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

  更多相關(guān)文章： IL-11 MDSC mTORC1 STAT3

【摘要】：目的:我們前期在膿毒血癥的臨床和實(shí)驗(yàn)動(dòng)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(fù)時(shí)間和延長膿毒癥小鼠的生存時(shí)間。動(dòng)物實(shí)驗(yàn)證明在肝臟等器官中髓系來源的免疫抑制性細(xì)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(dǎo)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(jìn)一步發(fā)展,從而縮短患者康復(fù)時(shí)間和延遲動(dòng)物死亡時(shí)間。IL-11和IL-6具有類似的信號(hào)轉(zhuǎn)導(dǎo)途徑。已有文獻(xiàn)報(bào)道IL-6能誘導(dǎo)MDSC的分化,且發(fā)現(xiàn)STAT3參與其信號(hào)轉(zhuǎn)導(dǎo),但是詳細(xì)機(jī)制尚未闡明。我們通過生物信息學(xué)分析發(fā)現(xiàn)STAT3分子中存在著哺乳動(dòng)物雷帕霉素靶蛋白復(fù)合物1(Mammalian Target of Rapamycin Complex 1,mTORC1)中Raptor的作用靶點(diǎn)TOS Motif,因此我們推測在IL-6/IL-11誘導(dǎo)MDSC中,mTORC1參與了對(duì)STAT3的直接調(diào)控。本研究旨在闡明IL-11誘導(dǎo)小鼠骨髓細(xì)胞分化為MDSC的機(jī)制,為拓展IL-11的臨床應(yīng)用奠定基礎(chǔ)。方法:根據(jù)文獻(xiàn)報(bào)道的IL-6聯(lián)合GM-CSF體外誘導(dǎo)骨髓細(xì)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(dǎo)小鼠骨髓細(xì)胞4天,用流式細(xì)胞術(shù)檢測誘導(dǎo)的體系中表面標(biāo)志為CD11b和Gr-1細(xì)胞的百分比。通過與IL-6聯(lián)合GM-CSF或者GM-CSF單獨(dú)誘導(dǎo)骨髓細(xì)胞分化為MDSC情況的比較,證明IL-11是否具有誘導(dǎo)CD11b+Gr-1+細(xì)胞的潛力。為檢測IL-11誘導(dǎo)的CD11b+Gr-1+細(xì)胞具有免疫抑制功能,將IL-11聯(lián)合GM-CSF誘導(dǎo)的CD11b+Gr-1+細(xì)胞與經(jīng)OVA肽活化的OT-1小鼠的CD8+T細(xì)胞共培養(yǎng),觀察其對(duì)活化的CD8+細(xì)胞的增殖抑制效應(yīng)。為了分析mTORC1和STAT3在IL-11誘導(dǎo)MDSC分化發(fā)育過程中的作用,在誘導(dǎo)體系中分別加入mTORC1抑制劑Rapamycin和STAT3抑制劑Stattic,4天后流式細(xì)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(jìn)一步驗(yàn)證假說,采用Western blot檢測IL-11對(duì)mTORC1的活性的調(diào)控作用以及IL-11和mTORC1對(duì)STAT3的磷酸化水平的影響,然后運(yùn)用負(fù)向調(diào)控mTORC1的TSC2基因敲除的MEF(TSC2-/-)細(xì)胞進(jìn)一步驗(yàn)證mTORC1對(duì)STAT3磷酸化水平的調(diào)節(jié)作用。最后為了證實(shí)mTORC1是否通過TOS Motif直接調(diào)控STAT3,我們在HEK-293T細(xì)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(xì)胞術(shù)分析MDSC亞群和MDSC對(duì)OVA257-264活化的OT-1小鼠CD8+T細(xì)胞增殖抑制試驗(yàn)結(jié)果表明:IL-11聯(lián)合GM-CSF誘導(dǎo)骨髓細(xì)胞分化發(fā)育為MDSC的能力強(qiáng)于GM-CSF單獨(dú)誘導(dǎo)MDSC的能力。在誘導(dǎo)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(duì)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(jìn)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(jìn)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(jìn)mTORC1活化。Rapamycin和mTORC1/2抑制劑Torin1明顯抑制IL-11誘導(dǎo)的STAT3的活化,同時(shí)在MEF(TSC2-/-)細(xì)胞中進(jìn)一步證實(shí)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(jìn)STAT3的磷酸化。最后免疫沉淀技術(shù)驗(yàn)證了Raptor可直接結(jié)合STAT3,這一結(jié)果表明mTORC1可直接促進(jìn)STAT3的磷酸化。結(jié)論:IL-11/IL-11R/gp130-mTORC1-STAT3信號(hào)軸促進(jìn)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(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R459.7

