高表达衰老标记蛋白30对高钙诱导人晶状体上皮细胞株SRA01/04氧化应激的影响
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摘要
【目的】探讨在高钙诱导细胞氧化应激下,高表达衰老标记蛋白30(SMP30)对人晶状体上皮细胞(HLEC)株SRA01/04增殖活力及抗氧化能力的影响。【方法】实验分3组:SMP30高表达组(OE,实验组)、NCOE组(阴性对照组)及SRA01/04组(空白对照组)。用OE及NCOE慢病毒载体分别转染SRA01/04细胞。15 mmol/LCaCl2处理细胞24 h建立高钙诱导模型。BrdU法检测细胞增殖、超氧化物歧化酶(SOD)、氧化型谷胱甘肽(GSSG)/总谷胱甘肽(T-GSH)法检测细胞氧化指标。【结果】荧光显微镜下可见各组转染细胞有大量绿色荧光蛋白(GFP)表达,转染效率接近80%,提示SMP30高表达SRA01/04细胞模型构建成功。在高钙状态下,OE组细胞相对增殖活力(3.89±0.20)和SOD活力(U/mg,47.5±4.3)均高于NCOE组(2.82±0.34、30.6±4.2)及SRA01/04组(2.96±0.25、26.8±1.5),OE组GSSG/T-GSH比值(2.36±0.51)低于NCOE组(16.36±2.48)及SRA01/04组(20.12±2.54),上述差异均有统计学意义(n=3,P<0.05),NCOE组与SRA01/04组相比差异均无统计学意义(n=3,P>0.05)。【结论】高表达SMP30增加SRA01/04(HLEC)细胞增殖活力、SOD活力及下降GSSG/T-GSH水平,提示SMP30在一定程度上可缓解高钙诱导细胞氧化损伤的进程,可能对HLEC有保护作用。
【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.
【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.
引文
[1]杨义,鲁建华,张文芳,等.钙蛋白酶抑制剂E-64d对高钙诱导的白内障的保护作用[J].国际眼科杂志,2015,15(6):972-975.Yang Y,Lu JH,Zhang WF,et al. Protective effectof E-64d on calcium-induced cataract[J]. Int EyeSci,2015,15(6):972-975.
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[10]Zhou D,Yin D,Xiao F,et al. Expressions ofsenescence-associatedβ-galactosidase andsenescence marker protein-30 are associated withlens epithelial cell apoptosis[J]. Med Sci Monit,2015,21:3728-3735.
[11]陈曦,韩子豪,李舒凝,等.高钙状态对人晶状体上皮细胞株SRA01/04氧化应激水平的影响[J].眼科新进展,2017,37(10):906-910.Chen X,Han ZH,Li SN,et al. Effects of calciumelevation on intracellular oxidative stress in humanlens epithelial cells SRA01/04[J]. Rec AdvOphthal,2017,37(10):906-910.
[12]Goo MJ, Park JK, Hong IH, et al. Increasedsusceptibility of radiation-induced intestinalapoptosis in SMP30 KO mice[J]. Int J Mol Sci,2013,14(6):11084-11095.
[13]Zhang SC,Liang MK,Huang GL,et al. Inhibitionof SMP30 gene expression influences the biologicalcharacteristics of human Hep G2 cells[J]. AsianPac J Cancer Prev,2014,15(3):1193-1196.
[14]Kadowaki S, Shishido T, Sasaki T, et al.Deficiency of senescence marker protein 30exacerbates cardiac injury after ischemia/reperfusion[J]. Int J Mol Sci,2016,17(4):542.
[15]Yamaguchi M. Regulatory role of regucalcin in heartcalcium signaling:insight into cardiac failure(Review)[J]. Biomed Rep,2014,2(3):303-308.
[16]Son YS,Ullah HMA,Elfadl AK,et al. Preventiveeffects of vitamin C on diethylnitrosamine-inducedhepatotoxicity in SMP30 knockout mice[J]. InVivo,2018,32(1):93-99.
[17]赖伟霞,谭少健,李霞,等.衰老标记蛋白30在不同年龄白内障患者晶状体上皮细胞中的表达变化[J].中华实验眼科杂志,2014,32(6):521-524.Lai WX, Tan SJ, Li X, et al. Comparison ofexpression levels of senescence marker protein 30 inlens epithelial cells among different ages of cataractpatients[J]. Chin J Exp Ophthalmol,2014,32(6):521-524.
