OSI-027治疗大鼠肝移植后急性移植物抗宿主病及其机制的研究

发布时间：2018-02-10 00:46

本文关键词： 肝移植急性移植物抗宿主病调节性T细胞雷帕霉素 OSI-027　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：第一部分新型mTOR通路抑制剂OSI-027治疗大鼠肝移植后急性移植物抗宿主病的研究实验目的肝移植后急性移植物抗宿主病(LTx-aGVDH)是肝移植的一种严重并发症,虽然其发病率仅为1-2%,但是死亡率却高达80-100%。目前该疾病发生发展的机制尚不清楚,临床上缺乏有效的治疗手段。在前期研究中,本课题组成功建立了世界上首例大鼠LTx-aGVHD模型,并发现疾病的发生发展与大鼠外周血单个核细胞(PBMCs)中调节性T细胞(T-regs)比例的变化有一定的相关性,雷帕霉素(RAPA)能够上调LTx-aGVHD大鼠PBMCs出T-regs的比例,降低大鼠的死亡率。OSI-027是一种新型的mTOR通路抑制剂,在本实验中,第一次将其应用于免疫学领域,探索OSI-027对大鼠LTx-aGVHD的作用。材料与方法建立大鼠LTx-aGVHD模型并分为三组,于肝移植术后第七天开始,分别经尾静脉连续注射生理盐水、RAPA和OSI-027七天。观察大鼠的健康状况和生存时间,流式细胞分析技术检测大鼠体内供体来源细胞比例和PMBCs中T-regs比例的变化,病理学和免疫组织化学(IHC)检测皮肤组织中单核细胞的浸润情况,ELISA检测血清中炎性细胞因子的变化。结果OSI-027组大鼠生存率高于RAPA组和对照组,长期存活(100天)的大鼠无明显的LTx-aGVHD症状。流式细胞学检测发现RAPA与OSI-027都能够增加大鼠PBMCs中T-regs的比例,OSI-027的作用强于RAPA.在抑制供体来源细胞增殖、单核细胞浸润和血清炎性因子水平等方面,OSI-027的作用也明显优于RAPA.结论在大鼠LTx-aGVHD的治疗中,OSI-027的效果明显优于RAPA。第二部分OSI-027治疗大鼠肝移植后急性移植物抗宿主病机制的研究实验目的急性移植物抗宿主病(LTx-aGVHD)是肝脏移植后致命的并发症,目前临床中缺乏有效的治疗手段。在前期研究中本我们发现LTx-aGVHD的发生发展与模型大鼠PBMCs出T-regs比例的变化相关,新型mTOR通路抑制剂OSI-027上调大鼠PBMCs中T-regs的比例、提高大鼠生存率的作用明显优于RAPA.在本研究中,我们探索OSI-027与RAPA对mTOR通路作用的异同,进而揭示导致两药对LTx-aGVHD治疗效果差异的分子机制。材料与方法大鼠LTx-aGVHD模型建立后,将其分为三组,于肝移植术后第七天开始,分别经尾静脉连续注射生理盐水、RAPA和OSI-027七天。流式细胞分技术检测大鼠PBMCs由T-regs比例的变化,Western Blot检测PBMCs中mTOR通路表达的差异；体外淋巴细胞混合培养(MLC)研究mTOR通路影响T-regs的机制；并在体外应用OSI-027诱导获得T-regs,回输给大鼠验证T-regs对LTx-aGVHD的治疗作用。结果OSI-027与RAPA都能够增加大鼠PBMCs出T-regs的比例,OSI-027的效果明显优于RAPA.模型大鼠PBMCs中供体来源淋巴细胞mTOR通路的表达水平较lewis大鼠的高。RAPA能够抑制mTOR1复合体的活性,但对mTOR2复合体的作用较弱,OSI-027能够快速有效的同时抑制mTOR1/mTOR2复合体, mTOR1/mTOR2复合体抑制后能够促使CD4+CD25T细胞向CD4+CD25+Foxp3+T-regs转化。将体外培养获得的CD4+CD25+Foxp3+T-regs输给大鼠,能够防止LTx-aGVHD的发生。结论OSI-027能够同时快速有效的抑制mTOR1/mTOR2复合体,促进CD4+CD25-T细胞向T-regs转化,从而更加有效的治疗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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R657.3

【相似文献】