【作者】 王娜；

【导师】 刘杞；

【作者基本信息】 重庆医科大学 ， 内科学， 2013， 博士

【摘要】 第一部分肝再生增强因子对PBMC增殖的影响与调节性T细胞的关系’目的：肝再生增强因子(Augmenter of liver regeneration, ALR)具有免疫调节作用,但其发挥负性免疫调控作用的机制尚未明确。本部分课题拟通过观察人肝再生增强因子(Human augmenter of liver regeneration, hALR)在体外对ConA刺激的正常人外周血单个核细胞(Peripheral blood mononuclear cells, PBMC)的增殖和凋亡的影响以及对其中CD4+CD25+FoxP3+调节性T细胞(Regulatory T cell, Treg)亚群分化增殖的影响,探讨hALR免疫抑制作用的可能机制。方法：以ConA刺激的健康人PBMC为实验细胞,分别设Control组、ConA组和ConA+hALR组。体外培养60h后用MTS法检测各组细胞增殖率,流式及免疫印迹法检测各组细胞的早期细胞凋亡率及凋亡相关蛋白表达,实时荧光定量PCR检测各组FoxP3的表达,流式检测各组细胞CD4+CD25+FoxP3+Treg的比例及细胞周期变化。结果：hALR能够明显抑制ConA刺激的PBMC增殖(ConA组vs ConA+hALR组,P<0.01),且hALR降低ConA引起的细胞凋亡(ConA组vs ConA+hALR组,P<0.01)。hALR在抑制细胞增殖的同时,上调FoxP3基因的表达,提高Treg的比例(ConA组vs ConA+hALR组,P<0.01),并且能够解除ConA导致的Treg亚群细胞周期G2/M期阻滞(ConA组vs ConA+hALR组,P<0.01)。结论：hALR能够显著抑制ConA刺激的PBMC增殖,但不是通过诱导PBMC凋亡实现的,而是具有抗凋亡作用,hALR同时上调CD4+CD25+FoxP3+Treg的比例,提示hALR可能通过诱导Treg来抑制ConA刺激的PBMC增殖。第二部分肝再生增强因子诱导调节性T细胞发挥免疫抑制作用的机制目的：细胞因子在Treg的分化增殖及免疫抑制作用中发挥重要作用,本研究拟通过观察hALR对各组细胞上清中细胞因子(TGF-β1、 IL-10、IL-2)水平的影响,以及对各组细胞信号通路分子(ERK1/2、 NF-κB)舌性及表达的影响,探讨hALR促进Treg分化增殖以及发挥免疫抑制作用的可能机制。方法：以ConA刺激的健康人PBMC为实验细胞,分别设Control组、ConA组和ConA+hALR组。用ELISA方法分别检测培养16h和40h各组细胞上清中TGF-β1、IL-10、IL-2的水平,用免疫印迹法检测培养60h的各组细胞ERK1/2的活性和NF-κB (p65)的表达。结果：hALR能够显著增加细胞培养上清中TGF-β1和IL-10的水平,降低IL-2的水平(ConA组vs ConA+hALR组,P<0.01)。hALR能够显著抑制ERK1/2的活性和NF-κB (p65)的表达(ConA组vsConA+hALR组,P<0.01)。结论：hALR可以促进TGF-β1和IL-10产生而抑制IL-2产生,能够抑制ERK1/2的活性和NF-κB (p65)的表达,提示hALR对细胞因子的调控可能既是hALR上调Treg的条件也是其发挥免疫抑制作用的机制,在分子机制上则表现为对信号通路分子的调控。第三部分肝再生增强因子对大鼠腹腔肝细胞移植免疫排斥的影响目的：hALR体外能够上调Treg并抑制ConA活化的免疫细胞增殖,本部分研究拟通过观察腹腔内同种异体肝细胞移植(Hepatocyte transplantation, HCT)联合hALR治疗急性肝衰竭(Acute liver failure,ALF)大鼠的疗效,以及hALR对大鼠免疫排斥反应的影响,探讨hALR体内的免疫抑制作用及机制。方法：以D-氨基半乳糖(D-galactosamine, D-GalN)诱导ALF的S-D大鼠为动物模型,半原位胶原酶灌注法新鲜分离的大鼠肝细胞腹腔内移植联合hALR治疗ALF大鼠,分为生理盐水(PS)组、HCT组、HCT+hALR组、hALR组。观察各组大鼠生存率,组织学分析腹腔内移植肝细胞存活情况,免疫组化检测大网膜免疫细胞CD68、CD4、CD8及Foxp3阳性率,流式检测腹腔内大网膜免疫细胞中CD4+CD25+FoxP3+Treg的比例,实时荧光定量PCR检测移植物周围大网膜免疫细胞TGF-β1、IL-10mRNA表达水平,ELISA检测腹水及血清中TNF-a和IL-1β水平。结果：hALR联合HCT治疗能够显著提高大鼠生存率(P<0.05),且hALR明显改善腹腔移植物存活情况,减少炎症细胞浸润。hALR明显降低大网膜免疫细胞CD68、CD4、CD8阳性率,增加Foxp3阳性率,上调大网膜免疫细胞中Treg的比例和TGF-β1、IL-10mRNA的表达,降低前炎症介质TNF-a和IL-1β水平(HCT组vs HCT+hALR组,P<0.05)。结论：hALR能够通过上调腹腔HCT的ALF大鼠腹腔大网膜内Treg的比例,抑制机体炎症反应和对腹腔移植物的免疫排斥反应,改善腹腔移植物存活,最终提高ALF大鼠存活率。这提示hALR在ALF大鼠体内同样具有免疫抑制作用,能够抑制机体对移植物的免疫排斥反应,促进移植耐受,而上调Treg可能是hALR发挥免疫调节作用的机制。更多还原