[18]刘兰,蔡小军,余爱华,等.不同类型年龄相关性白内障晶状体上皮细胞衰老标记蛋白30的表达及与细胞凋亡的关系[J].中华实验眼科杂志,2012,30(6):529-533.Liu L,Cai XJ,Yu AH,et al. The expression ofsenescence marker protein-30 in different types ofage-related cataracts and its relation to apoptosis oflens epithelial cell[J]. Chin J Exp Ophthalmol,2012,30(6):529-533.
[19]滕贺,黄亮瑜,田芳,等.衰老标记蛋白30高表达对紫外线诱导人晶状体上皮细胞凋亡的影响[J].中华眼科杂志,2017,53(11):835-841.Teng H,Huang LY,Tian F,et al. Effects of SMP-30 overexpression on apoptosis of human lensepithelial cells induced by ultraviolet B irradiation[J]. Chin J Ophthalmol,2017,53(11):835-841.
[20]Ishikawa Y,Hashizume K,Kishimoto S,et al.Effect of vitamin C depletion on UVR-B inducedcataract in SMP30/GNL knockout mice[J]. Exp EyeRes,2012,94(1):85-89.
[21]Sumerkina VA,Popov GK,Voronova LV. In vitrostudy of the role of lenticular epithelial calciumchannels and aquaporins in the development ofcataract[J]. Bull Exp Biol Med,2008,145(2):184-187.
[22]胡建章,徐国兴.自由基与白内障的关系研究进展[J].国外医学:眼科学分册,2003,27(1):45-49.Hu JZ,Xu GX. Progresses on relationship betweenfree radicals and cataract[J]. Foreign Med Sci(Section Ophthalmol),2003,27(1):45-49.
[23]鲍玉洲.晶状体与谷胱甘肽的研究进展[J].眼科研究,2000,18(6):571-573.Bao YZ. Current research on lens and glutathione[J]. Chin J Exp Ophthalmol,2000,18(6):571-573.
[24]Sanderson J,Marcantonio JM,Duncan G. A humanlens model of cortical cataract:Ca2+-inducedprotein loss,vimentin cleavage and opacification[J]. Invest Ophthalmol Vis Sci,2000,41(8):2255-2261.
[25]Matsushima H,Mukai K,Yoshida S,et al. Effectsof calcium on human lens epithelial cells in vitro[J]. Jpn J Ophthalmol,2004,48(2):97-100
[26]秦双立,官志忠.线粒体损伤和氧化应激的关系[J].国外医学(医学地理分册),2013,34(3):197-201.Qin SL, Guan ZZ. Correlation betweenmitochondrial injury and oxidative stress[J].Foreign Med Sci(Section Medgeogr),2013,34(3):197-201.
[27]Matrai J,Chuah MK,Van den Driessche T. Recentadvances in lentiviral vector development andapplications[J]. Mol Ther,2010,18(3):477-490.
[28]Nasri M, Karimi A, Allahbakhshian FM.Production,purification and titration of a lentivirus-based vector for gene delivery purposes[J].Cytotechnology,2014,66(6):1031-1038.
[29]Fujita T,Inoue H,Kitamura T,et al. Senescencemarker protein-30(SMP30)rescues cell death byenhancing plasma membrane Ca2+-pumping activityin Hep G2 cells[J]. Biochem Biophys ResCommun,1998,250(2):374-380.
[30]Son TG,Kim SJ,Kim K,et al. Cytoprotective rolesof senescence marker protein 30 against intracellularcalcium elevation and oxidative stress[J]. ArchPharm Res,2008,31(7):872-877.
[31]Ganea E,Harding JJ. Glutathione-related enzymesand the eye[J]. Curr Eye Res,2006,31(1):1-11.
[32]田黎明,彭圆,胡荣毅,等.衰老标记蛋白30对过氧化氢所致人皮肤成纤维细胞衰老表型的影响[J].新乡医学院学报,2016,33(6):452-456.Tian LM, Peng Y, Hu RY, et al. Effect ofsenescence marker protein 30 on senescentphenotypes in hydrogen peroxide-inducedsenescence human skin fibroblasts[J]. J XinxiangMed Coll,2016,33(6):452-456.
