人羊膜间充质干细胞(hAMSCs)的分离、体外培养及诱导分化  

Isolation and Culture of Human Amniotic Mesenchymal Stem Cells(hAMSCs) and Its Differentiation Potential In vitro

作　　者：丛姗[1] 宋瑾[1] 张惠娟[1] 李岩[1] 白立恒 曹贵方[1] 

机构地区：[1]内蒙古农业大学兽医学院/动物组织胚胎学与发育生物学实验室,呼和浩特010018 [2]内蒙古妇幼保健医院妇产科,呼和浩特010020

出　　处：《农业生物技术学报》2015年第1期20-31,共12页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863计划)(No.2008AA101005)

摘　　要：人羊膜间充质干细胞(human amniotic mesenchymal stem cells,h AMSCs)属于成体干细胞,具有来源广、低免疫原性、增殖快等特点。本研究旨在建立h AMSCs的体外分离培养方法,探索其向三胚层分化潜能。无菌条件机械分离人羊膜并剪碎,经过两次胰蛋白酶消化去除上皮细胞后,再用Ⅰ型胶原酶消化分离得到h AMSCs。观察细胞形态,生长规律,染色体分裂中期相;通过二氨基联苯胺(diaminobenzidine staining,DAB)染色、免疫荧光、流式细胞仪、定向诱导三胚层分化和RT-PCR检测等方法进行鉴定。结果表明,从人羊膜中分离得到纯度较高的间充质干细胞,经过免疫组化波形丝蛋白(vimentin)DAB染色呈阳性;免疫荧光检测表面标记vimentin和阶段特异表达抗原4(stage specific embryonic antigens,SSEA-4)均呈阳性;h AMSCs表达CD29、CD49d和CD73表面抗原,不表达CD34、CD45和HLA-DR;RT-PCR检测干细胞特异基因POU5f1转录因子4(octamer-binding transcription factor-4,OCT4)、性别决定基因相关转录因子2(SRY-related high-monility-group(HMG)-box protein-2,SOX2)和NANOG均在m RNA水平上表达;经过免疫组化和RT-PCR检测在体外特定条件下可成功诱导分化为成脂、成骨、神经和胰岛样细胞。结果表明,h AMSCs可在体外分离培养并保持稳定的遗传学特性,不仅具有间充质干细胞特性,还具有向三胚层分化潜能。本研究为组织工程和再生医学的临床应用提供实验基础。Human amniotic mesenchymal stem cells （hAMSCs） are adult stem cells, and have the characteristics of wide source, low immunogenicity and rapid proliferation. The isolation and culture condition of hAMSCs and its differentiation potency of three germ layers were investigated in this study. The amnion was mechanically separated from the placenta obtained by cesarean section after 38-40 weekpregnancy. The hAMSCs were isolated from human amnion digested twice with 0.05% trypsin-EDTA for 30 min and once with collaenase type I for 1 h. Therefore, the obtained cells were high purity human amniotic mesenchymal stem cells . The morphololgy of hAMSCs was observed under the microscope. The growth curve of hAMSCs was checked by cell counting. Chromosome analysis was checked, then the flow cytometry, immunofluorescence and reverse transcription-PCR techniques were adopted to identify the surface molecular markers and stem cell characteristics. Further more, hAMSCs were able to differentiate into multi-potentialability when cultured in the special inducing medium. The results showed that high-purified hAMSCs isolated from amnion in the most appropriate culture condition was established. The morphology of hAMSCs was identical with fibroblast and the cells showed active proliferative ability. Results of immunocytochemistry and immunofluorescence showed that they were positive for stage specific embryonic antigens （SSEA-4） and vimentin and had a strong proliferation and multipotential capacity. By FASC and RT-PCR analysis, the following cell surface antigens including CD29, CD49d and CD73 expressed in hAMSCs, but negatived for CD34, CD45 and HLA-DR. The stem cell special gene markers such as OCT4, SOX2 and NANOG expressed constantly in these cells, and these cells could differentiate into fat cells, osteoblasts cells, neuron-like cells and insulin secreting cells; special marker genes such as PPARy2, Osteocalcin, MAP-2, PDX1 expressed in insulin secreting cells, hAMSCs could be successfully isolated, cultured and prolifer

关 键 词：人羊膜间充质干细胞(hAMSCs) 体外培养 细胞鉴定 细胞分化 

分 类 号：S858.28[农业科学—临床兽医学]