【Abstract】 Part Ⅰ The Effect of hALR on Proliferation of PBMC and the Relationship to TregObjective:Augmenter of liver regeneration (ALR) has effect of immunoloregulation, but the underlying mechanism is not completely understood. The objective of this study is to observe the effect of hALR on proliferation of ConA-stimulated PBMC and on differentiation and proliferation of CD4+CD25+FoxP3+Tregs, and to investigate the underlying mechanism of immunosuppressive role of hALR.Methods:The PBMC from healthy donors was isolated and stimulated with ConA, then cultured in three groups(Control、ConA and ConA+hALR) in vitro for60h. MTS assay was used to determine the cellular viability. Flow cytometry (FCM) and western blot assay were used to detect the early apoptotic ratios and the main proteins of apoptosis. FoxP3mRNA was detected by Real-time fluorescent quantitation PCR, and the ratios of CD4+CD25+FoxP3+Treg and the mitotic cycle of Treg were observed by FCM.Results:The proliferation of ConA-stimulated PBMC was obviously inhibited by hALR (ConA group vs ConA+hALR group, P<0.01), and the apoptosis of ConA-stimulated PBMC was decreased by hALR (ConA group vs ConA+hALR group, P<0.01). Meanwhile, hALR increased the expression of FoxP3mRNA and up-regulated the percentage of Tregs (ConA group vs ConA+hALR group, P<0.01). In addition, hALR could relieve the G2/M cell cycle arrest of Treg induced by ConA (ConA group vs ConA+hALR group,P<0.01)Conclusions:The data demonstrated that hALR inhibited the proliferation of ConA-stimulated PBMC and up-regulated the percentages of Treg. It indicated us that hALR inhibited the proliferation of ConA-stimulated PBMC through the pathway of induction of Tregs possibly. Part Ⅱ The Mechanism of Induction of Treg and the Pathways of Immunosuppression of hALRObjective:Cytokines played important roles in the differentiation and proliferation of Treg. In this study, we detected the levels of the cytokines (TGF-β1、IL-10、IL-2) in the supernatant and observed the main signaling pathway related molecular (ERK1/2、NF-κB), then discussed the possible mechanism of hALR on induction of Treg and the underlying signaling pathway of immunosuppressive role.Methods:The PBMC from healthy donors was isolated and stimulated with ConA, then cultured in three groups(Control、ConA and ConA+hALR) in vitro. The levels of TGF-β1, IL-10and IL-2in the supernatant were determined by ELISA, and the expressions of the main signaling pathway related molecular were detected by western blot assay.Results:The levels of TGF-β1and IL-10in the supernatant were significantly increased by hALR. On the contrary, the level of IL-2was decreased notably (ConA group vs ConA+hALR group, P<0.01) Meanwhile, hALR inhibited the activation of ERK1/2and the expression of NF-κB (p65) obviously (ConA group vs ConA+hALR group, P<0.01).Conclusions:The levels of the cytokines in the supernatant were regulated by hALR, increasing the production of TGF-β1and IL-10, but decreasing IL-2. Notably, hALR inhibited the activation or expression of the signaling pathway relative molecular. On one hand, it was indicated that cytokines regulated by hALR might be the mechanism of induction of Treg and the approach of immunosuppressive role. On the other hand, the molecular mechanism of hALR on immunosuppressive role was the regulation of the signaling pathway molecular. Part Ⅲ The Effect of hALR on Immunological Rejection of HCT in ALF RatsObjective:As hALR up-regulated Treg and inhibited proliferation of immunocytes in vitro, in this part of study, we investigated the potential effect and mechanism of hALR on ALF rats treated with intraperitoneal HCT in vivo.Methods:ALF rats induced by D-GalN were studied, and were intraperitoneal injected with or without hepatocytes and hALR24h after the induction. The hepatocytes were isolated from healthy S-D rats with an improved two-step semi-situ recirculating collagenase perfusion method. The ALF rats were divided in four groups (PS group, HCT group、 HCT+hALR group and hALR group). Animal survival was assessed, and the levels of TNF-α and IL-1β were detected by ELISA. Histological examination was performed and immunological responses were identified by immunohistochemistry assay. FCM was used to determinate the percentages of Treg in immunocytes in the greater omentum. The expressions of TGF-β1and IL-10mRNA were detected by Real-time fluorescent quantitation PCR.Results:It showed that hALR improved the survival of ALF rats treated with HCT (P<0.05). The inflammatory damage was reduced significantly in the transplanted islets by hALR after being strained with PAS. Markedly lower percentages of CD68, CD4and CD8positive immunocytes and a higher percentage of Foxp3positive immunocytes in the greater omentum were observed in HCT+hALR group. Additionally, hALR up-regulated the percentage of Treg of the immunocytes in the greater omentum, and increased the expression of TGF-β1and IL-10mRNA, decreased pro-inflammation cytokines (HCT group vs HCT+hALR group, P<0.05)。Conclusions:In the treatment of ALF rats, hALR up-regulated Treg, inhibited the inflammation, attenuated the damage of immunological rejection, and improved the overall survival. It indicated that hALR inhibited the immunological rejection, promoted the transplantation tolerance of ALF rats, and the underlying mechanism was the up-regulation of Treg by hALR.更多还原