α晶状体蛋白对脂多糖诱导的视网膜小胶质细胞增生及TNF-α表达的影响  

Effect of α-crystallin on lipopolysaccharide-induced retinal microglia and expression of TNF-α

作　　者：吴楠[1] 王艳华[1] 王蕊[1] 刘东宁[1] 王晓芹[1] 王一[1] 

机构地区：[1]第三军医大学西南医院眼科,重庆400038

出　　处：《眼科研究》2007年第12期912-915,共4页Chinese Ophthalmic Research

基　　金：国家重点基础研究发展计划项目(973)分题(2007CB512203);全军医学科研"十一五"攻关项目(06G072)资助

摘　　要：目的观察α晶状体蛋白对脂多糖(LPS)诱导的视网膜小胶质细胞增生及肿瘤坏死因子α(TNF-α)生物学活性的影响。方法原代培养视网膜小胶质细胞,并采用CD11b细胞免疫荧光及流式细胞仪鉴定纯度,加入α晶状体蛋白及LPS后,通过MTT(四氮唑蓝比色细胞增生测定)检测α晶状体蛋白对LPS活化的小胶质细胞增生能力的影响,并采用ELISA、RT-PCR检测TNF-α蛋白水平及mRNA水平表达的变化。结果原代培养的小胶质细胞经免疫组织化学鉴定及流式细胞仪鉴定纯度分别达到95.8%和91.4%,经10-4g/Lα晶状体蛋白预处理后,LPS诱导的小胶质细胞增生被抑制(P<0.01);与未处理组比较,TNF-α蛋白质量浓度及mRNA表达量明显降低(P<0.05)。结论α晶状体蛋白可以减少LPS诱导的原代培养视网膜小胶质细胞增生及TNF-α表达。Objective As one of lentogenic factors,alpha-crystallin can promote axon regeneration after optic nerve injury. The microenvironment around retinal ganglion cells is also important. Present study was to investigate the effect of α-crystallin on the activity of rat retinal microglia and the expression of stimulated TNF-α with lipopolysaccharides（LPS）. Methods Retinal microglia cells were obtained from the newborn Long Evans rat （ 〈 3 days） and were premarily cultivated and passaged. The 3rd generation of cells were collected and identified using CD11 b with immunofluorescence and flow cytometry（FCM）. 10^-6g/L LPS was added in cultured microglia cells in LPS group,and 10^-4g/L alpha-crystallin and 10^-6g/L LPS was used in alpha-crystallin ＋ LPS group. Two groups of microglia cells were detected by MTT, and expression of TNF-α mRNA and protein was assessed with ELISA kit and RT-PCR. Results Cultured microglia cells showed red fluorescence in cell membrane for CDllb. Purity rate of cultued microglia cells was 95.8% and 91.4% by cell immunochemistry identification and FCM of CD11 b, respectively. After pretreatment with alpha-crystallin （10^-4g/L） , the total number of microglia was 1.91 ±0. 08 （ OD value） , indicating a obvious decrease in comparison with LPS group （2.43 ±0. 06） （t =51.38 ,P =0. 001 ） ,and the expression of TNF-α mRNA and protein was significantly inhibited in alpha-crystallin ＋ LPS group compared with LPS group （0. 64 ±0. 05,407.5 ± 50 versus 0.82±0.07,603.7±78 respectively）（t=5.74,P=0. 029;t=45.51,P=0. 001）. Conclusion Alpha-crystallin inhibits the proliferation of microglia and expression of TNF-α by blocking the activity of microglia induced by LPS. It suggests that alphacrystallin may have the protective effect on retinal ganglion cell.

关 键 词：小胶质细胞 脂多糖 α晶状体蛋白 肿瘤坏死因子α 

分 类 号：R774.1[医药卫生—眼科]