【作者】 董薇；

【导师】 蔡美英；

【作者基本信息】 四川大学 ， 免疫学， 2004， 博士

【摘要】 [目的] 联合应用GM-CSF+IL-4从健康人外周血单核细胞中诱导DC，经LPS进一步诱导其成熟。通过不同途径鉴定两种Dc的成熟程度，为LPS促进DC的进一步成熟提供了实验依据。半定量RT-PCR检测TLRs mRNA在DC上的表达水平，激光共聚焦扫描显微镜检测NF-κ B在核内外的分布情况，从基因及分子水平探讨LPS促进DC成熟的可能机制。从HBV DNA阳性肝癌患者外周血单核细胞中诱导HCC DC，加入LPS促进其成熟，比较HCC DC与健康人DC在形态及功能的差异，分析HCC DC上TLRs mRNA的水平，提出了肝癌患者的TLRs信号传导通路缺失学说，为HCC的发病提供了新的解释。 [方法] 联合应用GM-CSF+IL-4从健康人外周血PBMC中诱导DC，在培养的第6天加入促成熟因子LPS，继续培养24h。收获细胞待检。分别采用倒置相差显微镜、扫描电镜、透射电镜观察DC的形态；流式细胞仪检测DC表面的分子标志；HRP吞噬实验测定DC的吞噬能力；MTT法检测DC刺激同种异体淋巴细胞增殖的能力；ELISA检测DC培养上清中IL-12、TNF-α以及MLR中IFN-γ的水平。通过形态以及功能等鉴定DC的成熟程度。流式细胞仪检测DC表面CD14的表达；半定量RT-PCR分析DC上TLR2、3、4mRNA的水平；激光共聚焦扫描显微镜检测NF-κ B在核内外的分布情况。从分子水平及基因水平探索LPS促进DC进一步成熟的机制。从HBV DNA阳性肝癌患者外周血中分离单核细胞，经联合应用GM-CSF+IL-4诱导DC，培养的第6天加入LPS，24h后收获细胞，并与健康人DC加以比较。采用前面所述的检测方法从形态、功能方面鉴定HCC DC在经LPS刺激前后的成熟程度；从分子、基因水平探索HCC DC中TLRs信号传导通路的情况；分析LPS经TLRs信号传导通路作用于HCC DC，对其进一步成熟有无促进作用。更多还原

【Abstract】 [Objective] Induce DC from PBMC of the healthy men with granulocyte monocyte colony stimulating factor(GM-CSF) and IL-4, LPS induce DC to be more mature. Identify the maturation degree of DC through different approaches. Provide experimental evidence for LPS inducing the maturation of DC. Detect the TLRs mRNA expression in DC by semi-quantitative RT-PCR. Detect the distribution of NF- κB in the nuclear. Study the mechanism of LPS inducing mature DC from gene and molecular level. Induce HCC DC from PBMC of HBV DNA- positive HCC patients, adding LPS to induce it to be mature. Compare the difference of HCC DC and normal DC from the morphology and function. Analyze the level of TLRs mRNA on HCC DC. Put forward the theory of TLRs signaling transduction pathway defection of HCC patients. Offer new explanation for hepatocellular carcinoma pathogenesis.[Methods] Induce DC from PBMC of the healthy men with granulocyte monocyte colony stimulating factor(GM-CSF) and IL-4. Adding LPS on the day 6 of culture, cultivate for another 24h. Harvest the cells to examine. To observe the morphology of DC by inverted phase contrast microscope, electron microscope and laser scanning confocal microscope; Analyze the phenotype of DC with flow cytometry; Investigate the endocytosis ability of DC as a group by Horseradish peroxidase endocytosis assay; Appraise the allogeneic mixed lymphocytes reaction of DC by MTT reduction assay; Analyze the levels of IL-12、TNF-α in the culture medium of DC and IFN-γ in MLR by ELISA; Decide the maturation degree of DC from the更多还原