本文關(guān)鍵詞： 肝移植 急性移植物抗宿主病 調(diào)節(jié)性T細胞 雷帕霉素 OSI-027　出處：《浙江大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分新型mTOR通路抑制劑OSI-027治療大鼠肝移植后急性移植物抗宿主病的研究實驗?zāi)康母我浦埠蠹毙砸浦参锟顾拗鞑?LTx-aGVDH)是肝移植的一種嚴重并發(fā)癥,雖然其發(fā)病率僅為1-2%,但是死亡率卻高達80-100%。目前該疾病發(fā)生發(fā)展的機制尚不清楚,臨床上缺乏有效的治療手段。在前期研究中,本課題組成功建立了世界上首例大鼠LTx-aGVHD模型,并發(fā)現(xiàn)疾病的發(fā)生發(fā)展與大鼠外周血單個核細胞(PBMCs)中調(diào)節(jié)性T細胞(T-regs)比例的變化有一定的相關(guān)性,雷帕霉素(RAPA)能夠上調(diào)LTx-aGVHD大鼠PBMCs出T-regs的比例,降低大鼠的死亡率。OSI-027是一種新型的mTOR通路抑制劑,在本實驗中,第一次將其應(yīng)用于免疫學(xué)領(lǐng)域,探索OSI-027對大鼠LTx-aGVHD的作用。材料與方法建立大鼠LTx-aGVHD模型并分為三組,于肝移植術(shù)后第七天開始,分別經(jīng)尾靜脈連續(xù)注射生理鹽水、RAPA和OSI-027七天。觀察大鼠的健康狀況和生存時間,流式細胞分析技術(shù)檢測大鼠體內(nèi)供體來源細胞比例和PMBCs中T-regs比例的變化,病理學(xué)和免疫組織化學(xué)(IHC)檢測皮膚組織中單核細胞的浸潤情況,ELISA檢測血清中炎性細胞因子的變化。結(jié)果OSI-027組大鼠生存率高于RAPA組和對照組,長期存活(100天)的大鼠無明顯的LTx-aGVHD癥狀。流式細胞學(xué)檢測發(fā)現(xiàn)RAPA與OSI-027都能夠增加大鼠PBMCs中T-regs的比例,OSI-027的作用強于RAPA.在抑制供體來源細胞增殖、單核細胞浸潤和血清炎性因子水平等方面,OSI-027的作用也明顯優(yōu)于RAPA.結(jié)論在大鼠LTx-aGVHD的治療中,OSI-027的效果明顯優(yōu)于RAPA。第二部分OSI-027治療大鼠肝移植后急性移植物抗宿主病機制的研究實驗?zāi)康募毙砸浦参锟顾拗鞑?LTx-aGVHD)是肝臟移植后致命的并發(fā)癥,目前臨床中缺乏有效的治療手段。在前期研究中本我們發(fā)現(xiàn)LTx-aGVHD的發(fā)生發(fā)展與模型大鼠PBMCs出T-regs比例的變化相關(guān),新型mTOR通路抑制劑OSI-027上調(diào)大鼠PBMCs中T-regs的比例、提高大鼠生存率的作用明顯優(yōu)于RAPA.在本研究中,我們探索OSI-027與RAPA對mTOR通路作用的異同,進而揭示導(dǎo)致兩藥對LTx-aGVHD治療效果差異的分子機制。材料與方法大鼠LTx-aGVHD模型建立后,將其分為三組,于肝移植術(shù)后第七天開始,分別經(jīng)尾靜脈連續(xù)注射生理鹽水、RAPA和OSI-027七天。流式細胞分技術(shù)檢測大鼠PBMCs由T-regs比例的變化,Western Blot檢測PBMCs中mTOR通路表達的差異；體外淋巴細胞混合培養(yǎng)(MLC)研究mTOR通路影響T-regs的機制；并在體外應(yīng)用OSI-027誘導(dǎo)獲得T-regs,回輸給大鼠驗證T-regs對LTx-aGVHD的治療作用。結(jié)果OSI-027與RAPA都能夠增加大鼠PBMCs出T-regs的比例,OSI-027的效果明顯優(yōu)于RAPA.模型大鼠PBMCs中供體來源淋巴細胞mTOR通路的表達水平較lewis大鼠的高。RAPA能夠抑制mTOR1復(fù)合體的活性,但對mTOR2復(fù)合體的作用較弱,OSI-027能夠快速有效的同時抑制mTOR1/mTOR2復(fù)合體, mTOR1/mTOR2復(fù)合體抑制后能夠促使CD4+CD25T細胞向CD4+CD25+Foxp3+T-regs轉(zhuǎn)化。將體外培養(yǎng)獲得的CD4+CD25+Foxp3+T-regs輸給大鼠,能夠防止LTx-aGVHD的發(fā)生。結(jié)論OSI-027能夠同時快速有效的抑制mTOR1/mTOR2復(fù)合體,促進CD4+CD25-T細胞向T-regs轉(zhuǎn)化,從而更加有效的治療LTx-aGVHD。
[Abstract]:The first part of the new mTOR inhibitor OSI-027 treatment on experimental liver transplantation after liver transplantation in rats of acute graft-versus-host disease after acute graft-versus-host disease (LTx-aGVDH) is a serious complication of liver transplantation, although its incidence rate is only 1-2%, but the mortality rate is as high as 80-100%. pathogenesis of the disease is unclear, the lack of effective therapy in clinic. In previous studies, our research group has successfully established the first LTx-aGVHD rat model in the world, and found that the blood mononuclear cells and the occurrence and development of rat peripheral disease (PBMCs) in regulatory T cells (T-regs) have a certain correlation ratio changes of rapamycin (RAPA) can increase LTx-aGVHD rats PBMCs ratio of T-regs, reduce the mortality of rats.OSI-027 is a novel inhibitor of mTOR pathway, in this experiment, the first time will be applied to free The field of epidemiology, to explore the effect of OSI-027 on rat LTx-aGVHD. Materials and methods to establish a rat model of LTx-aGVHD were divided into three groups, on the seventh day after transplantation, respectively by intravenous