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 徐積恩;Rapamycin證明免疫系統(tǒng)和肝細(xì)胞增殖的聯(lián)系[J];國外醫(yī)藥(抗生素分冊);1992年04期

2 孫丹鳳;;Activation and its clinical significance of the mammalian target of rapamycin pathway and its downstream proteins in gastric cancer[J];China Medical Abstracts(Internal Medicine);2013年02期

3 周梅先;氮源對(duì)吸水鏈霉菌合成rapamycin的影響[J];國外醫(yī)藥(抗生素分冊);1999年06期

4 Yun Jeung Kim;Eaum Seok Lee;Seok Hyun Kim;Heon Young Lee;Seung Moo Noh;Dae Young Kang;Byung Seok Lee;;Inhibitory effects of rapamycin on the different stages of hepatic fibrosis[J];World Journal of Gastroenterology;2014年23期

5 徐積恩;FK506和Rapamycin的體內(nèi)藥理學(xué)[J];國外醫(yī)藥(抗生素分冊);1992年05期

6 ;Effects of rapamycin in liver transplantation[J];Hepatobiliary & Pancreatic Diseases International;2008年01期

7 郝衛(wèi)民;氨基酸對(duì)吸水鏈霉菌產(chǎn)生的rapamycin生物合成的影響[J];國外醫(yī)藥(抗生素分冊);1997年01期

8 趙存源;;mTOR抑制劑Rapamycin對(duì)細(xì)胞株786-O增殖和凋亡的影響[J];臨床合理用藥雜志;2013年28期

9 Feng Jiang;Li-Ming Hua;Yun-Lu Jiao;Pin Ye;Jin Fu;Zhi-Jun Cheng;Gang Ding;Yong-Hua Ji;;Activation of mammalian target of rapamycin contributes to pain nociception induced in rats by BmK I, a sodium channel-specific modulator[J];Neuroscience Bulletin;2014年01期

10 袁莉;姚亞民;呂丹丹;王麗君;湯曉琴;景玉宏;;Rapamycin保護(hù)大鼠缺血性腦損傷和特定腦區(qū)神經(jīng)新生有關(guān)[J];神經(jīng)解剖學(xué)雜志;2011年03期

相關(guān)會(huì)議論文 前10條

1 Lingyan Wang;Duojiao Wu;Yanjing Cao;Ming Xu;Ruiming Rong;Yinlong Guo;Xiangdong Wang;Tongyu Zhu;;Rapamycin Promotes Expansion of CD4+CD25+FOXP3+ Regulatory T Cells by Modulating Fatty Acid Metabolism?[A];2013中國器官移植大會(huì)論文匯編[C];2013年

2 ;The effects of rapamycin on lens epithelial cell proliferation,migration, and matrix formation: An in vitro study[A];第十屆全國中西醫(yī)結(jié)合眼科學(xué)術(shù)會(huì)議暨第五屆海峽眼科學(xué)術(shù)交流會(huì)論文匯編[C];2011年

3 ;Design, Synthesis of New FKBP12 Inhibitors With Potential Neurotrophic Efficiency[A];第九次全國生物物理大會(huì)學(xué)術(shù)會(huì)議論文摘要集[C];2002年

4 ;Antinociceptive effects of rapamycin on nociception and pain hypersensitivity:roles of mTOR signaling pathway?[A];Proceedings of the 8th Biennial Conference of the Chinese Society for Neuroscience[C];2009年

