An experimental investigation on Influence of APE1 overexpression on immune function of CD4+ CD25+regulatory T cells
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摘要: 目的 通过构建脱嘌呤/脱嘧啶核酸内切酶1(APE1)过表达CD4+CD25+调节性T细胞细胞模型,研究在PGE2体外刺激条件下,过表达APE1对CD4+CD25+调节性T细胞免疫功能的影响。 方法 选取BALB/C小鼠,分离培养CD4+CD25+调节性T细胞。构建APE1过表达慢病毒载体,并转染至CD4+CD25+调节性T细胞,建立APE1过表达CD4+CD25+调节性T细胞细胞模型,作为实验组;将空载体慢病毒载体转染至等量CD4+CD25+调节性T细胞,作为对照组。利用外源性PGE2分别与2组细胞共培养,检测2组细胞IL-10、TGF-β、APE1 mRNA和蛋白水平,并对结果进行t检验统计分析,P<0.05为差异具有统计学意义。 结果 成功制备APE1基因慢病毒载体,对分离培养的CD4+CD25+调节性T细胞进行APE1基因慢病毒载体转染。实验组细胞TGF-β、IL-10表达量(1.17±0.42,1.02±0.12)明显低于对照组(2.69±0.21,2.25±0.31),差异均具有统计学意义;实验组APE1 mRNA的相对表达量(6.57±0.49)明显高于对照组细胞(2.24±0.23),差异具有统计学意义;APE1蛋白表达量也呈同样结果。 结论 在外源性PGE2刺激下,当CD4+CD25+调节性T细胞过表达APE1时,其分泌的细胞因子即IL-10、TGF-β下调,可能导致其免疫抑制作用的下降。Abstract: Objective To research the impact of APE1 overexpression on the immune function of CD4+CD25+Treg cells with the stimulation of PGE2 in vitro. Methods We collected BALB/C mice,isolated and cultured CD4+CD25+Treg cells,constructed APE1 overexpression lentivirus vector and transfected into CD4+CD25+Treg cells,establish APE1 overexpression of CD4+ CD25+Treg cells,as the experimental group cells;the empty vector lentiviral vectors were transfected into the same amount of CD4+CD25+Treg cells,as the control group.In vitro,two groups were stimulated by PEG2 for 72 h,IL-10,TGF-βand APE1 mRNA levels were tested,the results were analyzed using t-test,P<0.05 was considered statistically significant. Results The APE1 gene lentiviral vector was constructed,APE1 gene lentiviral vector transfected the CD4+CD25+Treg successfully.After stimulation with PGE2 to transfected APE1 overexpressed CD4+CD25+Treg and empty vector transfected CD4+CD25+Treg in vitro,The levels of TGF-β and IL-10 (1.17±0.42,1.02±0.12) were significantly lower in transfected APE1 overexpressed CD4+CD25+Treg group as compared with the other group of cells(2.69±0.21,2.25±0.31),all with statistical significance;APE1 mRNA relative expression was significantly higher in APE1 overexpressed CD4+CD25+Treg(6.57±0.49) than the other group(2.24±0.23),with significant difference;APE1 protein expression also showed the same results. Conclusion PGE2 stimulation can result in the expression of APE1 of CD4+CD25+Treg cells,and decline the immunosuppression function of the CD4+CD25+Treg,which may be related to the downregulation of IL-10 and TGF-β.
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