens,CD29 and CD44,were negative for weak five positively in the 1st,3rd and 5th passage cells,but CD31 expression were negative.The expression of CD34 was weakly positive in the 1st and 3rd passage cells,and were examined with colorimetry.The cell growth curve showed that a latency from days 1 to 3,logarithmic phase on day 4 and a peak on day 10.The cell cycle studies revealed that there were 85.93% of the cells in G0/G1,7.24% in S and 6.83 % in G2/M.α-Sarcomeric Actin,myosin heavy chain and Troponin I were positively expressed 28 d after induction by immunocytochemistry.The 14th days NKX2.5 were positive by RT-PCR.Conclusion ADMSCs can be isol

关 键 词：间质干细胞 细胞培养 细胞分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]