中文摘要：

目的探讨Rho／ROCK信号传导通路在人类免疫缺陷病毒-1型反式转录激活因子（HIV-1 transactivator of transcription，HIV-1 Tat）诱导血脑屏障破坏及对β淀粉样蛋白（Amyloid—beta，Aβ）沉积中的作用。方法将培养的人脑微血管内皮细胞（human cerebral microvascular endothelial cells，hCMEC／D3）予以不同浓度的Rho／ROCK信号传导通路抑制剂盐酸法舒地尔（Hydroxyfasudil，HF）刺激细胞，并设立对照组，观察其对细胞活力的影响，分别予以HIV-1 Tat、HF刺激细胞，以蛋白免疫印迹法和实时反转录聚合酶链式反应（real—time reverse transcription polymerase chain reaction，RT-PCR）检测hCMEC／D3中紧密连接蛋白Occludin、晚期糖基化终产物受体（receptor for advanced glycation end products。RAGE，转移血液Aβ到脑组织）的蛋白和mRNA表达的变化。结果HF在30μmol／L（0．17±0．02）以下时对hCMEC／D3活力无明显影响（P〉0．05）。HIV-1 Tat能抑制hCMEC／D3的Occludin蛋白（0．71±0．03、P〈0．001）与mRNA（0．41±0．05、P〈0．05）表达，同时上调RAGE蛋白（0．83±0．01、P〈0．001）和mRNA（1．93±0．66、P〈0．05）表达。HF预处理细胞2h后与HIV-1 Tat共培养可以显著增加Occludin蛋白与mRNA的表达（0．93±0．03、P〈0．001；1．04±0．45、P〈0．05），同时可以明显减少RAGE蛋白与mRNA的表达（0．54±0．01、P〈0．001，1．08±0．43、P〈0．05）。结论HIV-1 Tat可使紧密连接蛋白Occludin蛋白和mRNA表达下调导致血脑屏障破坏，诱导A13转运受体RAGE的蛋白和mRNA过度表达，从而可能导致Aβ在脑内沉积增加；阻断Rho／ROCK信号传导通路可抑制HIV-1 Tat诱导血脑屏障破坏作用，阻止RAGE蛋白和mRNA的过度表达。

英文摘要：

Objective To evaluate the role of Rho/ROCK signaling pathway in HIV-1 Tat-induced Occludin dysfunction and Amyloid-beta deposition in human cerebral microvascular endothelial cells （hCMEC/D3）. Methods hC- MEC/D3 viability was tested by MTT assay ,with the exposure of different concentrations of Rho/ROCK inhibitor hydroxyfasudil （HF）. Respectively Western blot and real-time reverse transcription polymerase chain reaction （qRT-PCR） assay Occludin and advanced glycation end product receptor （RAGE,which regulates Aβ transfer from the blood stream into the central nervous system） protein and mRNA expression in hCMEC/D3. Results Hydroxyfasudil at 50 μmol/L （0.17 ± 0.02） or less had no significant effect on the hCMEC/D3 cell viability as determined by the MTT assay （ P 〉 0.05 ）. Treatment with HIV-1 Tat decreased protein expression of Occludin （0.71 ±0.03,P 〈0. 001 ） in hCMEC/D3 as well as mRNA （0.41±0.05,P 〈0.05） levels, and also activated the expression of RAGE （0.83 ± 0.01,P 〈 0. 001 in protein levels, 1.93 ±0.66,P 〈 0.05 in mRNA levels）. While inhibition of Rho by HF effectively protected against HIV-1 Tat-induced downregulation of Occludin protein （0.93 ± 0.03,P 〈 0. 001 ） and mRNA （ 1.04 ± 0.45,P 〈 0. 05 ） expression. At the same time, the effect on RAGE protein （ 0.54 ± 0.01 ,P 〈 0. 001 ） and mRNA （ 1.08 ± 0.43 ,P 〈 0.05 ） were attenuated by pretreatment with hydroxyfasudil. Conclusion HIV-1 Tat induces dysregulation of Occludin and Aβ transport RAGE receptor both in protein and mRNA levels. These effects were abolished by the addition of HF. These results show that Rho/ ROCK signaling pathway plays an important role in HIV-1 Tat-induced Occludin dysfunction and Amyloid-beta deposition in cerebral microvascular endothelial cells.