RESUMO
@#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. <p>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. <p>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)(<i>P</i><0.05); there was no significant difference between NCKD group and CON group(<i>P</i>>0.05).<p>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.
RESUMO
@#【Objective】To explore the effects of overexpressed senescence marker protein 30 （SMP30） on cell proliferation and antioxidative activity in human lens epithelial cell（HLEC）line SRA01/04 under high-calcium mediated oxidative stress. 【Methods】There were 3 groups in this experiment：SMP30 overexpressed group （OE，experimental group），NCOE group （negative control group） and SRA01/04 group （blank control group）. OE and NCOE lentiviral vectors were used to transfect SRA01/04 respectively. A high- calcium- mediated- stress cell model was established by culturing cells with medium containing 15 mmol/L CaCl2 for 24 h. BrdU assay was used to measure cell proliferation. SOD assay kit and GSSG/T- GSH assay kit were used to detect the level of intracellular oxidative stress. 【Results】Green fluorescence protein could be observed in all transfected cell groups under fluorescence microscope and the transfection efficiency was close to 80% ，suggesting that OE cell model was constructed successfully. Under the high calcium culture conditions，the activity of relative cell proliferation and SOD in OE group［（3.89 ± 0.20）and（47.5 ± 4.3 U/mg）］were significantly higher than that in NCOE group［（2.82 ± 0.34）and（30.6 ± 4.2 U/mg）］and SRA01/04 group［（2.96 ± 0.25）and（26.8 ± 1.5 U/mg）］，the ratio of GSSG/T-GSH in OE group（2.36 ± 0.51）was significantly lower than that in NCOE group（16.36 ± 2.48）and SRA01/04 group（20.12 ± 2.54）（n=3，P<0.05）；there was no significant difference between NCOE group and SRA01/04 group （n=3，P>0.05）. 【Conclusions】Overexpression of SMP30 increased the activity of cell proliferation and SOD，but decreased the ratio of GSSG/T- GSH in SRA01/04 cell（HLEC），indicating that SMP30 may alleviate the progression of high-calcium-mediated oxidative cell damage and possess the cytoprotective functions in HLEC.