骨髓间充质干细胞诱导分化为组织工程血管用种子细胞的研究  

Experiment on seed cells for tissue engineered blood vessels derived from canine bone marrow mesenchymal stem cells

作　　者：段红永[1] 管强[1] 武欣[2] 梁宁[1] 杨笑非[1] 韩锋[1] 王振峰[1] 刘增庆[1] 汪忠镐[2] 

机构地区：[1] 山西省人民医院 山西医科大学附属人民医院血管外科,太原030012 [2] 首都医科大学宣武医院血管外科

出　　处：《中华实验外科杂志》2015年第4期748-752,共5页Chinese Journal of Experimental Surgery

基　　金：山西省自然科学基金资助项目（2013011058-2）

摘　　要：目的 探讨利用犬骨髓间充质干细胞在体外诱导分化为血管内皮样细胞及平滑肌样细胞的实验方法.方法 无菌条件下获得犬骨髓后采用密度梯度离心和贴壁培养的方法分离、纯化及培养犬骨髓间充质干细胞,使用流式细胞术鉴定其表面标志物.分别使用合适的培养基加入适当的诱导因子对纯化和扩增后的骨髓间充质干细胞进行诱导,使其在体外分化成血管内皮样细胞及平滑肌样细胞,然后进行鉴定.结果 第3代骨髓间充质干细胞表面CD34表达阴性细胞数为99.98％;CD44表达阳性率为99.36％.利用第2代或第3代骨髓间充质干细胞,向血管内皮样细胞及平滑肌样细胞诱导分化,内皮样细胞呈“铺路石”样改变,血管性假性血友病因子（vWF）及CD31免疫荧光染色呈阳性;平滑肌样细胞具有“波峰-波谷”形状,α-肌动蛋白（α-actin）、钙调理蛋白（calponin）及平滑肌重链（SMMHC）免疫荧光染色为阳性.结论 利用犬骨髓间充质干细胞,使用适当的细胞因子进行诱导,在体外可以成功地将其分化为血管内皮样细胞及平滑肌样细胞.Objective To investigate the experimental approach by which the vascular endothelial-like cells （ECs） and smooth muscle-like cells （SMCs） can be derived from the canine bone marrow mesenchymal stem cells （MSCs）.Methods Density gradient centrifugation and adherent culture were adopted to isolate,purify and culture the canine MSCs.The surface markers of MSCs were determined by flow cytometry.Then the MSCs were induced to ECs and SMCs in appropriate medium plus inducing factors.ECs and SMCs were identified by immunofluorescence staining.Results CD34 negative and CD44 positive cells of MSCs at passage 3 account for 99.98% and 99.34%,respectively.ECs induced from MSCs at passage 2 or 3 shows typical ＆quot;cobblestone＆quot; appearance and positive immunofluorescence staining for yon Willebrand factor （vWF） and CD31.SMCs induced from MSCs at passage 2 or 3 were confirmed by their ＆quot;hill and valley＆quot; morphology and positive immunofluorescence staining for α-actin,calponin and smooth muscle myosin heavy chain （SMMHC）.Conclusion Canine MSCs can be induced to ECs and SMCs cells in appropriate medium plus inducing factors in vitro.

关 键 词：骨髓间充质干细胞 血管内皮样细胞 血管平滑肌样细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]