5 ;Regulation of gastric hormones by systemic rapamycin[A];中國生理學(xué)會(huì)第23屆全國會(huì)員代表大會(huì)暨生理學(xué)學(xué)術(shù)大會(huì)論文摘要文集[C];2010年

6 Shunmei Huang;Zhitao Song;Baoju Wang;Junzhong Wang;Zhenni Zhu;Bin Zhu;Xuemei Feng;Mengji Lu;Dongliang Yang;;Rapamycin modulates the course of hepatitis B virus(HBV)infection in hydrodynamic injection mouse model:the role of Treg in HBV persistence[A];中華醫(yī)學(xué)會(huì)第十六次全國病毒性肝炎及肝病學(xué)術(shù)會(huì)議論文匯編[C];2013年

7 曾玲暉;;Paradoxical effect of rapamycin on S6 phosphorylation in rats with or without seizures[A];第十五屆中國神經(jīng)精神藥理學(xué)學(xué)術(shù)會(huì)議論文摘要[C];2012年

8 潘登;李艷;李哲;王亞柱;王萍萍;梁穎;蔡大利;高然;蘇楠;徐贏東;王蕾;林娜;;急性髓細(xì)胞白血病中Hedgehog-gli通路活化及rapamycin對(duì)其調(diào)節(jié)作用的研究[A];中華醫(yī)學(xué)會(huì)血液學(xué)分會(huì)第十三屆全國血栓與止血學(xué)術(shù)會(huì)議暨“血栓栓塞性疾病（血栓與止血）基礎(chǔ)與臨床研究進(jìn)展”論文摘要匯編及學(xué)習(xí)班講義[C];2011年

9 ;Rapamycin attenuates the development of chronic hypoxia-induced pulmonary hypertension in mice[A];中華醫(yī)學(xué)會(huì)呼吸病學(xué)年會(huì)——2011（第十二次全國呼吸病學(xué)學(xué)術(shù)會(huì)議）論文匯編[C];2011年

10 Feng JIANG;Li-Ming HUA;Yun-Lu JIAO;Pin YE;Jin FU;Zhi-Jun CHENG;Gang DING;Yong-Hua JI;;Activation of mammalian target of rapamycin contributes to rat nociceptive behaviors[A];中國神經(jīng)科學(xué)學(xué)會(huì)第十屆全國學(xué)術(shù)會(huì)議論文摘要集[C];2013年

相關(guān)碩士學(xué)位論文 前8條

1 彭韶;雷帕霉素對(duì)人外周血B細(xì)胞IM-9增殖和產(chǎn)生IgG/IgM抗體的影響[D];南華大學(xué);2015年

2 王勇;Rapamycin聯(lián)合SAHA對(duì)人肺腺癌細(xì)胞的放療增敏效應(yīng)及機(jī)制[D];南昌大學(xué);2016年

3 司小琴;Rictor在血管新生及成熟中的作用機(jī)制研究[D];云南中醫(yī)學(xué)院;2017年

4 劉月芳;IL-11-mTORC1-STAT3信號(hào)軸調(diào)節(jié)M-MDSC的分化發(fā)育[D];江蘇大學(xué);2017年

5 教瑩瑩;Rapamycin通過抑制內(nèi)質(zhì)網(wǎng)應(yīng)激途徑保護(hù)光損傷中視網(wǎng)膜感光細(xì)胞[D];吉林大學(xué);2015年

6 劉晶;Rapamycin與As_2O_3聯(lián)合誘導(dǎo)NB4細(xì)胞凋亡及其信號(hào)轉(zhuǎn)導(dǎo)機(jī)制的研究[D];大連醫(yī)科大學(xué);2005年

7 胡莉莉;雷帕霉素和LY294002對(duì)負(fù)重游泳訓(xùn)練大鼠骨骼肌生長的影響[D];上海體育學(xué)院;2011年

8 蔣蓉;自噬激活劑/抑制劑對(duì)小鼠神經(jīng)干細(xì)胞增殖和分化潛能的影響[D];山西醫(yī)科大學(xué);2017年

，

本文編號(hào)：1383365