低表达衰老标记蛋白30对高钙状态下人晶状体上皮细胞增殖和氧化的影响

目的:探讨高钙状态下衰老标记蛋白30(SMP30)低表达对人晶状体上皮细胞(LECs)系SRA01/04增殖、氧化应激的影响。方法:设计3条干扰SMP30靶向基因RGN表达的RNAi序列(KD1~3),以空载序列为阴性对照组(NCKD),构建慢病毒载体并感染SRA01/04细胞,同时以未感染的SRA01/04细胞作空白对照组(CON)。RT-PCR筛选干扰效率最高的慢病毒载体用于后续实验。采用含15mmol/L CaCl2的完全培养基处理细胞24h模拟发生白内障时人LECs的病理状态,通过BrdU-Elisa法检测细胞增殖活力,并检测细胞超氧化物歧化酶(SOD)活性及氧化型谷胱甘肽/总谷胱甘肽(GSSG/T-GSH)水平以评估细胞氧化应激水平。结果:成功构建KD1~3及NCKD慢病毒载体,感染SRA01/04细胞效率约80%。三种慢病毒载体(KD1~3)敲减效率分别为93%、60%、74%,故选择KD1慢病毒载体进行后续实验。高钙状态下,KD1组细胞相对增殖活力和SOD活力(2.42±0.08)和(11.69±0.52U/mg)]均低于NCKD组(2.95±0.08)和(31.10±2.24U/mg)]和CON组(2.96±0.25)和(26.33±1.04U/mg)],GSSG/T-GSH比值(70.80±2.34)高于NCKD组(15.93±3.47)和CON组(20.05±2.45)(均P<0.05),而NCKD组与CON组上述指标均无差异(P>0.05)。结论:高钙状态培养下,RGN-RNAi慢病毒载体介导的SMP30低表达SRA01/04细胞增殖活力及抗氧化应激能力减弱,提示SMP30可能具有调控细胞增殖、抗氧化应激的保护作用。

Effect of low expression of senescence marker protein 30 on proliferation and oxidation of human lens epithelial cells line SRA01/04 under high calcium conditions

·AIM:To explore the effect of low expression of senescence marker protein 30(SMP30) on proliferation and oxidative stress of human lens epithelial cells(LECs)line SRA01/04 under high calcium conditions.·METHODS:Three RNAi sequences were designed to knock down SMP30 target gene RGN expression(KD1-3),and the blank-load sequence was used as the negative control group(NCKD),all of which were used to construct lentiviral vectors to infect SRA01/04 cells.Meanwhile,the uninfected SRA01/04 cells was used as the blank control group(CON).After transfecting SRA01/04 cells,the lentiviral vector with the highest knockdown efficiency was selected by RT-PCR for subsequent experiments.Cells were treated with 15 mmol/L CaCl2 for 24 h to simulate a high calcium conditions.BrdU-Elisa assay was used to measure cell proliferation,superoxide dismutase(SOD) assay kit and oxidized glutathione/total glutathione(GSSG/T-GSH) assay kit were used to detect the level of intracellular oxidative stress.·RESULTS:KD1-3 and NCKD lentiviral vectors were successfully constructed to infect SRA01/04 cells with an infection efficiency of about 80%.The knockdown efficiency of KD1-3 group was 93%,60% and 74%,respectively,KD1 group was selected for follow-up experiment.Under the high calcium conditions,the activity of relative cell proliferation and SOD in KD1 group(2.42±0.08) and(11.69±0.52 U/mg)] were significantly lower than that in NCKD group (2.95±0.08) and(31.10±2.24 U/mg)] and CON group (2.96±0.25) and(26.33±1.04 U/mg)],the ratio of GSSG/T-GSH in KD1 group(70.80±2.34) was significantly higher than that in NCKD group(15.93±3.47) and CON group(20.05±2.45)(P<0.05);there was no significant difference between NCKD group and CON group(P>0.05).·CONCLUSION:Under high calcium conditions,SRA01/04 cells(HLECs) with low expression of SMP30 mediated by shRNA lentivirus resulted in the decrease of the proliferation activity and antioxidant capacity,suggesting that SMP30 may play a protective role in regulating cell proliferation and anti-oxidative stress in HLECs.