[2]姚瑶,徐国兴.晶状体蛋白与年龄相关性白内障研究进展[J].国际眼科杂志,2014,14(2):255-258.Yao Y,Xu GX. Advances of crystallins and age-related cataract[J]. Int Eye Sci,2014,14(2):255-258.
[3]唐雅婷,卢奕.年龄相关性白内障遗传流行病学研究进展[J].国际眼科纵览,2013,37(2):117-122.Tang YT,Lu Y. The genetic epidemiology of age-related cataract[J]. Int Rev Ophthalmol,2013,37(2):117-122.
[4] Wang S,Guo C,Yu M,et al. Identification of H2O2induced oxidative stress associated microRNAs inHLE-B3 cells and their clinical relevance to theprogression of age-related nuclear cataract[J].BMC Ophthalmol,2018,18(1):93.
[5] Gupta PD,Johar K,Vasavada A. Causative andpreventive action of calcium in cataracto-genesis[J]. Acta Pharmacol Sin,2004,25(10):1250-1256.
[6]杨洁,闾宏伟.线粒体钙单向转运体和线粒体应激关系的研究进展[J].生命科学研究,2016,20(2):178-182.Yang J,Lu HW. Progresses on relationship betweenmitochondrial calcium uniporter and mitochondrialoxidative stress[J]. Life Sci Res,2016,20(2):178-182.
[7] Scott SH,Bahnson BJ. Senescence marker protein30:functional and structural insights to its unknownphysiological function[J]. Biomol Concepts,2011,2(6):469-480.
[8] Li S,Chen X,Lai W,et al. Downregulation ofSMP30 in senescent human lens epithelial cells[J].Mol Med Rep,2017,16(4):4022-4028.
[9] Chen X,Li SM,Li YW,et al. Effect of senescencemarker protein 30 on the proliferation and apoptosisof human lens epithelial cells SRA01/04[J]. Int JOphthalmol,2018,11(4):553-558.
[10]Zhou D,Yin D,Xiao F,et al. Expressions ofsenescence-associatedβ-galactosidase andsenescence marker protein-30 are associated withlens epithelial cell apoptosis[J]. Med Sci Monit,2015,21:3728-3735.
[11]陈曦,韩子豪,李舒凝,等.高钙状态对人晶状体上皮细胞株SRA01/04氧化应激水平的影响[J].眼科新进展,2017,37(10):906-910.Chen X,Han ZH,Li SN,et al. Effects of calciumelevation on intracellular oxidative stress in humanlens epithelial cells SRA01/04[J]. Rec AdvOphthal,2017,37(10):906-910.
[12]Goo MJ, Park JK, Hong IH, et al. Increasedsusceptibility of radiation-induced intestinalapoptosis in SMP30 KO mice[J]. Int J Mol Sci,2013,14(6):11084-11095.
[13]Zhang SC,Liang MK,Huang GL,et al. Inhibitionof SMP30 gene expression influences the biologicalcharacteristics of human Hep G2 cells[J]. AsianPac J Cancer Prev,2014,15(3):1193-1196.
[14]Kadowaki S, Shishido T, Sasaki T, et al.Deficiency of senescence marker protein 30exacerbates cardiac injury after ischemia/reperfusion[J]. Int J Mol Sci,2016,17(4):542.
[15]Yamaguchi M. Regulatory role of regucalcin in heartcalcium signaling:insight into cardiac failure(Review)[J]. Biomed Rep,2014,2(3):303-308.
[16]Son YS,Ullah HMA,Elfadl AK,et al. Preventiveeffects of vitamin C on diethylnitrosamine-inducedhepatotoxicity in SMP30 knockout mice[J]. InVivo,2018,32(1):93-99.
[17]赖伟霞,谭少健,李霞,等.衰老标记蛋白30在不同年龄白内障患者晶状体上皮细胞中的表达变化[J].中华实验眼科杂志,2014,32(6):521-524.Lai WX, Tan SJ, Li X, et al. Comparison ofexpression levels of senescence marker protein 30 inlens epithelial cells among different ages of cataractpatients[J]. Chin J Exp Ophthalmol,2014,32(6):521-524.
