| Purpose:Studies have shown that the loss of calcium homeostasis in lens epithelial cells(LECs)caused by high calcium is closely related to cataract formation.Therefore,this study intends to explore the effects of changes in the expression of senescence marker protein 30(SMP30)on cell proliferation,apoptosis and oxidative stress of human LECs line SRA01/04 under high calcium culture conditions,and to further improve the basic research of SMP30 in the field of cataract.Methods:There were five groups in this experiment:SMP30 over-expression group(OE),negative control OE group(NCOE),SMP30 knock down group(KD),negative control KD group(NCKD),and blank control group(CON).Lentiviral vectors carrying OE,NCOE,KD and NCKD genes,respectively,were selected to change the expression of SMP30 in SRA01/04 cells by lentiviral transfection.Based on the pre-transfection experiments,the optimal transfection conditions consisted of normal complete culture medium and Polybrene transfection reagent(co-transfection reagent)(5μg/mL),and the multiplicity of infection(MOI)was 5.SRA01/04 cells were cultured in a complete culture medium containing 15 mmol/L CaCl2 for 24 hours to simulate the pathological state of high calcium/calcium disorder during cataract formation.Cell proliferation was detected by BrdU-Elisa assay,cell viability was detected by CCK8 assay,cell cycle distribution was measured by PI-FACS cell cycle kit,apoptotic rate was tested by Annexin V-APC single staining method through flow cytometry,total superoxide dismutase(SOD)activity was detected by SOD kit(WST-1 method),and the ratio of oxidative glutathione(GSSG)/total glutathione(T-GSH)was detected by the glutathione kit to assess the level of intracellular oxidative stress.Results:(1)Under the optimum transfection conditions,after lentiviral vector transfection with the target gene,a large number of green fluorescent protein(GFP)expression were observed under fluorescence microscope in all transfected cell groups,and the transfection efficiency was close to 80%.The cell morphology was similar to that of non-transfected SRA01/04 cells(CON group)under optical microscopy:the transfected cells grew well and the morphology was uniform and stable after several subculture,suggesting that OE or KD SRA01/04 cell models were constructed successfully by lentivirus vector transfection.(2)Under high calcium conditions(15 mmol/1 CaCl2,24 hours),most of the SRA01/04 and transfected cells appeared to have undergone shrinkage or severe distortion and cytoplasmic extension with protrusions.Some cells were found to exhibit various degrees of nuclear chromatin condensation and fragmentation.(3)Cell proliferation in each group:①By BrdU-Elisa assay,under the high calcium conditions,the ability of relative cell proliferation in OE group(3.89±0.20)was significantly higher than that in NCOE group(2.82±0.34,P=0.003)and CON group(2.96±0.25,P=0.006),there was no significant difference between NCOE group and CON group(P=0.551)(n=3,F=13.627,P=0.006);the ability of relative cell proliferation in KD group(2.42±0.08)was significantly lower than that in NCKD group(2.95±0.08,P=0.006)and CON group(P=0.006),there was no significant difference between NCKD group and CON group(P=0.971)(n=3,F=11.510,P=0.009).②CCK8 assay results indicated that the cell viability could grow exponentially(n=5,P=0.000)within 1-5 days under high calcium conditions.Except the comparisons between CON group and NCKD group(P=0.788),the pairwise comparison difference of cell viability between any two groups was statistically significant,with the OE group being the highest and the KD group the lowest(n=5,P=0.000).③Under high calcium conditions,the percentage of cells in the S phase in the OE group(40.40±2.46%)showed a significant increase when compared with that in the CON group(20.81±1.25%,P<0.05)in addition to a significant decrease in the G1 phase(46.90±2.17 vs 66.37±1.11%,P<0.05)(n=3,x1=30.594,P=0.000);the percentage of cells in the S phase in the KD group(15.25±0.88%)showed a significant decrease when compared with that in the NCKD group(25.97±0.77%,P<0.05)(n=3,x2=12.261,P=0.016).(4)Cell apoptosis in each group:The results of Annexin V-APC single stained method suggested that:under the high calcium conditions,the apoptotic rate in the OE group(1.66±0.20%)showed a significant decrease when compared with that in the NCOE group(2.36±0.22%,P=0.004)(n=3,F=29.136,P=0.001);the apoptotic rate in the KD group(6.11±0.03%)showed a significant increase when compared with that in the NCKD group(1.83±0.05%,P=0.000)and CON group(1.22±0.12%,P=0.000)(n=3,F=3810.28,P=0.000).(5)Oxidative stress in each group:① Under the high calcium conditions,the activity of SOD in OE group(47.49±4.34 U/mg)was significantly higher than that in NCOE group(30.55±4.15 U/mg,P=0.001)and CON group(26.76± 1.49 U/mg,P=0.000),there was no significant difference between NCOE group and CON group(P=0.241)(n=3,F=28.635,P=:0.001);the activity of SOD in KD group(11.699±0.53 U/mg)was significantly lower than that in NCKD group(31.10±2.24 U/mg,P=0.000)and CON group(P=0.000),and the activity of SOD in NCKD group was higher than that in CON group(P=0.015)(n=3,F=124.249,P=0.000)·②Under the high calcium conditions,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,P=0.000)and CON group(20.1±2.54,P=0.000),there was no significant difference between NCOE group and CON group(P=0.068)(n=3,F=61.306,P=0.000);the ratio of GSSG/T-GSH in KD group(70.80±2.34)was significantly higher than that in NCKD group(15.93±3.47,P=0.000)and CON group(P=0.000),there was no significant difference between NCKD group and CON group(P=0.119)(n=3,F=349.813,P=0.000).Conclusions:Under the high calcium culture conditions,overexpression of SMP30 increased the activity of cell proliferation and antioxidant capacity,but decreased the apoptotic rate and the level of oxidative stress in SRA01/04 cell(human LECs),indicating that increasing the expression of SMP30 may play a protective role in human LECs in regulating cell proliferation,inhibiting cell apoptosis,enhancing the antioxidant capacity,and may alleviate the process of cell damage induced by high calcium to a certain extent. |
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