雌激素对HIV-1 Tat诱导紧密连接蛋白破坏的抑制作用及其机制研究

发布时间：2019-03-18 18:29

【摘要】：第二章人脑微血管内皮细胞的培养【目的】探讨人脑微血管内皮细胞(human brain endothelial cells,hCMEC/D3)培养、传代、冻存、生长周期规律、HIV-1Tat对细胞毒性作用以及HIV-1Tat对细胞内活性氧含量的影响。观察hCMEC/D3的生物学特性和药物对其的抑制性作用，，为下一阶段提供hCMEC/D3进行药物的处理及蛋白的提取奠定基础。【方法】应用倒置相差显微镜观察hCMEC/D3传代后细胞的形态特征、生长情况、绘制hCMEC/D3的生长曲线。采用甲基噻唑基四唑(Matrixmetalloproteinases,MTT）比色法测定HIV-1Tat蛋白对hCMEC/D3活性的影响。应用荧光分光光度计，以2‘,7‘-二氯荧光素为荧光探针检测HIV-1Tat对细胞内活性氧含量的影响。【结果】传代培养的hCMEC/D3在显微镜下形态呈多角形或长梭形细胞，向四周伸出细长突起，互不重叠的单层“鹅卵石”状。hCMEC/D3的生长曲线显示，细胞经过3-4天进入指数生长期，生长速度加快。MTT法测定发现1ug/ml HIV-1Tat作用hCMEC/D324h后对细胞的抑制率分别为2.84%、7.80%、13.48%。用荧光分光光度计检测发现1μg/ml HIV-1Tat组活性氧含量的平均荧光强度（10.315±0.648），明显高于空白对照组（P0.001）。【结论】经传代后培养细胞生长能力强，性状稳定。给予1ug/ml HIV-1Tat处理细胞24h后对细胞生长的活性无明显影响。HIV-1Tat可诱导细胞内活性氧含量升高。第三章雌激素抑制HIV-1诱导hCMEC/D3间紧密连接蛋白表达的影响【目的】初步探讨雌激素、HIV-1Tat诱导hCMEC/D3紧密连接蛋白表达的作用。【方法】采用蛋白免疫印迹（Western blot, WB）技术检测hCMEC/D3中蛋白表达变化。同代人脑微血管内皮细胞（human brain endothelial cells，hCMEC/D3)分为培养基对照组(CTL组)给予无血清培养基处理；Tat组给予1μg/ml HIV-1Tat；17β-雌二醇（E2）组给予10-8mol/L17β-E2处理；Tat+E2组以浓度10-8mol/L17β-E2预处理2小时后，再加入1μg/ml HIV-1Tat共处理24小时。【结果】HIV-1Tat蛋白对人脑微血管内皮细胞处理24小时后，整合膜蛋白-1（Claudin-1）,带状闭合蛋白-1（zona occludens-1, ZO-1),带状闭合蛋白-2(zona occludens-2, ZO-2)蛋白表达下调，（P0.05，P0.01，P0.01）。E2可诱导Claudin-1,ZO-1的蛋白表达上调（P0.01，P0.05），同时抑制HIV-1Tat诱导Claudin-1,ZO-2蛋白表达下调（P0.05，P0.01），但对ZO-1蛋白表达调节无统计学意义（P0.05）。【结论】 HIV-1Tat诱导人脑微血管内皮细胞间紧密连接蛋Claudin-1,ZO-1,ZO-2降解。然而，雌激素可抑制HIV-1Tat诱导紧密连接蛋白Claudin-1,ZO-2降解。
[Abstract]:Chapter two: culture of human brain microvascular endothelial cells [objective] to investigate the regulation of culture, passage, freezing and growth cycle of human brain microvascular endothelial cells (human brain endothelial cells,hCMEC/D3). The cytotoxicity of HIV-1Tat and the effect of HIV-1Tat on the content of reactive oxygen species (Ros) in cells were studied. To observe the biological characteristics of hCMEC/D3 and the inhibitory effect of drugs on it, and to lay a foundation for providing hCMEC/D3 for drug treatment and protein extraction in the next stage. [methods] the morphological characteristics and growth of hCMEC/D3 cells were observed by inverted phase contrast microscope, and the growth curve of hCMEC/D3 was drawn. Methyl thiazolyl tetrazolium (Matrixmetalloproteinases,MTT) colorimetry was used to determine the effect of HIV-1Tat protein on hCMEC/D3 activity. The effect of HIV-1Tat on the content of reactive oxygen species (Ros) in cells was detected by fluorescence spectrophotometer. [results] the morphology of subcultured hCMEC/D3 was polygonal or long fusiform cells under microscope, protruding out a slender protuberance around it, and the growth curve of non-overlapping monolayer "cobblestone"-shaped hCMEC / D3 showed that there was no overlap in the growth curve of HCMEC / D3. The inhibition rate of 1ug/ml HIV-1Tat on hCMEC/D324h was 2.84%, 7.80% and 13.48%, respectively. The average fluorescence intensity of reactive oxygen species (Ros) in 1 渭 g / ml HIV-1Tat group (10.315 卤0.648) was significantly higher than that in blank control group (P0.001). [conclusion] after passage, the cell growth ability is strong and the characters are stable. 1ug/ml HIV-1Tat-1Tat could induce the increase of reactive oxygen species (Ros) content in the cells after 24 h treatment. Chapter 3 the effect of estrogen on HIV-1-induced tight junction protein expression in hCMEC/D3 [objective] to explore the effect of estrogen and HIV-1Tat on hCMEC/D3 tight junctional protein expression. [methods] Western blot (Western blot, WB) was used to detect the expression of protein in hCMEC/D3. Human brain microvascular endothelial cells (human brain endothelial cells,hCMEC/D3) were divided into medium control group (CTL group) treated with serum-free medium, Tat group treated with 1 渭 g / ml HIV-1Tat;17 尾-estradiol (E2) group and 10-8mol/L17 尾-E2 group treated with 10-8mol/L17 尾-E2. Tat-E _ 2 group was pretreated with 10-8mol/L17 尾-E _ 2 for 2 hours, then treated with 1 渭 g / ml HIV-1Tat for 24 hours. [results] after 24 hours of treatment with HIV-1Tat protein, integrin-1 (Claudin-1), band-closure protein-1 (zona occludens-1, ZO-1) and band-closure protein-2 (zona occludens-2,) were observed in human microvascular endothelial cells. ZO-2) protein expression was down-regulated (P0.05, P0.01, P0.01). E2 induced up-regulation of Claudin-1,ZO-1 protein expression (P0.01, P0.05) and inhibited HIV-1Tat-induced Claudin-1,. The expression of ZO-2 protein was down-regulated (P0.05, P0.01), but the regulation of ZO-1 protein expression was not statistically significant (P0.05). [conclusion] HIV-1Tat induced the degradation of Claudin-1,ZO-1,ZO-2 in human brain microvascular endothelial cells. However, estrogen can inhibit HIV-1Tat-induced degradation of tight junction protein Claudin-1,ZO-2.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.91

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