IL-11-mTORC1-STAT3信号轴调节M-MDSC的分化发育

发布时间：2018-01-05 13:37

本文关键词：IL-11-mTORC1-STAT3信号轴调节M-MDSC的分化发育　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

更多相关文章： IL-11 MDSC mTORC1 STAT3

【摘要】：目的:我们前期在脓毒血症的临床和实验动物模型研究中发现IL-11能明显改善脓毒症患者临床症状,缩短康复时间和延长脓毒症小鼠的生存时间。动物实验证明在肝脏等器官中髓系来源的免疫抑制性细胞(myeloid derived suppressive cells,MDSC)数量明显增加,因此我们推测IL-11可能具有诱导MDSC分化发育的能力,在脓毒症早期具有炎症抑制作用,故而能防止脓毒症的进一步发展,从而缩短患者康复时间和延迟动物死亡时间。IL-11和IL-6具有类似的信号转导途径。已有文献报道IL-6能诱导MDSC的分化,且发现STAT3参与其信号转导,但是详细机制尚未阐明。我们通过生物信息学分析发现STAT3分子中存在着哺乳动物雷帕霉素靶蛋白复合物1(Mammalian Target of Rapamycin Complex 1,mTORC1)中Raptor的作用靶点TOS Motif,因此我们推测在IL-6/IL-11诱导MDSC中,mTORC1参与了对STAT3的直接调控。本研究旨在阐明IL-11诱导小鼠骨髓细胞分化为MDSC的机制,为拓展IL-11的临床应用奠定基础。方法:根据文献报道的IL-6联合GM-CSF体外诱导骨髓细胞分化发育为MDSC的方法,我们用IL-11替换IL-6,与GM-CSF联合诱导小鼠骨髓细胞4天,用流式细胞术检测诱导的体系中表面标志为CD11b和Gr-1细胞的百分比。通过与IL-6联合GM-CSF或者GM-CSF单独诱导骨髓细胞分化为MDSC情况的比较,证明IL-11是否具有诱导CD11b+Gr-1+细胞的潜力。为检测IL-11诱导的CD11b+Gr-1+细胞具有免疫抑制功能,将IL-11联合GM-CSF诱导的CD11b+Gr-1+细胞与经OVA肽活化的OT-1小鼠的CD8+T细胞共培养,观察其对活化的CD8+细胞的增殖抑制效应。为了分析mTORC1和STAT3在IL-11诱导MDSC分化发育过程中的作用,在诱导体系中分别加入mTORC1抑制剂Rapamycin和STAT3抑制剂Stattic,4天后流式细胞术分别检测MDSC的百分比以及MDSC亚群,即单核系MDSC(M-MDSC)和粒系MDSC(G-MDSC)。为了进一步验证假说,采用Western blot检测IL-11对mTORC1的活性的调控作用以及IL-11和mTORC1对STAT3的磷酸化水平的影响,然后运用负向调控mTORC1的TSC2基因敲除的MEF(TSC2-/-)细胞进一步验证mTORC1对STAT3磷酸化水平的调节作用。最后为了证实mTORC1是否通过TOS Motif直接调控STAT3,我们在HEK-293T细胞中转染Myc-Raptor和HA-STAT3,然后通过免疫共沉淀检测Raptor是否可以直接与STAT3结合。结果:流式细胞术分析MDSC亚群和MDSC对OVA257-264活化的OT-1小鼠CD8+T细胞增殖抑制试验结果表明:IL-11联合GM-CSF诱导骨髓细胞分化发育为MDSC的能力强于GM-CSF单独诱导MDSC的能力。在诱导体系中分别加入Rapamycin和Stattic,4天后流式检测结果发现Rapamycin对MDSC总体百分比没有显著影响,但明显抑制M-MDSC亚群分化发育,促进G-MDSC亚群的分化发育,Stattic完全抑制M-MDSC亚群分化发育,抑制大部分的G-MDSC亚群分化发育,这些结果表明mTORC1促进M-MDSC的分化发育,抑制G-MDSC的分化发育;STAT3完全调控M-MDSC的分化发育,部分调控G-MDSC的分化发育。Western blot技术检测发现IL-11可上调STAT3磷酸化水平和促进mTORC1活化。Rapamycin和mTORC1/2抑制剂Torin1明显抑制IL-11诱导的STAT3的活化,同时在MEF(TSC2-/-)细胞中进一步证实了mTORC1调控STAT3的磷酸化水平,这些结果表明IL-11可通过mTORC1调控STAT3,促进STAT3的磷酸化。最后免疫沉淀技术验证了Raptor可直接结合STAT3,这一结果表明mTORC1可直接促进STAT3的磷酸化。结论:IL-11/IL-11R/gp130-mTORC1-STAT3信号轴促进M-MDSC分化发育。
[Abstract]:Objective: our previous study found that in clinical and experimental animal model of sepsis in IL-11 can significantly improve the clinical symptoms of sepsis patients, shorten recovery time and prolong the survival time of mice with sepsis. Animal experiments showed that in liver and other organs and immune myeloid derived suppressor cells (myeloid derived suppressive cells, MDSC) a marked increase in the number, so we speculate that IL-11 may have the ability to induce MDSC differentiation, inflammation has inhibitory effect on the early stage of sepsis, so as to prevent the further development of sepsis, so as to shorten the recovery time of patients and the delay time of.IL-11 and IL-6 with the death of the animal similar signal transduction pathway. It has been reported that IL-6 can induce differentiation MDSC, and found that STAT3 involved in the signal transduction, but the detailed mechanism has not been elucidated. Our bioinformatics analysis showed the existence of STAT3 molecules The mammalian target of rapamycin complex 1 (Mammalian Target of Rapamycin Complex 1, mTORC1) in the Raptor targets of the TOS Motif, so we speculate that in the induction of IL-6/IL-11 in MDSC, mTORC1 is involved in the direct regulation of STAT3. The purpose of this study is to elucidate the IL-11 induced differentiation of mouse bone marrow cells for the mechanism of MDSC, and lay the foundation for the clinical application of IL-11 in vitro. Methods: according to IL-6 GM-CSF reported in the literature combined with