IL-4与SDF-1α协同激活ZAP-70激酶信号途径诱导Th2极化

发布时间：2018-05-03 06:05

  本文选题：IL-4 + SDF-1α/CXCL12　； 参考：《安徽医科大学》2005年硕士论文

【摘要】：目的:通过探讨IL-4和基质细胞源因子(SDF)-1α/CXCL12协同刺激下,Naive Th细胞Th1型、Th2型细胞因子的表达以及信号转导通路中关键分子的活化情况,判断协同刺激诱导的Th细胞极化方向,以及其信号转导通路可能的分子机制。 方法:使用流式细胞术和RT-PCR检测IL-4和SDF-1α协同刺激的脐带血fCB)CD4~+T细胞中Th1型/Th2型细胞因子的mRNA和胞内蛋白两个水平的表达情况;利用免疫复合物激酶分析法和免疫印记法,检测协同诱导Th2极化过程中Syk激酶和ZAP-70激酶的磷酸化情况以及Cb1家族和NFAT家族活化的情况;使用ZAP-70PNA技术阻断ZAP-70的表达,探讨ZAP-70激酶在协同刺激后Th细胞极化过程中的重要意义;使用EMSA技术进一步探讨NFAT转录因子家族成员的活化情况,明确参与细胞因子转录的关键转录因子。 结果:流式检测结果:用IL-4和SDF-1α/CXCL12刺激8天,CB CD4+T细胞(Naive Th)转换成Th2细胞模式(90.3% IL-4阳性);RT-PCR检测结果:新鲜分离的CBCD4+T细胞中的IFN-γ或者IL-4 mRNA大约7.3×10~2或者2.1×10~3拷贝,相应的,用IL-4+SDF-1α/CXCL12刺激的细胞中的IL-4 mRNA大约有1.3×10~4拷贝,IFN-γ mRNA约7.8×10~1拷贝;免疫复合物激酶分析法和免疫印记检测结果:IL-4和SDF-1α/CXCL12协同刺激的8天内,ZAP-70激酶和Cbl-b蛋白发生显著而稳
[Abstract]:Aim: to investigate the expression of Th1 type Th2 cytokines and the activation of key molecules in signal transduction pathway of naive Th cells after co-stimulation of IL-4 and stromal cell source factor SDF- 1 伪 / CXCL12, and to determine the polarization direction of Th cells induced by co-stimulation. And the possible molecular mechanism of its signal transduction pathway. Methods: flow cytometry and RT-PCR were used to detect the expression of mRNA and intracellular protein of Th1 type Th2 cytokines in IL-4 and SDF-1 伪 co-stimulated cord blood fCBC CD4T cells, and immunocomplex kinase assay and imprinting method were used to detect the expression of Th1 type Th2 cytokines. The phosphorylation of Syk kinase and ZAP-70 kinase and the activation of Cb1 family and NFAT family during co-induced Th2 polarization were detected, and the expression of ZAP-70 was blocked by ZAP-70PNA technique to explore the significance of ZAP-70 kinase in the process of co-stimulated Th cell polarization. The activation of family members of NFAT transcription factors was further investigated by EMSA technique, and the key transcription factors involved in cytokine transcription were identified. Results: IL-4 and SDF-1 伪 / CXCL12 were used to stimulate the transformation of CB CD4 T cells into Th2 cell model (90.3% IL-4 positive RT-PCR): IFN- 纬 or IL-4 mRNA was about 7.3 脳 10 ~ (-2) or 2.1 脳 10 ~ (-3) copies of CBCD4 T cells. There were about 1.3 脳 10 ~ (4) copies of IL-4 mRNA in cells stimulated with IL-4 SDF-1 伪 / CXCL12 and about 7.8 脳 10 ~ (-1) copy of IFN- 纬 mRNA. The results of immuno-complex kinase assay and immunological imprinting showed that ZAP-70 kinase and Cbl-b protein were remarkably stable in 8 days after co-stimulation of SDF-1 伪 -IL-4 and SDF-1 伪 / CXCL12.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
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本文编号：1837288