摘要
目的探索HIV-1gp120蛋白侵犯人血-视网膜屏障的机制。方法原代培养人血-视网膜屏障细胞（HBRBC），包括人视网膜微血管内皮细胞（HRCEC）、人视网膜微血管周细胞（HRCPC）、人视网膜色素上皮细胞（HRPE），以培养基作为对照。用MTT法观察7种不同浓度（0．01～0．15mg／L）的HIV-1gp120蛋白作用24h和0．08mg／LHIV-1gp120蛋白作用不同时间（4～72h）对3种细胞的生长抑制作用。0．08、0．1、0．12、0．15mg／L的HIV-1lgp120蛋白作用24h后，用流式细胞仪检测其对3种细胞凋亡率和线粒体膜电位（△ψm）的影响；用Western免疫印迹检测Cleavedcaspase-9蛋白的活化情况。用透射电镜观察经0．08mg／LHIV-1gp120蛋白处理24h前后3种细胞超微结构的变化。结果HIV-1gp120蛋白作用24h，低浓度（〈0．08mg／L）对3种细胞的活性均没有明显影响，而当浓度超过0．08mg／L时，对细胞的增殖活性有明显的抑制作用，呈浓度依赖性（HRCEC：r=-0．763，P〈0．01；HRCPC：r=-0．804，P〈0．01；HRPE：r=-0．698，P〈0．01）。HIV-1gp120蛋白（0．08mg／L）作用12h即可显著抑制细胞的增殖活性，24、48和72h抑制效应更明显，相对增殖率分别为HRCEC：84％、70％、41％、22％，HRCPC：80％、69％、38％、18％，HRPE：86％、73％、45％、26％，抑制效应呈时间依赖性（HRCEC：r=-0．833，P〈0．01；HRCPC：r=-0．784，P〈0．01；HRPE：r=-0．701，P〈0．01）。HIV-1gp120蛋白作用24h后与对照组相比，各浓度组3种细胞凋亡率增加、△ψm明显降低以及Cleavedeaspase-9蛋白表达增强，均呈浓度依赖性。透射电镜示0．08mg／LHIV-1gp120蛋白处理24h后，3种细胞均出现了线粒体肿胀、溶酶体增多等早期凋亡的微观改变。结论HIV-1gp120蛋白能够抑制人血-视网膜屏障细胞的增殖并具有诱导凋亡的作用，破坏线粒体的结构和功能是其可能的机制。
Objective To study the mechanism of human blood-retina barrier （BRB） destroyed by HIV-1 gpl20 protein. Methods Human blood-retina barrier cells （HBRBCs） including human retina capillary endothelial cells （HRCECs） , human retina capillary perieytes （ HRCPCs）, human retinal pigment epithelium （HRPE） were primarily cultured. Culture media were regarded as control. MTT method was used to observe the inhibition effect of HIV-1 gpl20 protein on cell viability at 7 different concentrations （0.01 to 0.15 rag/L） for 24 h, and at a fixed concentration（0.08 mg/L） for different times （4-72 h）. After 0.08, 0.1, 0.12 and 0.15 mg/L HIV-1 gpl20 protein were applied in those cells for 24 h, cell apoptotie rates and membrane potential of mitoehondria （ △ψ m） were measured by flow cytometry. Activation of Cleaved caspase-9 was detected by Western blot. Change of cell microstructure with 0.08mg/L HIV-1 gpl20 protein before and after 24 h was detected by transmission electron microscopy （TEM）. Results Concentration of HIV-1 gpl20 protein less than 0.08 mg/L did not influence cell viability at 24 h. But at the concentration of more than 0.08 mg/L, HIV-1 gpl20 protein could obviously inhibit HBRBCs proliferation with a concentration- dependent manner（HRCECs： r=-0.763, P〈0.01; HRCPCs： r=-0.804, P〈0.01 ; HRPE： r=-0.698, P〈0.01）. HIV-1 gpl20 protein（0.08 mg/L） significantly inhibited cells proliferation at 12 h, and this inhibition effect was more stronger at 24,48,72 h with a time-dependent increase（HRCECs： r=-0.833, P〈0.01 ; HRCPCs： r=-0.784, P〈0.01 ; HRPE： r=-0.701, P〈0.01 ）. The relative growth rates were HRCECs： 84%, 70%, 41%, 22%; HRCPCs： 80%, 69%, 38%, 18%; HRPE： 86%, 73%, 45%, 26% respectively. Compared with control group, the ratio of apoptotic cells of HBRBCs and expression level of Cleaved caspase-9 protein increased but △ψm decreased at different concentrations with HIV-1 gpl20 protein treatment for 24 h. The changes of these indexes were all concentration-dependent manner. Early apoptotic changes of HBRBCs such as mitochondrial swelling and lysosome increase were observed by TEM after treatment of 0.08 mg/L HIV- 1 gpl20 protein for 24 h. Conclusion HIV- 1 gpl20 protein can inhibit proliferation and induce apoptosis of HBRBCs. The mechanism is probably associted with the structure and function destruction of mitochondiral.
出处
《中华生物医学工程杂志》
CAS
2009年第1期20-26,共7页
Chinese Journal of Biomedical Engineering
基金
国家自然科学基金（30471851）
广东省科技计划项目（20088060600026）
