Tie-2单克隆抗体鉴定人骨髓间充质干细胞体外定向诱导分化的内皮细胞  

EVALUATION OF ENDOTHELIAL CELLS DIFFERENTIATED FROM MESENCHYMAL STEM CELLS OF HUMAN BONE MARROW WITH Tie-2 MONOCLONAL ANTIBODY BY IMUNOHISTOCHEMISTRY IN VITRO

作　　者：冯滨 刘迎龙[2] 冯凯[3] 龚茹[4] 陈虎[3] 

机构地区：[1]南充中心医院心脏外科,四川南充637000 [2]中国医学科学院中国协和医科大学阜外心血管病医院,北京100037 [3]军事医学科学院全军造血干细胞移植中心,北京100850 [4]第三军医大学成都军医学院,四川成都610083

出　　处：《中国应用生理学杂志》2005年第3期340-343,i0007,共5页Chinese Journal of Applied Physiology

基　　金：国家高技术发展计划(863)资助项目(2001AA216061);国家自然科学基金资助项目(30271289);中国博士后科学基金(2003033224)

摘　　要：目的:体外扩增和定向诱导成人骨髓间充质干细胞(MSCs)向内皮细胞分化,并探讨其可行性和条件.方法:利用Percoll(1 073 g/L)从正常成人骨髓中分离MSCs,用含10%FBS的LG-DMEM培养基进行纯化和扩增培养,流式细胞仪分析鉴定MSCs的纯度.用含VEGF(10μg/L)的HGDMEM培养基诱导MSCs向内皮细胞定向分化,Tie-2单克隆抗体的免疫组化法和透射电镜(TEM)鉴定其细胞的性质.结果:5.0×105个MSCs在体外扩增15代后,获得了8.0×1012个MSCs,扩增了约1.6×107倍.加入诱导培养体系培养14～21 d,光镜下可观察到内皮细胞呈典型的'鹅卵石'样:90%的细胞Tie-2免疫组化呈阳性反应;TEM下可观察到胞浆内有Weible-palade小体.结论:成人骨髓MSCs在体外具有定向诱导分化为内皮细胞的潜能,为构建心脏组织工程瓣种子细胞的来源提供了可能性.Aim： To amplify mesenchymal stem cells（MSCs） from human bone marrow （HBM） and to induce MSCs differentiated into endothelial cells（ECs） in vitro, which possibility and conditions were to be discussed. Methods： MSCs were separated by gradient centrifugation on Percoll （density 1. 073 g/ml） from HBM, and incubated for purification and amplification in DMEM （low glucose） with 10 96 fetal bovine serum（FBS）. MSCs, which their phenotypic characteristics were analyzed by flow cytometry, were incubated for orientation differentiated into ECs in DMEM（high glucose） with 20% FBS and VEGF（ 10 ng/ml） for about 14 - 21 days. Afterwards, the cells differentiated were evaluated by inmmnohistochemistry with Tie-2 monoclonal antibody and by transmission electron microscopy（TEM） for observation Weible-palade corpuscle in the cytoplasm. Results： The quantity of MSCs was increased from 5.0 - 105 in the primary culture to 8.0 × 10^12 , or to increase 1.6 × 10^7 times after 15 generations incubated. The purity of MSCs was above 95 % and 98 96 homogeneous at passages 2 and 3, respectively. The typical “cobblestone” of these cells presented from MSCs differentiation culture after 14 -21 days was observed by light microscopy. More than 90% of the ceils were positive stain for Tie-2 related antigen by immunohistochemistry assay. The Weible-palade corpuscle, which form is typical morphology of ECs, was also observed by TEM in the cytoplasm. Conclusion ： MSCs from HBM has the capability in differentiation into ECs in vitro, which is possibile to provide the seed cells for fabrication of tissue-engineering heart valve.

关 键 词：骨髓 间充质干细胞 内皮细胞 TIE-2 

分 类 号：R336[医药卫生—人体生理学]