【作者】 周华；

【导师】 唐晓文；

【作者基本信息】 苏州大学 ， 血液内科（专业学位）， 2014， 硕士

【摘要】 目的：研究JAK-STAT信号转导通路在IFN-α促进及加速单核细胞分化产生高度活化的、部分成熟的DC（IFN-DC）发生发展过程中所起的作用。方法：采集正常供体动员的外周造血干细胞（G-PBSC），Ficoll法分离获得单个核细胞，采用磁珠分离获得CD14+单核细胞，根据培养条件的不同分为三组，分别为CD14+单核细胞(对照组),CD14+单核细胞+800U/mlGM-CSF(GM-CSF组)和CD14+单核细胞+1000U/mlIFN-α2b+800U/mlGM-CSF(IFN-α2b+GM-CSF组)。进行如下实验：1.IFN-α2b+GM-CSF组和GM-CSF组分别于加入细胞因子0min、10min、30min、1h、2h、4h提取总蛋白，通过Westernblot实验，观察两组在不同时间点下相关信号蛋白（JAK1,p-JAK1,STAT1及p-STAT1）的表达情况；并检测IFN-α2b+GM-CSF组和GM-CSF组分别加入JAK-STAT信号通路阻断剂（AG490）后相关信号蛋白的表达高峰是否减弱；2.以CD14+单核细胞CD80、CD86及CD83的表达水平作为基线水平，分别检测IFN-α2b+GM-CSF组和GM-CSF组各培养三天后上述CD分子的表达水平，并检测在以上两组分别加入AG490各培养三天后上述CD分子的表达水平。结果：1.IFN-α2b+GM-CSF组培养三天后的IFN-DC表达DC较为特征性标志，包括CD11c、CD86、CD80、HLA-DR及CD209；CD83为DC的成熟标志之一，在IFN-DC上部分表达；同时在浆细胞样树突状细胞（pDC）高表达的CD123，在IFN-DC亦高表达；培养三天后的细胞形态上可见突起。2.Westernblot检测IFN-α2b+GM-CSF组在不同时间点相关蛋白表达显示：JAK1和STAT1磷酸化水平在30min表达达到高峰，后逐渐减弱。予AG490干预后，JAK1和STAT1磷酸化水平在30min表达减弱。同时检测IFN-α2b+GM-CSF组均加入AG490培养三天后CD80及CD86表达明显下降，CD83弱表达。IFN-α2b+GM-CSF组干预后CD分子表达下降均具有统计学显著性差异（P<0.05）。结论：1.IFN-α2b联合GM-CSF可诱导CD14+单核细胞分化为树突状细胞。2.JAK-STAT信号通路在IFN-DC的分化中起着重要的作用。更多还原

【Abstract】 Objective:To study the role of JAK/STAT signaling pathway in generation of high active partially mature dendritic cell from monocyte after a singe step of culture with IFN-a/GM-CSF.Methods: Peripheral blood mononuclear cells (PBMC) were obtained from heparinised blood of healthy donors by Ficoll density gradient centrifugation. Briefly, DCs were isolated from CD14+monocytes immunomagnetically. Three groups were divided according to different culture conditions. They were CD14+monocyte (Control group), CD14+monocyte+800U/ml GM-CSF (GM-CSF group) and CD14+monocyte+1000U/ml IFN-α2b+800U/ml GM-CSF+PBMC (IFN-α2b+GM-CSF group).1. After cultured for Omin,10min,30min,1h,2h,4h, the expressed of protein JAK1, p-JAK1, STAT1and p-STAT1in CD14+monocytes were detected by Western blot.2. Surface expression of CD80, CD86and CD83molecule in CD14+monocyte were detected as baseline value. Surface expression of CD80, CD86and CD83molecule were detected in cells acquired from GM-CSF group and IFN-α2b+GM-CSF group respectively after3days culture. After AG490added, the same molecules were detected from these two groups.Results:1. Typical DC lineage markers CDllc and CD209were highly expressed on IFN-DC. The expression HLA-DR of MHC molecules, co-stimulatory molecules (CD40, CD80and CD86) and CD123(surface marker of plasmacytiod DC) were also of moderate to high expression. The maturation marker CD83was partially expressed on IFN-DC. IFN-DC had a similar typical morphology of DC. IFN-DC was smaller, less granular and had a polarized distribution of cytoplasmic protrusions.2. Western Blot indicated p-JAK1and p-STAT1were increased quickly after cultured for10min, and p-JAK1, p-STAT1achieved the peak at30min in IFN-α2b+GM-CSF group, and then attenuated gradually. Western Blot indicated that after AG490inhibition, p-JAK1and p-STAT1level decreased. Reduced expression of CD80and CD86was found in IFN-α2b+GM-CSF group after AG490added, similar as CD83. The differences were statistically significant(P<0.05). Conclusion:1. IFN-DC diaplays a characteristic morphology and immunotype of DC with intermediate mature state.2. JAK-STAT pathway plays a key role in the differentiation of DC.更多还原