论文部分内容阅读
目的:研究慢病毒介导siRNA靶向沉默Gata3基因表达对妊娠哮喘模型小鼠骨髓来源树突状细胞(DC)抗原递呈及其免疫生物学特性的影响。方法:以脂质体DNA沉淀法将重组慢病毒干扰质粒与包装质粒共转染AD293细胞,包装生成慢病毒感染体外诱导DC,RT-PCR、Western blot分析其对siGata3基因的整合转录效果;流式细胞术观察细胞表型变化。通过尾静脉注射将pSH1/siGata3转移至小鼠体内,ELISA法测定其肺泡灌洗液(BALF)中的炎性因子含量;MTT法验证混合淋巴细胞反应(MLR)后T细胞增殖能力。结果:经重组、包装携有Gata3特异siRNA慢病毒感染DC成功,其siRNA靶点Gata3 mRNA及蛋白的转录表达分别下调87.30%和81.33%(P<0.05);同时降低DCs膜表面CD86荧光强度(P<0.05)。减少pSH1/siGata3组小鼠BALF中IL-5、IL-17分泌而提升IL-12水平(P<0.05);MLR证实,共培养感染DC激活T细胞的能力明显不足(P<0.05)。结论:siRNA干扰可有效抑制模型小鼠免疫微环境特定Gata3通路,阻遏T细胞及其亚群分化由此来诱导DC免疫耐受,为妊娠期哮喘防治奠定基础并提供新的思路和手段。
OBJECTIVE: To study the effect of lentivirus-mediated targeted silencing Gata3 gene on the presentation of bone marrow-derived dendritic cells (DCs) and the immunological characteristics of asthmatic mice. METHODS: AD293 cells were co-transfected with the recombinant lentiviral interference plasmids and packaging plasmids by lipofection method. The recombinant plasmids were packaged and induced into DCs by lentivirus infection. RT-PCR and Western blotting were used to analyze the integration of siGata3 gene. Cytometry observed cell phenotype changes. PSH1 / siGata3 was transplanted into mice by tail vein injection, and the inflammatory cytokines in BALF were measured by ELISA. The proliferation of T lymphocytes after mixed lymphocyte reaction (MLR) was tested by MTT assay. Results: The recombinant adenovirus was successfully transfected into DCs carrying Gata3 specific siRNA lentivirus. The transcriptional targets of Gata3 mRNA and protein in siRNA target were down-regulated by 87.30% and 81.33%, respectively (P <0.05), and the CD86 fluorescence intensity P <0.05). The level of IL-12 in BALF secreted from pSH1 / siGata3 mice was decreased (P <0.05). MLR demonstrated that the ability of co-cultured DCs to activate T cells was significantly less (P <0.05). CONCLUSION: siRNA interference can effectively inhibit immune GABA-specific Gata3 pathway, repress T-cells and their sub-population differentiation in order to induce DC immune tolerance and lay a foundation for the prevention and treatment of asthma in pregnancy and provide new ideas and means.