本文關(guān)鍵詞：IL-11-mTORC1-STAT3信號軸調(diào)節(jié)M-MDSC的分化發(fā)育　出處：《江蘇大學》2017年碩士論文　論文類型：學位論文

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【摘要】：目的:我們前期在膿毒血癥的臨床和實驗動物模型研究中發(fā)現(xiàn)IL-11能明顯改善膿毒癥患者臨床癥狀,縮短康復時間和延長膿毒癥小鼠的生存時間。動物實驗證明在肝臟等器官中髓系來源的免疫抑制性細胞(myeloid derived suppressive cells,MDSC)數(shù)量明顯增加,因此我們推測IL-11可能具有誘導MDSC分化發(fā)育的能力,在膿毒癥早期具有炎癥抑制作用,故而能防止膿毒癥的進一步發(fā)展,從而縮短患者康復時間和延遲動物死亡時間。IL-11和IL-6具有類似的信號轉(zhuǎn)導途徑。已有文獻報道IL-6能誘導MDSC的分化,且發(fā)現(xiàn)STAT3參與其信號轉(zhuǎn)導,但是詳細機制尚未闡明。我們通過生物信息學分析發(fā)現(xiàn)STAT3分子中存在著哺乳動物雷帕霉素靶蛋白復合物1(Mammalian Target of Rapamycin Complex 1,mTORC1)中Raptor的作用靶點TOS Motif,因此我們推測在IL-6/IL-11誘導MDSC中,mTORC1參與了對STAT3的直接調(diào)控。本研究旨在闡明IL-11誘導小鼠骨髓細胞分化為MDSC的機制,為拓展IL-11的臨床應(yīng)用奠定基礎(chǔ)。方法:根據(jù)文獻報道的IL-6聯(lián)合GM-CSF體外誘導骨髓細胞分化發(fā)育為MDSC的方法,我們用IL-11替換IL-6,與GM-CSF聯(lián)合誘導小鼠骨髓細胞4天,用流式細胞術(shù)檢測誘導的體系中表面標志為CD11b和Gr-1細胞的百分比。通過與IL-6聯(lián)合GM-CSF或者GM-CSF單獨誘導骨髓細胞分化為MDSC情況的比較,證明IL-11是否具有誘導CD11b+Gr-1+細胞的潛力。為檢測IL-11誘導的CD11b+Gr-1+細胞具有免疫抑制功能,將IL-11聯(lián)合GM-CSF誘導的CD11b+Gr-1+細胞與經(jīng)OVA肽活化的OT-1小鼠的CD8+T細胞共培養(yǎng),觀察其對活化的CD8+細胞的增殖抑制效應(yīng)。為了分析mTORC1和STAT3在IL-11誘導MDSC分化發(fā)育過程中的作用,在誘導體系中分別加入mTORC1抑制劑Rapamycin和STAT3抑制劑Stattic,4天后流式細胞術(shù)分別檢測MDSC的百分比以及MDSC亞群,即單核系MDSC(M-MDSC)和粒系MDSC(G-MDSC)。為了進一步驗證假說,采用Western blot檢測IL-11對mTORC1的活性的調(diào)控作用以及IL-11和mTORC1對STAT3的磷酸化水平的影響,然后運用負向調(diào)控mTORC1的TSC2基因敲除的MEF(TSC2-/-)細胞進一步驗證mTORC1對STAT3磷酸化水平的調(diào)節(jié)作用。最后為了證實mTORC1是否通過TOS Motif直接調(diào)控STAT3,我們在HEK-293T細胞中轉(zhuǎn)染Myc-Raptor和HA-STAT3,然后通過免疫共沉淀檢測Raptor是否可以直接與STAT3結(jié)合。結(jié)果:流式細胞術(shù)分析MDSC亞群和MDSC對OVA257-264活化的OT-1小鼠CD8+T細胞增殖抑制試驗結(jié)果表明:IL-11聯(lián)合GM-CSF誘導骨髓細胞分化發(fā)育為MDSC的能力強于GM-CSF單獨誘導MDSC的能力。在誘導體系中分別加入Rapamycin和Stattic,4天后流式檢測結(jié)果發(fā)現(xiàn)Rapamycin對MDSC總體百分比沒有顯著影響,但明顯抑制M-MDSC亞群分化發(fā)育,促進G-MDSC亞群的分化發(fā)育,Stattic完全抑制M-MDSC亞群分化發(fā)育,抑制大部分的G-MDSC亞群分化發(fā)育,這些結(jié)果表明mTORC1促進M-MDSC的分化發(fā)育,抑制G-MDSC的分化發(fā)育;STAT3完全調(diào)控M-MDSC的分化發(fā)育,部分調(diào)控G-MDSC的分化發(fā)育。Western blot技術(shù)檢測發(fā)現(xiàn)IL-11可上調(diào)STAT3磷酸化水平和促進mTORC1活化。Rapamycin和mTORC1/2抑制劑Torin1明顯抑制IL-11誘導的STAT3的活化,同時在MEF(TSC2-/-)細胞中進一步證實了mTORC1調(diào)控STAT3的磷酸化水平,這些結(jié)果表明IL-11可通過mTORC1調(diào)控STAT3,促進STAT3的磷酸化。最后免疫沉淀技術(shù)驗證了Raptor可直接結(jié)合STAT3,這一結(jié)果表明mTORC1可直接促進STAT3的磷酸化。結(jié)論:IL-11/IL-11R/gp130-mTORC1-STAT3信號軸促進M-MDSC分化發(fā)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.

【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R459.7

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