三种细胞因子体外联合诱导兔骨髓间充质干细胞向血管内皮细胞的分化

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背景:已证实血管内皮细胞种植于组织工程材料上在体内可增殖分化形成血管,但获取内皮细胞时创伤较大,且来源极其有限。目的:观察在血管内皮细胞生长因子、碱性成纤维细胞生长因子、胰岛素样生长因子1体外联合诱导下,兔骨髓间充质干细胞能否向血管内皮细胞分化。设计、时间及地点:2006-11/2007-06在辽宁医学院附属第一医院中心实验室完成的细胞对照观察。材料:清洁级8周龄日本大耳白兔1只用于骨髓间充质干细胞的分离培养。血管内皮细胞生长因子、碱性成纤维细胞生长因子、胰岛素样生长因子1均为Peprotech公司产品。方法:兔麻醉后抽取骨髓液,贴壁法+胰酶消化分离培养获得足够数量的骨髓间充质干细胞,分为两组,诱导组向含10%胎牛血清的DMEM培养液中加入10μg/L血管内皮细胞生长因子、1μg/L碱性成纤维细胞生长因子、2μg/L胰岛素样生长因子1,诱导2周。对照组单纯加入含10%胎牛血清的DMEM培养液。主要观察指标:对诱导后细胞进行形态观察、NO含量测定、Ⅷ因子相关抗原免疫组织化学染色以及电镜观察Weibel-Palade小体形成情况。结果:诱导5d后细胞呈集落样分布,形态趋于圆形;未诱导细胞均匀散在分布,呈长梭形和三角形。诱导14d后,诱导组细胞培养上清液中NO含量明显高于对照组(t=3.75,P<0.05)。诱导组表达血管内皮细胞特有表面标志Ⅷ因子,超微结构可见Weibel-Palade小体;对照组Ⅷ因子相关抗原呈阴性表达。结论:兔骨髓间充质干细胞经上述3种细胞因子体外联合诱导后,可向血管内皮细胞方向分化。 BACKGROUND: Vascular endothelial cells have been implanted in tissue engineering materials to proliferate and differentiate to form blood vessels in the body. However, the endothelial cells are traumatized and their sources are extremely limited. OBJECTIVE: To observe whether rabbit bone marrow mesenchymal stem cells can differentiate into vascular endothelial cells induced by vascular endothelial growth factor, basic fibroblast growth factor and insulin-like growth factor 1 in vitro. DESIGN, TIME AND SETTING: The cellular control study performed at the Central Laboratory of the First Affiliated Hospital of Liaoning Medical College from November 2006 to June 2007 was performed. MATERIALS: One 8-week-old Japanese white rabbits were used for the isolation and culture of bone marrow-derived mesenchymal stem cells. Vascular endothelial growth factor, basic fibroblast growth factor, and insulin-like growth factor 1 are all Peprotech products. Methods: Bone marrow was extracted after anesthesia in rabbits, and a sufficient number of bone marrow mesenchymal stem cells were obtained by adherent method and trypsin digestion. Two groups were divided into two groups. In the induction group, DMEM medium containing 10% fetal bovine serum was added with 10μg / L vascular endothelial cell growth factor, 1μg / L basic fibroblast growth factor, 2μg / L insulin-like growth factor 1, induction for 2 weeks. Control group was added DMEM containing 10% fetal bovine serum. MAIN OUTCOME MEASURES: Morphological observation, NO content determination, Ⅷ factor-related antigen immunohistochemical staining and electron microscopy were used to observe the formation of Weibel-Palade bodies. Results: After 5 days of induction, the cells showed colony-like distribution with a round shape. The non-induced cells were evenly scattered and distributed in long fusiform and triangular shapes. After induction for 14 days, the content of NO in the supernatant of the induction group was significantly higher than that of the control group (t = 3.75, P <0.05). Induced group expressed vascular endothelial cell specific surface marker factor VIII, weibel-Palade ultrastructure can be seen; the control group was negative for factor-related antigen. CONCLUSION: Rabbit mesenchymal stem cells can differentiate into vascular endothelial cells induced by the above three kinds of cytokines in vitro.
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