【摘要】：第二章人腦微血管內(nèi)皮細胞的培養(yǎng) 【目的】探討人腦微血管內(nèi)皮細胞(human brain endothelial cells,hCMEC/D3)培養(yǎng)、傳代、凍存、生長周期規(guī)律、HIV-1Tat對細胞毒性作用以及HIV-1Tat對細胞內(nèi)活性氧含量的影響。觀察hCMEC/D3的生物學(xué)特性和藥物對其的抑制性作用，，為下一階段提供hCMEC/D3進行藥物的處理及蛋白的提取奠定基礎(chǔ)。 【方法】應(yīng)用倒置相差顯微鏡觀察hCMEC/D3傳代后細胞的形態(tài)特征、生長情況、繪制hCMEC/D3的生長曲線。采用甲基噻唑基四唑(Matrixmetalloproteinases,MTT）比色法測定HIV-1Tat蛋白對hCMEC/D3活性的影響。應(yīng)用熒光分光光度計，以2‘,7‘-二氯熒光素為熒光探針檢測HIV-1Tat對細胞內(nèi)活性氧含量的影響。 【結(jié)果】傳代培養(yǎng)的hCMEC/D3在顯微鏡下形態(tài)呈多角形或長梭形細胞，向四周伸出細長突起，互不重疊的單層“鵝卵石”狀。hCMEC/D3的生長曲線顯示，細胞經(jīng)過3-4天進入指數(shù)生長期，生長速度加快。MTT法測定發(fā)現(xiàn)1ug/ml HIV-1Tat作用hCMEC/D324h后對細胞的抑制率分別為2.84%、7.80%、13.48%。用熒光分光光度計檢測發(fā)現(xiàn)1μg/ml HIV-1Tat組活性氧含量的平均熒光強度（10.315±0.648），明顯高于空白對照組（P0.001）。 【結(jié)論】經(jīng)傳代后培養(yǎng)細胞生長能力強，性狀穩(wěn)定。給予1ug/ml HIV-1Tat處理細胞24h后對細胞生長的活性無明顯影響。HIV-1Tat可誘導(dǎo)細胞內(nèi)活性氧含量升高。 第三章雌激素抑制HIV-1誘導(dǎo)hCMEC/D3間緊密連接蛋白表達的影響 【目的】初步探討雌激素、HIV-1Tat誘導(dǎo)hCMEC/D3緊密連接蛋白表達的作用。 【方法】采用蛋白免疫印跡（Western blot, WB）技術(shù)檢測hCMEC/D3中蛋白表達變化。同代人腦微血管內(nèi)皮細胞（human brain endothelial cells，hCMEC/D3)分為培養(yǎng)基對照組(CTL組)給予無血清培養(yǎng)基處理；Tat組給予1μg/ml HIV-1Tat；17β-雌二醇（E2）組給予10-8mol/L17β-E2處理；Tat+E2組以濃度10-8mol/L17β-E2預(yù)處理2小時后，再加入1μg/ml HIV-1Tat共處理24小時。 【結(jié)果】HIV-1Tat蛋白對人腦微血管內(nèi)皮細胞處理24小時后，整合膜蛋白-1（Claudin-1）,帶狀閉合蛋白-1（zona occludens-1, ZO-1),帶狀閉合蛋白-2(zona occludens-2, ZO-2)蛋白表達下調(diào)，（P0.05，P0.01，P0.01）。E2可誘導(dǎo)Claudin-1,ZO-1的蛋白表達上調(diào)（P0.01，P0.05），同時抑制HIV-1Tat誘導(dǎo)Claudin-1,ZO-2蛋白表達下調(diào)（P0.05，P0.01），但對ZO-1蛋白表達調(diào)節(jié)無統(tǒng)計學(xué)意義（P0.05）。 【結(jié)論】 HIV-1Tat誘導(dǎo)人腦微血管內(nèi)皮細胞間緊密連接蛋Claudin-1,ZO-1,ZO-2降解。然而，雌激素可抑制HIV-1Tat誘導(dǎo)緊密連接蛋白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 鹵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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R512.91

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