Si-GATA-3对AR小鼠PBMCs中Th1/Th2细胞亚群功能的体外调节

In Vitro Regulation of Th1/Th2 Cell of PBMCs in AR Mice by si-GATA-3

目的研究RNAi介导的GATA-3基因慢病毒载体si-GATA-3在体外对变应性鼻炎(allergic rhinitis,AR)小鼠PBMCs中Th2、Th1细胞亚群功能的调节作用。方法构建RNAi介导的GATA-3基因慢病毒载体siGATA-3,并进行包装、纯化和滴定。BALB/C小鼠随机分为AR组和正常对照组。用卵蛋白构建BALB/C小鼠变应性鼻炎模型,抽取其外周血并分离外周血中单个核细胞(PBMCs),随机分为3组,即si-GATA-3组、siNC组和AR组。进行细胞培养,然后分别用si-GATA-3、si-NC和生理盐水对3组变应性鼻炎小鼠的PBMCs进行干预72 h,用生理盐水代替si-GATA-3干预正常对照组小鼠的PBMCs。干预结束分离PBMCs和细胞培养上清液。分别用荧光定量qPCR和Western bloting方法检测PBMCs中GATA-3 mRNA、T-bet mRNA和GATA3、T-bet蛋白的相对表达量;用ELISA技术检测细胞培养上清液中IL-4和IFN-γ的含量。结果构建出RNAi介导的GATA-3基因慢病毒载体si-GATA-3,其滴度为5×10^(8) TU/mL。制备出BALB/C小鼠的AR模型。Si-GATA-3组PBMCs中GATA-3 mRNA、GATA-3蛋白的相对表达量和上清液中IL-4的含量均明显低于AR组和si-NC组的表达量(P<0.01),AR组和si-NC组中3者的表达量均明显高于正常对照组(P<0.01);AR组3者表达量和si-NC组无差异(P>0.05)。si-GATA-3组PBMCs中T-bet mRNA及其蛋白的相对表达量明显高于AR组和si-NC组的表达量(P<0.01),AR组和si-NC组中二者的表达量均明显低于对照组(P<0.01);AR组的二者表达量和si-NC组无差异(P>0.05)。但si-GATA-3组细胞培养上清液中IFN-γ的含量高于正常对照组(P<0.05),AR组与si-NC组中IFN-γ明显高于正常对照组和si-GATA-3组(P<0.01);AR组与si-NC组之间IFN-γ的表达,差异无统计学意义(P>0.05)。结论si-GATA-3能有效的下调Th2细胞中GATA-3 mRNA、GATA-3蛋白和IL-4的表达,并上调Th1细胞中T-bet mRNA和T-bet蛋白的表达,体外能有效地纠正小鼠AR模型中PBMCs中Th2/Th1的免疫失衡。

Objective To investigate regulatory effect of the Lentivirus-mediated-GATA-3 RNAi(siGATA-3)on the Th1/Th2 imbalance in a murine model of allergic rhinitis in vitro.Methods The lentiviral vector SI-GATA-3 with GATA-3 gene mediated by RNAi was developed and packaged,purified and titrated.BALB/C mice were randomly divided into AR group and normal control group.The allergic rhinitis model of BALB/C mice was developed by using ovalbumin(OVA).The peripheral blood of BALB/C mice was collected and their mononuclear cells(PBMCs)were isolated.The PBMCs were randomly divided into three groups,namely,si-GATA-3 group,si-NC group and AR group.si-GATA-3,si-NC and normal saline were used to intervene PBMCs in allergic rhinitis mice for 72 h,and normal saline was used to replace si-GATA-3 to intervene PBMCs in normal control mice.PBMCs and cell culture supernatant were separated after the intervention.The relative expression levels of GATA-3 mRNA,T-bet mRNA and GATA-3,T-bet protein in PBMCs were detected by fluorescence quantitative qPCR and Western bloting,respectively.The contents of IL-4 and IFN-γin supernatant of cell culture were determined by ELISA.Results The RNAi mediated lentiviral vector si-GATA-3 with a titer of 5×10^(8) TU/mL was successfully deeloped.The allergic rhinitis mice models were established successfully.The relative expression levels of GATA-3 mRNA and GATA-3 protein and the content of IL-4 in supernatant of PBMCs in si-GATA-3 group were significantly lower than those in AR group and si-NC group(P<0.01),and the expression levels of GATA-3 in AR group and si-NC group were significantly higher than those in normal control group(P<0.01).There was no difference between AR group and si-NC group(P>0.05).The relative expression of T-bet mRNA and T-bet protein in si-GATA-3 group was significantly higher than that in AR group and si-NC group(P<0.01),and the expression of T-bet mRNA and T-bet protein in AR group and si-NC group was significantly lower than that in control group(P<0.01).There was no difference between AR group and si-NC group(P>0.05).However,the content of IFN-γin cell culture supernatant of si-GATA-3 group was higher than that of normal control group(P<0.05),and the content of IFN-γin AR and si-NC groups was significantly higher than that of normal control group and si-GATA-3 group(P<0.01).There was no significant difference in IFN-γexpression between AR group and si-NC group(P>0.05).Conclusions si-GATA-3 could down-regulate the relative expression of GATA-3 mRNA,GATA-3 protein and IL-4 level in Th2 cells,and up-regulate the relative expression of T-bet mRNA and T-bet protein in Th1 cells.Therefor,it can effectively correct the immune imbalance of Th2/Th1 in PBMCs in mouse AR model.