[18]刘兰,蔡小军,余爱华,等.不同类型年龄相关性白内障晶状体上皮细胞衰老标记蛋白30的表达及与细胞凋亡的关系[J].中华实验眼科杂志,2012,30(6):529-533.Liu L,Cai XJ,Yu AH,et al. The expression ofsenescence marker protein-30 in different types ofage-related cataracts and its relation to apoptosis oflens epithelial cell[J]. Chin J Exp Ophthalmol,2012,30(6):529-533.
[19]滕贺,黄亮瑜,田芳,等.衰老标记蛋白30高表达对紫外线诱导人晶状体上皮细胞凋亡的影响[J].中华眼科杂志,2017,53(11):835-841.Teng H,Huang LY,Tian F,et al. Effects of SMP-30 overexpression on apoptosis of human lensepithelial cells induced by ultraviolet B irradiation[J]. Chin J Ophthalmol,2017,53(11):835-841.
[20]Ishikawa Y,Hashizume K,Kishimoto S,et al.Effect of vitamin C depletion on UVR-B inducedcataract in SMP30/GNL knockout mice[J]. Exp EyeRes,2012,94(1):85-89.
[21]Sumerkina VA,Popov GK,Voronova LV. In vitrostudy of the role of lenticular epithelial calciumchannels and aquaporins in the development ofcataract[J]. Bull Exp Biol Med,2008,145(2):184-187.
[22]胡建章,徐国兴.自由基与白内障的关系研究进展[J].国外医学:眼科学分册,2003,27(1):45-49.Hu JZ,Xu GX. Progresses on relationship betweenfree radicals and cataract[J]. Foreign Med Sci(Section Ophthalmol),2003,27(1):45-49.
[23]鲍玉洲.晶状体与谷胱甘肽的研究进展[J].眼科研究,2000,18(6):571-573.Bao YZ. Current research on lens and glutathione[J]. Chin J Exp Ophthalmol,2000,18(6):571-573.
[24]Sanderson J,Marcantonio JM,Duncan G. A humanlens model of cortical cataract:Ca2+-inducedprotein loss,vimentin cleavage and opacification[J]. Invest Ophthalmol Vis Sci,2000,41(8):2255-2261.
[25]Matsushima H,Mukai K,Yoshida S,et al. Effectsof calcium on human lens epithelial cells in vitro[J]. Jpn J Ophthalmol,2004,48(2):97-100
[26]秦双立,官志忠.线粒体损伤和氧化应激的关系[J].国外医学(医学地理分册),2013,34(3):197-201.Qin SL, Guan ZZ. Correlation betweenmitochondrial injury and oxidative stress[J].Foreign Med Sci(Section Medgeogr),2013,34(3):197-201.
[27]Matrai J,Chuah MK,Van den Driessche T. Recentadvances in lentiviral vector development andapplications[J]. Mol Ther,2010,18(3):477-490.
[28]Nasri M, Karimi A, Allahbakhshian FM.Production,purification and titration of a lentivirus-based vector for gene delivery purposes[J].Cytotechnology,2014,66(6):1031-1038.
[29]Fujita T,Inoue H,Kitamura T,et al. Senescencemarker protein-30(SMP30)rescues cell death byenhancing plasma membrane Ca2+-pumping activityin Hep G2 cells[J]. Biochem Biophys ResCommun,1998,250(2):374-380.
[30]Son TG,Kim SJ,Kim K,et al. Cytoprotective rolesof senescence marker protein 30 against intracellularcalcium elevation and oxidative stress[J]. ArchPharm Res,2008,31(7):872-877.
[31]Ganea E,Harding JJ. Glutathione-related enzymesand the eye[J]. Curr Eye Res,2006,31(1):1-11.
[32]田黎明,彭圆,胡荣毅,等.衰老标记蛋白30对过氧化氢所致人皮肤成纤维细胞衰老表型的影响[J].新乡医学院学报,2016,33(6):452-456.Tian LM, Peng Y, Hu RY, et al. Effect ofsenescence marker protein 30 on senescentphenotypes in hydrogen peroxide-inducedsenescence human skin fibroblasts[J]. J XinxiangMed Coll,2016,33(6):452-456.