bone marrow cells induced by the differentiation and development of MDSC method, we replace IL-6 with IL-11 and GM-CSF induced bone marrow cells of mice induced by 4 days, the surface markers were detected by flow cytometry in the system as a percentage of CD11b and Gr-1 cells with IL-6. Combined with GM-CSF or GM-CSF alone induced differentiation of bone marrow cells for MDSC, whether IL-11 CD11b+Gr-1+ cells could be induced by the potential for the detection of IL-11 induction. CD11b+Gr-1+ cells have immunosuppressive functions, IL-11 combined with GM-CSF in CD11b+Gr-1+ cells induced by OVA peptide and activated OT-1 mice were co cultured with CD8+T cells, observe the activation of CD8+ cell proliferation inhibition effects. In order to analyze the mTORC1 and STAT3 induced MDSC differentiation and function in the process of IL-11, mTORC1 inhibitor Rapamycin and STAT3 inhibitor Stattic were added in the induction system, were used to detect the percentage of MDSC and MDSC subsets after 4 days of flow cytometry, namely monocytic MDSC (M-MDSC) and myeloid MDSC (G-MDSC). In order to verify the hypothesis, influence by Western blot detection of IL-11 on the activity of mTORC1 and IL-11 and the regulatory effect of mTORC1 on the level of phosphorylation of STAT3, and then use the negative regulation of TSC2 gene knockout of mTORC1 MEF (TSC2-/-) cells to further verify the effect of mTORC1 on the phosphorylation of STAT3. At last In order to confirm whether mTORC1 TOS Motif through direct regulation of STAT3, we transfected Myc-Raptor and HA-STAT3 in HEK-293T cells, followed by immunoprecipitation to detect whether Raptor can directly bind to STAT3. Results: the analysis of MDSC subsets and MDSC OT-1 on the proliferation of mouse CD8+T cells activated by OVA257-264 inhibition test results showed that flow cytometry: IL-11 combined with GM-CSF bone marrow cells induced by differentiation ability of MDSC is stronger than GM-CSF alone induced MDSC. Rapamycin and Stattic were added in the induction system, 4 days after the flow cytometry results showed that Rapamycin had no significant effect on the overall MDSC percentage, but significantly inhibited M-MDSC subsets differentiation and development, promote the differentiation of G-MDSC subsets in the development of Stattic completely inhibited M-MDSC subsets differentiation, inhibition of most of the G-MDSC subsets differentiation, these results suggest that mTORC1 promotes the differentiation and development of M-MDSC, Inhibition of the differentiation and development of G-MDSC; STAT3 complete regulation of the differentiation and development of M-MDSC, part of the development, differentiation and regulation of G-MDSC detection.Western blot technology found that IL-11 can promote the activation of mTORC1 and activation of.Rapamycin and mTORC1/2 inhibitor Torin1 significantly inhibited IL-11 induced upregulation of STAT3 phosphorylation of STAT3, while MEF (TSC2-/-) cells further confirmed the regulation of mTORC1 STAT3 the phosphorylation level, these results suggest that IL-11 can be regulated by mTORC1 STAT3, promote the phosphorylation of STAT3. Finally, immunoprecipitation proved that Raptor can be directly combined with STAT3, the results show that the mTORC1 can directly promote the phosphorylation of STAT3. Conclusion: IL-11/IL-11R/gp130-mTORC1-STAT3 signal axis promotes the differentiation and development of M-MDSC.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7

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