α-硫辛酸对高压条件下培养大鼠视网膜神经节细胞的保护作用  

Neuroprotective effect of α-lipoic acid on cultured retinal ganglion cells under elevated pressure in vitro

作　　者：刘兵[1] 郭大东[1] 毕宏生[1] 郭媛媛[1] 陈宁虹[1] 马晓华[1] 

机构地区：[1]山东中医药大学附属眼科医院,济南250014

出　　处：《中华眼底病杂志》2012年第5期501-506,共6页Chinese Journal of Ocular Fundus Diseases

摘　　要：目的观察α-硫辛酸对高压条件下培养大鼠视网膜神经节细胞（RGc）的保护作用。方法将RGC分为正常对照组、阴性对照组、单纯加压组、加压联合不同浓度α-硫辛酸干预组。正常对照组和单纯加压组不做任何处理；阴性对照组仅接受200μmol／L α-硫辛酸预处理；加压联合不同浓度的α-硫辛酸干预组分别用50、100、200μmol／L等3种不同浓度的α-硫辛酸对其进行干预。α-硫辛酸预处理1h后，单纯加压组和各干预组置于50mmHg（1mmHg=0．133kPa）下培养24h；正常对照组、阴性对照组置于常压下培养24h。分别用噻唑蓝比色法检测各组细胞的活性；4，6-联脒-2-苯基吲哚（DAPI）染色观察各组细胞核形态改变；实时聚合酶链反应（Real—timePCR）及蛋白免疫印迹法（Westernblot）检测并比较各组细胞中锰超氧化物歧化酶（MnSOD）mRNA和蛋白表达水平的变化。结果单纯加压组细胞活性为（65．64-3．4）％，50、100、200μmol／L3种浓度的α-硫辛酸干预组细胞活性分别为（75．14-3．3）％、（81．8±2．9）％、（87．9±3．1）％，均较单纯加压组显著增高（t=5．108、10．007、12．513，P〈0．05）。DAPI染色显示，加压后RGC-5细胞核可见空泡、染色质高度凝聚、边缘化等典型的凋亡特征，而α-硫辛酸干预组仅有部分细胞核染色质表现出浓缩现象。Real—timePCR及Westernblot检测结果显示，加压培养后细胞内MnSODmRNA和蛋白相对表达量较正常对照组显著降低（t=22．045、26．979，P％0．01）；与单纯加压组相比，α-硫辛酸干预组细胞内MnSODmRNA和蛋白相对表达量均显著增高，差异有统计学意义（t=9．171、12．267、23．567、7．723、12．009、28．198，P〈0．05）；不同浓度的α-硫辛酸干预组之间，随α-硫辛酸浓度增加，MnSODmRNA和蛋白相对表达量呈升高趋势，且差异有统计学意义（F=134．273、194．597，P〈0．0Objective To observe the neuroprotective effect of α-lipoic acid （ALA） on cultured retinal ganglion cells （RGC-5） under elevated pressure in vitro. Methods Cultured RGC-5 cells were divided randomly into 4 groups, including normal control group （group A）, negative control group （group B）, elevated pressure group （group C） and elevated pressure α- ALA group （group D）. The cells of group A and C were not intervened with ALA. The cells of group B were treated with 200 μmol/L ALA. The cells of group D were treated with different concentrations of ALA （50, 100, 200 μmol/L） for one hour. Then cells of group C and D were exerted to 50 mm Hg （1 mm Hg：0. 133 kPa） for 24 hours, while the cells of group A and B were exerted to normal pressures for 24 hours. The cell viability was measured using the methyl thiazolyl tetrazolium （MTT） assay and apoptosis was evaluated using 4 ,6-diamidino-2-pbenylindole （DAPI） staining. Expression of MnSOD was determined by real-time polymerase chain reaction （RT-PCR） and Western blot, respectively. Results The cell viability of group B was （65.6±3.4）G, which lower than that in group D of three concentrations of ALA[（75.1±3. 3） %, （81.8±2.9） % ,（87.9±3.1）%], the differences were significantly （t=5.108, 10.007, 12.513; P〈0.05）. DAPI staining revealed that characteristic apoptotic changes, such as chromatin condensation, convoluted nuclei with cavitations, fragmentation of the nucleus, and apoptotic bodies appeared in RGC-5 cells after 24 ours pressure. There was almost no evidence of apoptosis in group D. RT-PCR and Western blot analysis revealed that the expression of MnSOD mRNA and protein were weakly expressed in group C compared with control A （t= 22. 045,26. 979; P（0.01）. Compared group C with group D, the level of MnSOD mRNA and protein in group D increased significantly （t = 9. 171, 12. 267, 23. 567, 7. 723, 12. 009, 28. 198; P （0.05）. In addition, the presence of ALA was found to inhibit hydrostatic

关 键 词：视网膜神经节细胞/病理学 硫辛酸/治疗应用 细胞 培养的 动物实验 

分 类 号：R774.6[医药卫生—眼科] R775[医药卫生—临床医学]