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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[1] Bousquet J,Anto J M,Bachert C,et al. Allergic rhinitis[J]. Nat Rev Dis Primers,2020,6(1):95. doi: 10.1038/s41572-020-00227-0 [2] Pallasaho P,Juusela M,Lindqvist A,et al. Allergic rhinoconjunctivitis doubles the risk for incident asthma-results from a population study in Helsinki,Finland[J]. Respir Med,2011,105(10):1449-1456. doi: 10.1016/j.rmed.2011.04.013 [3] Brozek J L,Bousquet J,Baena Cagnani C E,et al. Allergic rhinitis and ita impact on asthma (ARIA) guidelines:2010 revision[J]. J Allergic Clin Immunol,2010,126(3):466-476. doi: 10.1016/j.jaci.2010.06.047 [4] Wang X D,Zheng M,Lou H F,et al. An increased prevalence of self-reported allergic rhinitis in major chineae cities from 2005 to 2011[J]. Allergy,2016,71(8):1170-1180. doi: 10.1111/all.12874 [5] 刘阳,刘争. Ⅱ型固有淋巴细胞作用、调节及在慢性上呼吸道炎性疾病中的研究[J]. 中华耳鼻咽喉头颈外科杂志,2017,52(2):130-135. doi: 10.3760/cma.j.issn.1673-0860.2017.02.017 [6] Yao Y,Wang Z C,Wang N,et al. Allergen immunotherapy improves defective follicular T cells in patiens with allergic rhintis[J]. J Allergy Clin Immunol,2019,144(1):118-128. doi: 10.1016/j.jaci.2019.02.008 [7] Yao Y,Chen C L,Wang N,et al. Correlation of allergen-specific T follicular helper cell counts with specific IgE levels and efficacy of allergen immunotherapy[J]. J Allergy Clin Immunol,2018,142(1):321-324. doi: 10.1016/j.jaci.2018.03.008 [8] Jutel M,Akdis C A. T-cell subset regulation in atopy[J]. Curr Allergy Asthma Rep,2011,11(2):139-145. doi: 10.1007/s11882-011-0178-7 [9] Yagi R,Zhu J,Paul W E. An updated view on transcription factor GATA3-mediated regulation of Th1 and Th2 cell differentiation[J]. Int Immunol,2011,23(7):415-420. doi: 10.1093/intimm/dxr029 [10] Naito T,Tanaka H,Naoe Y,et al. Transcriptional control of T-cell development[J]. Int Immunol,2011,23(11):661-668. doi: 10.1093/intimm/dxr078 [11] Finotto S,Neurath M F,Glickman J N,et al. Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet[J]. Science,2002,295(5553):336-338. doi: 10.1126/science.1065544 [12] Zeng W P. All things considered':Transcriptional regulation of T helper type 2 cell differentiation from precursor to effector activation[J]. Immunology,2013,140(1):31-38. doi: 10.1111/imm.12121 [13] 陈冬,李玉晓,李添应,等. 大蒜新素对变应性鼻炎大鼠转录因子T-bet和GATA-3表达的影响[J]. 中华耳鼻咽喉头颈外科杂志,2010,45(2):133-138. doi: 10.3760/cma.j.issn.1673-0860.2010.02.011 [14] Son H L,Park H R,Park Y J,et al. Effect of retinoic acid in a mouse model of allergic rhinitis[J]. Allergy Asthma Immunol Res,2015,7(6):590-598. doi: 10.4168/aair.2015.7.6.590 [15] Zhang Y,Feng J,Sun J,et al. H2-Eb1 expression is upregulated in the nasal mucosa of allergic rhinitis[J]. Asian Pac J Allergy Immunol,2014,32(4):308-315. [16] Laurent S,Boutry S,Mahieu I,et al. Iron oxide based MR contrast agents:From chemistry to cell labeling[J]. Curr Med Chem,2009,16(35):4712-4727. doi: 10.2174/092986709789878256 [17] 李玉晓,何晓光,姚永刚,等. 小鼠GATA-3基因RNA干扰慢病毒载体的构建与鉴别[J]. 临床耳鼻咽喉头颈外科杂志,2017,38(8):627-632. doi: 10.13201/j.issn.1001-1781.2017.08.014 [18] 李林格,冯娟,胡斌,等. 实验变应性鼻炎模型小鼠构建以及与Th1/Th2失衡的相关性[J]. 中国组织工程研究,2015,19(40):6515-6519. doi: 10.3969/j.issn.2095-4344.2015.40.022 [19] Raby B A,Hwang E S,Van Steen K,et al. T-bet polymorphisms are associated with asthma and airway hyperresponsiveness[J]. Am J Respir Crit Care Med,2006,173(1):64-70. doi: 10.1164/rccm.200503-505OC [20] Zhu J,Yamane H,Cote-Sierra J,et al. GATA-3 promotes Th2 responses through three different mechanisms:Induction of Th2 cytokine production,selective growth of Th2 cells and inhibition of Th1 cell-specific factors[J]. Cell Res,2006,16(1):3-10. doi: 10.1038/sj.cr.7310002 [21] Malmhall C,Bossios A,Pullerits T,et al. Effects of pollen and nasal glucocorticoid on FOXP3+,GATA-3+ and T-bet+ cells in allergic rhinitis[J]. Allergy,2007,62(9):1007-1013. doi: 10.1111/j.1398-9995.2007.01420.x [22] Finotto S,De Sanctis G T,Lehr HA,et al. Treatment of allergic airway inflammation and hyperresponsiveness by antisense-induced local blockade of GATA-3 expression[J]. J EXP Med,2001,193(11):1247-1260. doi: 10.1084/jem.193.11.1247 [23] Huang H Y,Lee C C,Chiang B L,et al. Small interfering RNA against interleukin-5 decreases airway eosinophilia and hyperresponsiveness[J]. Gene Therapy,2008,15(9):660-667. doi: 10.1038/gt.2008.15 [24] Holgate S T,Polosa R. Treatment strategies for allergy and asthma[J]. Nat Rev Immunol,2008,8(3):218-230. doi: 10.1038/nri2262 [25] Behera A K, Kumar M, Lockey R F, et al. Adenovirus-mediated interferon gamma gene therapy for allergic asthma: involvement of interleukin12 and STAT4 signaling[J].Hum Gene Ther, 2002, 13（14）: 1697-1709. [26] Yanyun Luo,Yuqin Deng,Zezhang Tao,et al. Regulatory effect of microRNA-135a on the Th1/Th2 imbalance in a murine model of allergic rhinitis[J]. Experimental and Therapeutic Medicin,2014,8(4):1105-1110. doi: 10.3892/etm.2014.1855 [27] Zhu X Y,Wang X P,W ang Y,et al. The regulatory network among CircHIPK3,LncGAS5,and miR-495 promotes Th2 differentiation in allergic rhinitis[J]. Cell Death Dis,2020,11(4):216-228. doi: 10.1038/s41419-020-2394-3 [28] Koh D M,Brown G,Collins D J. Nanoparticles in rectal cancer imaging[J]. Cancer Biomark,2009,5(2):89-98. doi: 10.3233/CBM-2009-0544 -
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