In Vitro Regulation of Th1/Th2 Cell of PBMCs in AR Mice by si-GATA-3
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摘要:
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si-GATA-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、si-NC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GATA-3 、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5×108 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。 Abstract:Objective To investigate regulatory effect of the Lentivirus-mediated-GATA-3 RNAi （si-GATA-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-γ in supernatant of cell culture were determined by ELISA. Results The RNAi mediated lentiviral vector si-GATA-3 with a titer of 5×108 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-γ in cell culture supernatant of si-GATA-3 group was higher than that of normal control group （ P < 0.05）, and the content of IFN-γ in 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-γ expression 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. -
Key words:
- Rhinitis /
- Allergic /
- RNA interference /
- GATA-3 /
- T-bet /
- Lentiviral vector /
- Model /
- Mouse
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图 1 病黏膜病理（HE）。AR组小鼠鼻黏膜中嗜酸性粒细胞较正常组小鼠增多（×200）
A ：正常组；B：AR组。
Figure 1. Nasal mucosa pathology in normal mice and allergic rhinitis mice （×200）
图 2 si-GATA-3感染AR小鼠外周血PBMCs效率的检测
A： si-GATA-3感染AR小鼠外周血PBMCs的倒置荧光显微镜图像（×100） ；B： LV3NC的阴性对照图像（×100） ；C： qPCR检查测si-GATA-3的转染效率。***P < 0.01。
Figure 2. Detection of transfecting effeciency of si-GATA-3 for PBMCs in allergic rhinitis mouse
图 3 si-GATA-3对AR小鼠外周血PBMCs中GATA-3 mRNA和T-bet mRNA表达的调控
A ： GATA-3 mRNA相对表达量；B ：T-bet mRNA相对表达量。与对照组比较，***P < 0.01；与si-NC组比较，###P < 0.01。
Figure 3. Regulation of si-GATA-3 on relative expressions of GATA-3 and T-bet mRNA in PBMCs of allergic rhinitis mouse
图 4 si-GATA-3对AR小鼠外周血PBMCs中GATA-3和T-bet蛋白表达的调控
A： Western blotting 灰度图像显示：B ： GATA-3蛋白相对表达量；C ：T-bet 蛋白的相对表达量。与对照组比较，*P < 0.05；与si-NC组比较，**P < 0.05。
Figure 4. Regulation of si-GATA-3 on Relative expressions of GATA-3 and T-bet protein in PBMCs of allergic rhinitis mouse
图 5 si-GATA对AR小鼠外周血PBMCs培养上清液液中IL-4和IFN-γ的调控
A ：上清液中IL-4含量；B 上清液中IFN-γ的含量。与对照组比较，**P < 0.01；与si-NC组中IFN-γ含量的比较，##P < 0.01；与si-NC组中IL-4含量的比较，###P < 0.01。
Figure 5. Regulation of si-GATA-3 on Relative expressions of IL-4 and IFN-γ in supernatant of the cultured PBMCs of allergic rhinits mouse
表 1 AR小鼠症状评分标准
Table 1. Symptom scoring criteria for AR mice
分级积分 喷嚏（个） 鼻分泌物量 挠鼻次数 0 0 无 无 1 1～3 前鼻孔有鼻分泌物 轻微挠鼻几次 2 4～10 鼻分泌物超过前鼻孔 双爪反复挠鼻 3 > 11 面部布满鼻分泌物 挠鼻面部不止，四处摩擦 
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表 2 GATA-3和T-bet mRNA引物
Table 2. The primer of GATA-3 and T-bet mRNA
引物 序列 大小 GATA-3 mRNA F：CCACGGGAGCCAGGTATG 169 bp R：CGGAGGGTAAACGGACAGAG T-bet mRNA F：GGACCCAACTGTCAACTGC 490 bp R：TGTCGCCACTGGAAGGAT β-actin mRNA F：GAGACCTTCAACACCCCAGC 263 bp R：ATGTCACGCACGATTTCCC
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