人脐带间充质干细胞的原代培养及多向分化潜能的研究  

Primary culture and multiple differentiation potency of mesenchymal stem cells from human umbilical cord

作　　者：辛毅[1,2] 李娜[1,2] 黄益民[1,2] 崔巍[1,3] 刘飒[1,3] 许秀芳[1,2] 张兆光[4] 

机构地区：[1]首都医科大学附属北京安贞医院 [2]北京市心肺血管疾病研究所分子生物学研究室,北京100029 [3]北京市心肺血管疾病研究所实验中心,北京100029 [4]首都医科大学附属北京安贞医院心外科,北京100029

出　　处：《细胞与分子免疫学杂志》2013年第10期1087-1093,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81070077);省部共建重点教育部心血管重塑相关疾病重点实验室项目(110267);北京市优秀人才培养资助项目(2012D003034000009);北京市自然科学基金(7072012)

摘　　要：目的探讨人脐带间充质干细胞（hUCMSC）的体外分离培养鉴定方法及多向分化潜能。方法应用Ⅱ型胶原酶和透明质酸酶联合消化法及组织贴块培养法对hUCMSC进行体外培养。在倒置显微镜下观察不同方法获得的hUCMSC的生长特点；台盼蓝法测定细胞传代成活率；采用生长曲线、MTT法测定hUCMSC的增殖、流式细胞术（FCM）测定细胞周期变化及细胞免疫表型变化；采用相应试剂盒鉴定其向成脂细胞、成骨细胞的分化，采用免疫荧光细胞化学技术检测向成心肌样细胞的分化、采用荧光标记技术检测hUCMSC向血管内皮细胞的分化。结果酶消化法分离培养的细胞1d后，在倒置相差显微镜下可见细胞贴壁呈圆形生长，4d后生长迅速呈长梭形，7d后由中心向周围生长且增殖明显，10d后达80％融合即可传代；贴块法培养的细胞7d后，可见细胞从组织块边缘长出，10d后细胞数量逐渐增多，16d后细胞生长迅速呈单层致密排列即可传代，细胞传代成活率均为96％以上。2种方法获得的第3代细胞生长曲线近似“S”形、MTr法显示3～5d细胞增殖较明显；酶消化法培养的细胞G0／G1期和s＋G2／M期所占比例分别为88．78％和10．21％，组织贴块法培养的细胞G0／G1期和S＋G2／M期所占比例分别为84．82％和13．87％，细胞DNA周期无明显差异；2种方法获得的细胞经FCM检测CD90、CD105、CD73阳性率均为99％以上为高表达；CD45、CD34、CD14、CD11b、CD79a、CD19、HLA—DR低表达；2种方法获得的细胞在体外诱导都能向成骨细胞、成脂细胞、成心肌样细胞及血管内皮细胞分化，其诱导阳性率均为90％以上。结论酶消化法和贴块法均可高效获得hUCMSC，与贴块法培养相比较，酶消化法能更有效的获得扩增迅速、细胞成分单一的细胞。Objective To establish a reliable method of isolation, culture and characterization of human umbilical cordderived mesenchymal stem cells （hUCMSCs） and study its multiple differentiation potency. Methods HUCMSCs were isolated and cultured using Trypsin-type ]/ collagen and hyaluronidase digestion method and tissue explant culture method, respectively. The cell growth of hUCMSCs was observed under an inverted microscope. Cell viability rate of the different passages was evaluated by trypan blue staining. The proliferation profile of hUCMSCs was analyzed by growth curve and MTT assay. Flow cytometry was used to study the cell cycle and immunophenotypage change. The differentiation potency of hUCMSCs towards the osteoblasts, adipocytes was assayed using the differentiation kits. The differentiation towards the cardiomyocytes and endothelial cells was tested by immunofluoresence staining with the specific markers. Results After l-day culture of the enzyme digested cells, under the inverted microscope, the adherent cells were round, and 4 days later, they grew quickly and presented fusiform. Seven days later, the cells proliferated from the center to the peripheral and fused by 80% on day 10. With the tissue explant culture method, the cells started to proliferate gradually from the periphery of the tissue and grew quickly and arrayed closely in monolayer after 10 days. The cell viability in both isolation methods were more than 96% as tested by trypan blue staining. The growth curve of the third passage presented an ＂S＂ shape. MTT assay showed that the optimal cell proliferation occured on day 3 to 5. The ratios of G0/G] phase and S ＋ G2/M phase was 88.78% and 10.21% respectively by enzyme digestion, and 84.82% and 13.87% respectively by explant culture method. There was no significant difference in cell cycle. The positive rates of CD90, CD105, CD73 were more than 99% and the expressions of CD45, CD34, CD14, CD11b, CD79a, CD19, HLA-DR were lower than 1%. The hUCMSCs isolated by the two methods could effici

关 键 词：人脐带间充质干细胞 原代培养 诱导分化 

分 类 号：R392-33[医药卫生—免疫学] Q254[医药卫生—基础医学]