continuous injection of saline, RAPA and OSI-027 for seven days. To observe the rat health status and survival time, flow cytometry the analysis was used to detect the changes in rat donor derived cells and T-regs ratio in PMBCs, pathology and immunohistochemistry (IHC) infiltration of mononuclear cells were detected in skin tissue, changes of inflammatory cytokines ELISA in serum. Results the survival rate of rats in OSI-027 group was higher than that of RAPA group and control group, long-term the survival (100 days) of the rats had no obvious symptoms of LTx-aGVHD. Flow cytometry showed that RAPA and OSI-027 can increase the proportion of PBMCs in T-regs rats, the effect of OSI-027 is stronger than RAPA. in inhibiting donor cell proliferation, single Infiltration of mononuclear cells and serum inflammatory factor level, the effect of OSI-027 is significantly better than RAPA. conclusion in the treatment of LTx-aGVHD in rats, OSI-027 is better than RAPA. second OSI-027 treatment after liver transplantation in rats of acute graft versus host disease research mechanism of acute graft-versus-host disease (LTx-aGVHD) is a fatal complication after liver transplantation, the clinical lack of effective treatment. In the previous study, we found that the occurrence and development of rat model of PBMCs related changes in the proportion of T-regs LTx-aGVHD, mTOR T-regs way inhibitor OSI-027 model raised rat PBMCs proportion, improve the survival rate of the rats was superior to that of RAPA. in this study, we explore the similarities and differences between OSI-027 and RAPA on the mTOR pathway, and reveal the molecular mechanisms leading to two drug treatment effect on LTx-aGVHD of different materials and party. Method to establish rat model of LTx-aGVHD, which is divided into three groups on the seventh day after liver transplantation, respectively by intravenous continuous injection of saline, RAPA and OSI-027 for seven days. Changes of flow cytometry detection of rat PBMCs by T-regs proportion, the differential expression of mTOR pathway Western Blot detection in PBMCs mixed lymphocyte culture in vitro; (MLC) mTOR pathway influence T-regs mechanism; and induced by T-regs in vitro by OSI-027 infusion to rats to verify T-regs therapeutic effect on LTx-aGVHD. The results of OSI-027 and RAPA can increase the rat PBMCs T-regs ratio, high.RAPA is better than RAPA. in a rat model of PBMCs OSI-027 the expression level of donor lymphocytes from the mTOR pathway than Lewis rats can inhibit the activity of mTOR1 complex, but had less effect on the mTOR2 complex, OSI-027 can effectively suppress the mTO The R1/mTOR2 complex, mTOR1/mTOR2 complex can inhibit after transformation to CD4+CD25+Foxp3+T-regs CD4+CD25T cells cultured in vitro. To get the lost CD4+CD25+Foxp3+T-regs to rats, can prevent the occurrence of LTx-aGVHD. Conclusion OSI-027 can effectively inhibit mTOR1/ mTOR2 complex, promote the transformation of CD4+CD25-T cells into T-regs, so as to be more effective in the treatment of LTx-aGVHD.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R657.3

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