硫氧还蛋白-2 在氧化损伤的人晶状体上皮细胞中的表达及其意义

目的：探讨硫氧还蛋白-2(thioredoin-2，Trx-2)是否参与白内障的发病过程及其对过氧化氢(H2O2)诱导的 人晶状体上皮细胞氧化损伤的影响。方法：选取志愿者(因外伤行透明晶状体取出术患者)10例和行超声乳化白内 障手术患者(年龄大于60岁)30例的晶状体前囊膜，采用链霉素亲生物素蛋白-过氧化物酶标免疫组织化学法检测志 愿者和白内障患者晶状体上皮细胞中Trx-2蛋白的表达情况。体外培养SRA01/04细胞，根据不同处理分为空白对照 组、20 μmol/L H2O2组、50 μmol/L H2O2组、100 μmol/L H2O2组、阴性对照组(转染对照空pCMV6质粒并用100 μmol/L H2O2处理)和过表达Trx-2组(转染pCMV6-Trx-2过表达质粒并用100 μmol/L H2O2处理)。采用噻唑蓝3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide，MTT]法检测各组细胞活力；采用流式细胞术检测各组细胞的凋亡；采用 化学比色法检测各组细胞中超氧化物歧化酶(superoxide dismutase，SOD)和过氧化氢酶(catalase，CAT)活性，谷胱甘肽 (glutathione，GSH)和丙二醛(malondialdehyde，MDA)含量；采用Western印迹检测各组细胞中Trx-2以及B细胞淋巴瘤/白 血病-2蛋白(B-cell lymphoma 2 protein，Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein，Bax)和含半胱氨酸的天冬氨酸 蛋白水解酶-3(cysteinyl aspartate specific proteinase-3，caspase-3)的表达。结果：与志愿者相比，白内障患者晶状体上皮 细胞中Trx-2蛋白表达明显减少(P<0.05)。与空白对照组比较，20， 50，100 μmol/L H2O2组Trx-2蛋白表达量均明显降低 (均P<0.05)。与空白对照组相比，100 μmol/L H2O2组和阴性对照组细胞存活率降低、凋亡率增加，细胞内SOD和CAT 活性降低、GSH含量减少、MDA含量增加，Trx-2和Bcl-2蛋白表达降低，Bax和caspase-3蛋白表达增加(均P<0.05)。与 阴性对照组比较，过表达Trx-2组细胞存活率增加、凋亡率降低，SOD和CAT活性增加、GSH含量升高、MDA含量降 低，Trx-2和Bcl-2蛋白表达增加，Bax和caspase-3蛋白表达减少(均P<0.05)。结论：Trx-2参与白内障发病中上皮细胞的凋 亡，过表达Trx-2能够减少H2O2诱导的细胞凋亡，这可能与拮抗H2O2诱导的氧化损伤有关。

Expression of thioredoxin-2 in human lens epithelial cells with oxidative damage and its significance

Objective: To explore whether thioredoin-2 (Trx-2) is involved in the development of cataract and to study the effect of Trx-2 on hydrogen peroxide (H2O2)-induced injury in human lens epithelial cells. Methods: A total of 10 volunteers (removing the lens due totraumatism) and 30 patients received phacoemulsification (age more than 60 years) were selected. The expression of Trx-2 protein in lens epithelial cells from cataract patients and volunteers were detected by the immunohistochemical streptavidin-peroxidase (SP) method. SRA01/04 cells were cultured and were divided into six groups according to different treatment: a control group, H2O2-treated groups at 20, 50 or 100 μmol/L, a negative control group (transfected with pCMV6 plasmid plus 100 μmol/L H2O2), and a Trx-2 overexpression group (transfected with pCMV6-Trx-2 plasmid plus 100 μmol/L H2O2). Methyl thiazolyltetrazolium (MTT) assay and flow cytometry was performed to measure the cell viability and apoptosis for SRA01/04 cells, respectively. The activities of superoxide dismutase (SOD) and catalase (CAT), the content of glutathione (GSH) and malondialdehyde (MDA) in human lens epithelial cells were measured via chemical chromatometry. Western blot was used to measure the protein levels of Trx-2, B-cell lymphoma 2 protein (Bcl-2), Bcl-2 associated X protein (Bax) and caspase-3. Results: Compared with the volunteers, the expression of Trx-2 was significantly decreased in lens epithelial cells in patients with cataract (P<0.05). Compared with the control group, the expression of Trx-2 protein in the 20, 50 or 100 μmol/L H2O2 groups was decreased (all P<0.05). Compared with the control group, the cell survival rates were decreased in the 100 μmol/L H2O2 group and the negative control group (both P<0.05), along with enhanced apoptotic rates, inhibited cellular SOD activities and CAT activities, reduced GSH contents, augmented MDA contents, down-regulated Trx-2 and Bcl-2 expression and up-regulated Bax and caspase-3 expression (all P<0.05). Compared with the negative control group, the cell survival rate was increased in the Trx-2 overexpression group (P<0.05), along with suppressed apoptosis, increased SOD activities and CAT activities, elevated GSH contents, decreased MDA content, up-regulated Trx-2 and Bcl-2 expression and down-regulated Bax and caspase-3 expression (P<0.05). Conclusion: Trx-2 might be involved in the apoptosis of lens epithelial cells in patients with cataract. The overexpression of Trx-2 obviously attenuated H2O2-induced injury of human lens epithelial cells, which might be associated with the inhibition of H2O2-mediated oxidative stress.