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
- Author:
- No author available
- Journal Title:
- International Eye Science
- Issue:
- 2
- DOI:
- 10.3980/j.issn.1672-5123.2020.2.05
- Key Word:
- 衰老标记蛋白30;人晶状体上皮细胞;高钙状态;细胞增殖;氧化应激;白内障
Abstract： 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.
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