【摘要】 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。更多还原

【Abstract】 Objective To explore the mechanism modulating the function of dendritic cells( DCs) and promoting the T cell response by lipopolysaccharide( LPS). Methods Splenic DCs were purified with anti-CD11 c immunomagnetic beads. After DCs were stimulated with LPS,the expressions of co-stimulatory molecules CD80 and CD86 on the DCs were detected by flow cytometry. The protein levels of pro-inflammatory cytokines interleukin 4( IL-4),IL-5,IL-6,IL-12p40,IL-12p70 and tumor necrosis factor alpha( TNF-α) in the culture supernatant were measured by ELISA. The apoptotic levels of DCs which were labeled with annexinⅤ-FITC/PI were determined by flow cytometry. The phosphorylation level of nuclear factor κB P65( NF-κB P65) was assessed by phos-flow. The mRNA levels of variable genes in microarray were determined by real-time PCR. The proliferation of CD4+T cells which were co-cultured with OVA323-329-treated DCs was analyzed by flow cytometry. Results The purity of DCs reached over 93 % after isolation. LPS up-regulated the expressions of CD80 and CD86 and enhanced DCs-mediated proliferation of CD4+T cells. In addition,LPS increased the protein levels of IL-12 p40,TNF-α and IL-6,and inhibited the apoptosis of DCs through the NF-κB signaling pathway. Conclusion LPS could enhance DC-mediated proliferation of CD4+T cells by modulating the DCs survival and cytokine secretion.更多还原