【摘要】 目的探讨缺氧条件下体外培养人脑微血管内皮细胞VEGF基因表达与细胞超微结构变化。方法取材于手术中切除的脑动静脉畸形周围黏连的新鲜脑组织,采用匀浆、过滤和酶消化技术分离微血管内皮细胞。免疫组化方法检测第八因子相关抗原(FⅧ-RA)表达。选用RT-PCR技术观察静态和缺氧状态下(体积分数95%N2,5%CO2;2 h、4 h和8 h)内皮细胞VEGF mRNA表达,同时以ELISA方法检测每组细胞上清液中VEGF蛋白含量,透射电镜观察细胞超微结构的变化。结果相差显微镜下,培养的活细胞具有单层“卵石样”排列的典型特征,90%以上的细胞为FⅧ-RA阳性染色。缺氧4 h细胞VEGFmRNA和蛋白表达较对照组显著升高(P<0.01),8 h后表达下调,并出现线粒体肿胀、内质网扩张及溶酶体多囊泡形成。结论缺氧条件下脑血管内皮细胞的VEGF表达水平可能与其细胞超微结构变化有重要关系。更多还原

【Abstract】 Objective To investigate the gene expression of vascular endothelial growth factor（VEGF） and ultrastructural changes in cultured endothelial cells from human cerebral microvessels under hypoxic conditions.Methods Human cerebral microvessels were isolated from freshly obtained specimens of normal brain adherent to resected cerebral arteriovenous malformations.The expression of factor Ⅷ-relative antigen（FⅧ-RA） in cultured cells was observed with immunocytochemistry.The level of VEGFmRNA in cells and released VEGF protein in cell supernatant were determined by RT-PCR analysis and ELISA respectively when they were exposed to hypoxic conditions（95% N₂,5% CO₂;two hours,four hours,eight hours） or maintained in basal condition.Ultrastructural changes in cells were also observed by electron microscopy.Results In inverted microscope the cultured cells showed contact inhibition and a rounded cobblestone appearance.More than 90% of them were stained strongly with antibodies against FⅧ-RA.Significant VEGF mRNA and protein accumulated when these cells were exposed to hypoxia for 4 hours.However,their VEGF expression was down-regulated after hypoxia for 8 hours and a number of vesicles and swollen mitochondria were present in the cytoplasm.Conclusion The level of VEGF expression may havesignificant relationship with ultrastructural changes in human cerebral endothelial cells under hypoxic conditions.更多还原