VEGF对低糖性脑微血管内皮细胞损伤的作用研究

发布时间：2018-04-25 01:36

本文选题：低血糖 + 紧密连接　；参考：《上海交通大学》2015年硕士论文

【摘要】：低血糖是糖尿病患者采用胰岛素强化治疗时常见而严重的并发症。临床研究表明,低血糖诱发的脑水肿是低血糖性脑损伤的始动过程。脑微血管内皮细胞是血脑屏障重要组成成分,位于相邻内皮细胞间的紧密连接通过限制小分子物质通过进而维持脑组织内环境的稳定。但是,低血糖是否通过破坏紧密连接来影响脑微血管内皮细胞功能引起脑水肿的发生迄今还没有被研究。血管内皮生长因子(Vascular endothelial growth factor,VEGF),作为调节血管功能的一个潜在因子,在脑缺血中具有保护作用已经被证实,并且还能促进神经元的存活及突触的生长。这些报道促使我们研究VEGF对低血糖性脑内皮细胞功能是否具有保护作用。我们采用永生化的小鼠脑微血管内皮细胞作为研究对象,体外模拟血脑屏障。首先,研究低糖条件对脑微血管内皮细胞的影响。通过采用荧光素钠(Na-F)检测暴露于低糖条件的跨内皮细胞的通透性;Western blot和细胞免疫荧光(IF)检测紧密连接相关蛋白claudin-5,ZO-1和occludin的表达变化和分布改变;乳酸脱氢酶实验(LDH实验)检测低糖对内皮细胞的毒性作用。结果显示,与正常葡萄糖组相比,低糖条件组(0、0.5、1 mM)的跨内皮细胞通透性均明显升高,同时,紧密连接蛋白claudin-5的表达也相应下降。当较长时间(24 h)较低糖浓度(0、0.5 mM)时,脑内皮细胞毒性增加。进而,我们采用ELISA检测低糖条件下24 h后培养基中的VEGF的分泌量,发现低糖条件组中分泌的VEGF含量比正常葡萄糖条件组中高。最后,通过外加VEGF研究低糖条件下VEGF对脑内皮细胞是否有保护作用,我们检测了低糖和VEGF共存条件下跨内皮细胞通透性、紧密连接相关蛋白以及细胞毒性的变化。另外,我们还检测了转运葡萄糖的主要蛋白Glut-1和反映细胞凋亡程度的主要指标Bcl-2的表达变化。实验结果显示,与低糖组相比,低糖和VEGF共存条件下跨内皮细胞通透性降低、claudin-5表达相应增加以及内皮细胞的毒性显著下降。另外,Glut-1和Bcl-2蛋白表达水平比低糖组显著增加。综上所述,该研究发现低糖条件下紧密连接蛋白claudin-5表达下降,跨内皮细胞通透性增加,这可能是低血糖引起脑水肿的重要分子机制之一。同时,低糖时VEGF对脑微血管内皮细胞可能具有保护作用。
[Abstract]:Hypoglycemia is a common and serious complication of intensive insulin therapy in diabetic patients. Clinical studies have shown that brain edema induced by hypoglycemia is the starting process of hypoglycemic brain damage. The cerebral microvascular endothelial cells are important components of the blood brain barrier, and the close intercellular junction between adjacent endothelial cells is restricted by small molecular substances. Vascular endothelial growth factor (VEGF), as a potential factor in regulating vascular function, is in the brain. The protective effects of ischemia have been confirmed and also promote the survival of neurons and the growth of synapses. These reports have prompted us to study the protective effect of VEGF on hypoglycemic endothelial cell function. We use immortalized mouse brain microvascular endothelial cells as research objects, and in vitro mimic blood brain barrier. The effects of low sugar conditions on cerebral microvascular endothelial cells were investigated. The permeability of cross endothelial cells exposed to low glucose conditions was detected by fluorescein sodium (Na-F); Western blot and cell immunofluorescence (IF) detected the change and distribution of closely linked protein claudin-5, ZO-1 and occludin; lactate dehydrogenase experiment (LDH experiment) The toxicity of low sugar to endothelial cells was detected. The results showed that the permeability of the endothelial cells in the low glucose group (0,0.5,1 mM) increased significantly compared with the normal glucose group, and the expression of the close connexin claudin-5 decreased correspondingly. The toxicity of the endothelial cells increased when a longer time (24 h) was lower (0,0.5 mM). We used ELISA to detect the secretion of VEGF in the medium after 24 h in low sugar conditions and found that the VEGF content in the low sugar group was higher than that in the normal glucose group. Finally, the VEGF was used to study the protective effect of VEGF on the endothelial cells under low sugar conditions. We detected the cross endothelium under the coexistence of low sugar and VEGF. Cell permeability, close connection of related proteins and changes in cytotoxicity. In addition, we also detected the major protein Glut-1 of transshipment of glucose and the changes in the expression of Bcl-2, the main indicator of the degree of apoptosis. Experimental results showed that, compared with the low sugar group, the cross endothelial cell permeability decreased under the coexistence of low sugar and VEGF, and the claudin-5 table In addition, the expression level of Glut-1 and Bcl-2 protein was significantly higher than that in the low sugar group. To sum up, the study found that the expression of compact connexin claudin-5 decreased and the permeability of endothelial cells increased under low sugar conditions, which may be one of the important molecular mechanisms of brain edema caused by hypoglycemia. At the same time, VEGF may protect brain microvascular endothelial cells at low sugar level.

【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2;R743

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