原核pprⅠ基因活体转染对系统性红斑狼疮小鼠氧化应激的影响

Effect of oxidate stress on systemic lupus erythematosus mice by transfection of prokaryotic pprⅠ gene

目的:通过检测耐辐射奇球菌ppr I基因活体转染系统性红斑狼疮(SLE)小鼠的氧化应激和疾病活动性指标,探讨其对SLE小鼠氧化应激状态的影响。方法:62只纯品系B6. MRL-Faslpr NJU雌性小鼠随机分成3组,未转染组(20只),pEGFP-c1空载体转染组(20只),pEGFP-c1-pprⅠ转染组(22只),纯品系C57BL/6健康小鼠作为健康对照组(20只)。应用活体电转染技术将pEGFP-c1空载体及pEGFP-c1-pprⅠ基因重组质粒转入SLE小鼠股前肌肉。Western blot鉴定转染后SLE小鼠外周血pEGFP-c1和pEGFP-c1-pprⅠ融合基因表达。采用化学比色法检测各组小鼠外周血氧化应激标志物,ELISA法检测抗ds DNA抗体,免疫比浊法检测补体C4和C-反应蛋白。结果:(1)Western blot检测结果提示,质粒转染一周后均能在小鼠体内成功表达,且无毒副作用;(2)与未转染质粒和pEGFP-c1转染组SLE小鼠相比,pEGFP-c1-pprⅠ转染组SLE小鼠GSH含量显著升高(P<0. 01),丙二醛(MDA)、蛋白质羰基(Carbonyls)和氧化型谷胱甘肽(GSSG)含量显著降低(P<0. 01),超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性明显升高(P<0. 05),血浆抗ds DNA抗体滴度和CRP含量显著降低(P<0. 01),补体C4明显升高(P<0. 05),与健康组相比,各项指标差异无统计学意义(P>0. 05);(3)与未转染质粒和pEGFP-c1转染组SLE小鼠相比,pEGFPc1-ppr I转染组SLE小鼠淋巴细胞凋亡率和死亡率明显下降(P<0. 01),与健康小鼠相比,差异无统计学意义(P>0. 05)。结论:耐辐射奇球菌ppr I基因活体转染后能在SLE小鼠体内稳定表达且能调节体内的氧化应激状态,降低SLE小鼠的疾病活动度。

Objective:To explore the effect on oxidative stress state by detecting oxidative stress and disease activity index of Deinococcus radiodurans transfected systemic lupus erythematosus(SLE)mice.Methods:62 pure B6.MRL-Faslpr NJU female mice were randomly divided into three groups,the non transfected group,the pEGFP-c1 empty vector transfected and pEGFP-c1-pprI recombinant plasmid transfected group,the numbers of cases are 20,20 and 22 respectively.moreover,20 pure line C57BL/6 healthy mice were collected as the healthy control group.The pEGFP-c1 empty vector and recombinant plasmid pEGFP-c1-pprI were transferred into the anterior femur of SLE mice by the live electrotransfection technique.The oxidative stress markers in peripheral blood of each group mice were detected by chemical colorimetry.pEGFP-c1 and pEGFP-c1-pprI fusion gene expression in peripheral blood of SLE mice were identificated by Western blot.The anti-dsDNA antibody was detected by ELISA,and the complement C4 and C-reactive protein were detected by immunoturbidimetry.Results:①pEGFP-c1 and pEGFP-c1-pprI fusion gene expressed successfully in SLE mice identificated by Western blot,and had no toxic side effect.②Compared with non transfected and pEGFP-c1-pprI transfection group,plasma GSH level were significantly increased(P<0.01),MDA,GSSG and protein carbonyl content decreased significantly(P<0.01),SOD,CAT activity increased significantly(P<0.05),plasma anti-dsDNA antibody titers and the content of CRP decreased significantly(P<0.01),complement C4(P<0.05)increased significantly,compared with the healthy group,there was no statistical difference(P>0.05).③Compared with non transfected and pEGFP-c1-pprI transfection group SLE mice,the apoptosis rate and mortality of SLE mice lymphocytes in pEGFP-c1-pprI transfection group decreased significantly(P<0.01),and there was no significant difference compared with healthy mice(P>0.05).Conclusion:The Deinococcus radiodurans pprI gene can be stably expressed in SLE mice after being transfected,it can regulate the oxidative stress state in vivo and reduce the disease activity of SLE mice.