共表达sCD40LIg及CTLA4Ig双基因修饰原代人内皮细胞包被大鼠.docVIP

共表达sCD40LIg及CTLA4Ig双基因修饰原代人内皮细胞包被大鼠 　 作者：李钊伦, 薛武军, 田普训, 刘华, 冯新顺, 丁小明 【摘要】 　　目的: 建立以分离培养的原代人脐静脉内皮细胞包被大鼠胰岛细胞模型。方法: 分离培养原代人脐静脉内皮细胞, 并分别通过免疫组化试验检测人Ⅷ因子相关抗原, 透射电镜观察Weiblepalade小体, 荧光显微镜观察DiIAcLDL的吞噬效果, 对所分离的人脐静脉内皮细胞进行鉴定。分离大鼠胰岛细胞, 然后在肝素化培养皿中进行共表达sCD40LIg和CTLA4Ig双基因的内皮细胞包被。对包被后的胰岛细胞进行形态学观察并检测其包被前后的功能。结果: 分离培养的内皮细胞表达人Ⅷ因子相关抗原, 电镜下可以观察到Weiblepalade小体, 荧光显微镜下可以观察到内皮细胞对DiIAcLDL的吞噬。共表达sCD40LIg和CTLA4Ig双基因的内皮细胞包被胰岛模型可以观察到内皮细胞的绿色荧光, 未包被胰岛和内皮细胞包被胰岛的胰岛素释放指数分别为4.09和3.94, 与未包被胰岛实验组相比, 内皮细胞包被胰岛对高糖刺激反应无明显差异, 但胰岛素释放量有所减少。结论: 成功分离培养并鉴定了原代人脐静脉内皮细胞, 并成功建立内皮细胞包被胰岛细胞模型, 包被后胰岛功能正常。 　　AIM: To isolate and culture primary human umbilical vein endothelial cells (HUVECs) and then to coat rat islets in vitro. METHODS: Primary HUVECs were isolated and cultured, then they were identified by detecting human Ⅷ factor associated antigen with immunohistochemical method. Weiblepalade bodies were observed by transmission electron microscope and the phagocytosis for DiIAcLDL was detected by fluorescent microscopy. Rat islets were isolated and coated with HUVECs coexpressing sCD40LIg and CTLA4Ig in a heparinized culture dish. The coated islets were observed via morphology and high glucose challenge test. RESULTS: The human Ⅷ factor associated antigen, Weiblepalade bodies and the phagocytosis for DiIAcLDL were found in HUVECs. The islets coated by HUVECs coexpressing sCD40LIg and CTLA4Ig were identified by green fluorescence proteins. Compared with that of the untreated islets (4.09), the release index of the coated islets was 3.94. There was no difference in insulin release between coated islets and untreated islets under glucose challenge. CONCLUSION: HUVECs have been isolated, cultured and identified successfully. The rat islets were coated with HUVECs in vitro function well. 【关键词】 胰岛; 内皮细胞; 包被; CD40L; CTLA4Ig 　　目前, 胰腺移植或胰岛移植是治疗糖尿病最令人感兴趣的方法之一。在健康胰腺中, 为了解决低氧问题, 胰腺最大限度地促使胰岛和循环血液接触, 以利于胰岛细胞保持更好活力［1］。可是瑞典一个研究小组［2］的研究表明: 在移植早期人胰岛和受者血液接触后会触发即刻性血液介导的炎症反应（instant bloodmediated inflam