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。

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 15mmol/L CaCl₂ for 24h 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±0.52U/mg)\〗 were significantly lower than that in NCKD group \〖(2.95±0.08)and(31.10±2.24U/mg)\〗 and CON group \〖(2.96±0.25)and(26.33±1.04U/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.