CAJ | 학술논문

目的观察高表达的β联蛋白对体外过氧化氢(H2O2)所致正常人皮肤成纤维细胞(NHSF)衰老表型的影响.方法 NHSF分别转染pcDNA3.1-β联蛋白或空载体pcDNA3.1,然后150 μmol/L H2O2处理2h.实验分为转染H2O2处理组(NHSF+β联蛋白+H2O2)、转空载体H2O2处理组(NHSF+空载体+H2O2)以及转空载体组(NHSF+空载体).用RT-PCR和Western印迹分析β联蛋白的表达,显微镜观察细胞形态,试剂盒检测衰老相关的β半乳糖苷酶(SA-β-gal)、细胞活性氧(ROS)以及超氧化物歧化酶(SOD)的活性.所有数据均用SPSS 13.0软件进行统计分析,多组间比较采用ANOVA检验.结果 pcDNA3.1-β联蛋白明显上调了NHSF中β联蛋白的表达.NHSF+空载体+H202组β联蛋白mRNA和蛋白水平(与GAPDHmRNA和蛋白的比值分别为0.2900±0.0195和0.3130±0.0171)明显低于NHSF+空载体组(分别为0.5963±0.0400和0.6190±0.0090),两组比较,均P＜0.05;NHSF+β联蛋白+H2O2组β联蛋白表达水平(0.7953±0.0074)高于NHSF+空载体+H2O2组(P＜0.05).SA-β-gal染色率在NHSF+空载体组、NHSF+空载体+ H2O2组、NHSF+β联蛋白+H2O2组分别为(2.9667±0.2517)％、(37.70±0.9539)％、(29.330±0.6359)％,各组比较,差异有统计学意义(P＜0.05).NHSF+空载体、NHSF+空载体+H2O2、NHSF+β联蛋白+ H2O2组ROS活性分别为(50.9963±9.2688)％、(109.9190±11.5215)％、(75.1063±3.0138)％,SOD水平分别为(88.0856±3.9181)、(35.5585±3.4438)、(61.7029±3.1716) U/mg,各组比较,差异均有统计学意义(P＜0.05).结论高表达的β联蛋白能够抑制细胞衰老相关β-半乳糖甘酶(SA-β-gal)和细胞活性氧(ROS)的表达,同时上调了SOD的活性.
목적 관찰고표체적β련단백대체외과양화경(H2O2)소치정상인피부성섬유세포(NHSF)쇠로표형적영향.방법 NHSF분별전염pcDNA3.1-β련단백혹공재체pcDNA3.1,연후150 μmol/L H2O2처리2h.실험분위전염H2O2처리조(NHSF+β련단백+H2O2)、전공재체H2O2처리조(NHSF+공재체+H2O2)이급전공재체조(NHSF+공재체).용RT-PCR화Western인적분석β련단백적표체,현미경관찰세포형태,시제합검측쇠로상관적β반유당감매(SA-β-gal)、세포활성양(ROS)이급초양화물기화매(SOD)적활성.소유수거균용SPSS 13.0연건진행통계분석,다조간비교채용ANOVA검험.결과 pcDNA3.1-β련단백명현상조료NHSF중β련단백적표체.NHSF+공재체+H202조β련단백mRNA화단백수평(여GAPDHmRNA화단백적비치분별위0.2900±0.0195화0.3130±0.0171)명현저우NHSF+공재체조(분별위0.5963±0.0400화0.6190±0.0090),량조비교,균P＜0.05;NHSF+β련단백+H2O2조β련단백표체수평(0.7953±0.0074)고우NHSF+공재체+H2O2조(P＜0.05).SA-β-gal염색솔재NHSF+공재체조、NHSF+공재체+ H2O2조、NHSF+β련단백+H2O2조분별위(2.9667±0.2517)％、(37.70±0.9539)％、(29.330±0.6359)％,각조비교,차이유통계학의의(P＜0.05).NHSF+공재체、NHSF+공재체+H2O2、NHSF+β련단백+ H2O2조ROS활성분별위(50.9963±9.2688)％、(109.9190±11.5215)％、(75.1063±3.0138)％,SOD수평분별위(88.0856±3.9181)、(35.5585±3.4438)、(61.7029±3.1716) U/mg,각조비교,차이균유통계학의의(P＜0.05).결론 고표체적β련단백능구억제세포쇠로상관β-반유당감매(SA-β-gal)화세포활성양(ROS)적표체,동시상조료SOD적활성.
Objective To observe the effect of high expression of β-catenin on senescent phenotypes in normal human skin fibroblasts (NHSFs) induced by hydrogen peroxide (H2O2).Methods Cultured NHSFs were classified into three groups: β-catenin + H2O2 group transfected with a recombinant plasmid pcDNA3.1-β-catenin and treated with H2O2 of 150 μ mol/L for two hours,H2O2 group transfected with the empty vector pcDNA3.1 and treated by H2O2 of 150 μmol/L for two hours,and vector group transfected with the empty vector pcDNA3.1 and receiving no treatment.Reverse transcription (RT)-PCR and Western blot were performed to quantify the mRNA and protein expressions of β-catenin in these cells,microscopy to observe the morphological changes of cells.The activity of senescence-associated β-galactosidase (SA-β-Gal) and superoxide dismutase (SOD) as well as the level of reactive oxygen species (ROS) were detected by using commercial kits.Data were processed with the software SPSS 13.0,and analysis of variance (ANOVA) was conducted for multiple group comparisons.Results The expression of β-catenin was significantly upregulated in NHSFs transfected with the recombinant plasmid pcDNA3.1-β-catenin.Both the mRNA and protein expression levels of β-catenin described as β-catenin/ glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio were significantly lower in the H2O2 group compared with the vector group (0.2900 ± 0.0195 vs.0.5963 ± 0.0400,0.3130 ± 0.0171 vs.0.6190 ± 0.0090,both P ＜0.05),while the protein expression level of β-catenin was statistically higher in the β-catenin + H2O2 group than in the H2O2 group (0.7953 ± 0.0074 vs.0.3130 ± 0.0171,P ＜0.05).Significant differences were observed between the vector group,H2O2 group and β-catenin+ H2O2 group in the percentage of SA-β-gal-positive cells ((2.9667 ± 0.2517)％ vs.(37.70 ± 0.9539)％ vs.(29.330 ± 0.6359)％,P ＜0.05),ROS activity ((50.9963 ±9.2688)％ vs.(109.9190 ± 11.5215)％ vs.(75.1063 ± 3.0138)％,P ＜0.05),and SOD levels ((88.0856 ±3.9181) vs.(35.5585 ± 3.4438) vs.(61.7029 ± 3.1716) U/mg,P ＜0.05).Conclusion The overexpression of β-catenin can downr_egulate the activity of SA-β-Gal and ROS level,but enhance the activity of SOD.