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目的探索脂多糖(LPS)调节树突状细胞(DC)的功能,促进T细胞免疫反应的机制。方法应用CDllc~+免疫磁珠分离小鼠脾脏DC。LPS处理DC后。流式细胞术检测DC表面的共刺激分子CD80和CD86的表达,ELISA检测DC培养上清中白细胞介素4(IL-4)、IL-5、IL-6、IL-12p40、IL-12p70、肿瘤坏死因子α(TNF-α)的水平,异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)双染色结合流式细胞术检测DC凋亡,Phos-flow技术检测核因子κB P65(NF-κB P65)磷酸化水平,实时定量PCR检测基因芯片变化差异明显基因的mRNA水平,DC经OVA323-329多肽处理后与和CD4~+T细胞共培养,流式细胞术检测CD4~+T细胞的增殖。结果利用CD11 c磁珠分离,可获得纯度为93%的DC,LPS可以上调DC表面CD80和CD86的表达,增强DC介导的CD4~+T细胞的增殖。并且LPS能促进促炎细胞因子IL-12 p40、TNF-α和IL-6的分泌,同时通过NF-κB通路抑制DC的凋亡。结论 LPS通过调节DC的存活和细胞因子的产生促进DC介导的CD4~+T细胞的增殖。
Objective To explore the mechanism of lipopolysaccharide (LPS) regulating the function of dendritic cells (DCs) and promoting T cell immune response. Methods CD11c ~ + immunomagnetic beads were used to isolate mouse spleen DCs. After LPS processing DC. The expression of costimulatory molecules CD80 and CD86 on DCs were detected by flow cytometry. The levels of IL-4, IL-5, IL-6, IL-12p40 and IL- The levels of tumor necrosis factor-α (TNF-α) and DC were detected by flow cytometry after double-stained with fluorescein isothiocyanate-labeled AnnexinⅤ-FITC / PI. Flow technique was used to detect the phosphorylation level of nuclear factor kappa B P65 (NF-κB P65). Real-time quantitative PCR was used to detect mRNA levels of genes that were significantly different in gene chips. DCs were treated with OVA323-329 peptide and co-cultured with CD4 ~ + T cells Cytometry was used to detect the proliferation of CD4 ~ + T cells. RESULTS: DCs with purity of 93% were obtained by CD11c magnetic beads separation. LPS up-regulated the expression of CD80 and CD86 on DC surface and enhanced the proliferation of DC-mediated CD4 ~ + T cells. And LPS can promote the secretion of pro-inflammatory cytokines IL-12 p40, TNF-α and IL-6, at the same time, inhibit the apoptosis of DC through NF-κB pathway. Conclusion LPS can promote the proliferation of DC-mediated CD4 ~ + T cells by regulating the survival of DCs and the production of cytokines.