【作者】 王敏；

【导师】 沈关心；

【作者基本信息】 华中科技大学 ， 免疫学， 2007， 博士

【摘要】 【目的】未折叠或错误折叠蛋白质在内质网腔蓄积及细胞内稳态的破坏将导致内质网应激。适宜的应激有利于细胞内环境的恢复,然而严重而持久的内质网应激反应将导致内质网功能受损,诱导细胞凋亡。β细胞具有高度发达的内质网,是对内质网应激最敏感的细胞之一,内质网应激介导的β细胞凋亡参与了糖尿病的发生与发展。糖调节蛋白78(GRP78)是热休克蛋白70家族的成员之一,作为一种分子伴侣在蛋白质的折叠和转运过程及内质网应激反应中发挥重要作用,也被称为内质网应激标志蛋白。本实验研究GRP78在链脲佐菌素(STZ)损伤糖尿病小鼠胰岛β细胞不同时期表达变化,揭示其与胰岛β细胞内质网应激的关联;研究GRP78对胰岛β细胞的保护作用,及对STZ诱导I型糖尿病发生发展的影响。【方法】1.STZ诱导NIT-1细胞与糖尿病小鼠胰岛GRP78表达的检测Balb/c小鼠20只,雄性,随机分为模型组15只和对照组5只。体内采用STZ一次大剂量腹腔注射制备I型糖尿病模型,分为第1、7、14天三个实验组,取其胰岛备用;体外用STZ处理NIT-1细胞株(来源于非肥胖型糖尿病小鼠(NOD)胰岛瘤细胞株),分别在处理后2、6、24h收集细胞,柠檬酸缓冲液处理NIT-1细胞作为对照。应用实时定量聚合酶链反应(real-time PCR)方法检测GRP78 mRNA和CHOPmRNA表达变化,应用免疫斑点(western blot)方法检测其蛋白表达,流式细胞术(FCM)及荧光显微镜检测凋亡。比较STZ处理后未发生糖尿病小鼠与对照组小鼠的GRP78蛋白水平。2.GRP78转染NIT-1细胞及其凋亡的检测用含GRP78基因的质粒转染NIT-1,命名为NIT-GRP78,作为细胞模型研究GRP78保护胰岛β细胞抵抗STZ,细胞因子(IL-1β+IFN-γ)和细胞毒性T细胞(CTL)诱导的损伤作用。FCM检测凋亡,real-time PCR检测CHOPmRNA的表达,比色法检测NO浓度和SOD活性。3.CTL与细胞因子的检测转染或未转染GRP78基因的NIT-1细胞免疫Balb/c小鼠,取其脾细胞体外再次刺激后作为效应细胞,以NIT-GRP78和NIT-GFP细胞为靶细胞,CFSE与PI双染色FCM检测效应细胞对靶细胞的杀伤作用,ELISA检测效应细胞分泌IL-4和IFN-γ水平,并且将转染和未转染基因的NIT-1细胞移植入STZ诱导的糖尿病小鼠,观察移植前后的血糖、体重、糖刺激的胰岛素释放及生存时间。【结果】1.STZ诱导NIT-1细胞与糖尿病小鼠胰岛GRP78表达在受侵袭的胰岛β细胞GRP78表达早期增高,但晚期显著下降。随着GRP78的下降和CHOP表达增高,细胞凋亡逐渐增加。特别有意义的是STZ处理后未发生糖尿病小鼠的GRP78蛋白水平与对照组相比稳定增高,提示GRP78蛋白的表达可能对应激细胞具有一定程度的保护作用。2.GRP78对损伤因子诱导NIT-1细胞凋亡的抵抗作用及其机制在经过STZ和细胞因子处理后,与对照组相比,转染GRP78基因细胞培养上清中NO(P<0.05)水平降低,而SOD活性增高(P<0.05),细胞内CHOPmRNA水平亦较低(P<0.05),细胞凋亡率显著下降(P<0.01)。与对照组相比,转染GRP78基因的细胞对CTL的抵抗力显著增强。3.GRP78对STZ诱导I型糖尿病小鼠发生发展的影响通过转染或未转染GRP78基因的NIT-1体内外联合刺激获得的脾淋巴细胞,分别与不同靶细胞体外共培养后,CFSE与PI双染色FCM检测结果表明,NIT-GFP细胞诱导的脾淋巴细胞介导NIT-GRP78靶细胞中度坏死,而介导NIT-GFP细胞有重度坏死;NIT-GRP78细胞诱导的脾淋巴细胞介导NIT-GRP78靶细胞轻度坏死,而介导NIT-GFP细胞有中度坏死;ELISA检测结果表明,与NIT-GFP免疫获得的淋巴细胞相比,NIT-GRP78细胞免疫获得的淋巴细胞分泌IL-4水平显著增高(P<0.01);将转染和未转染基因的NIT-1细胞移植入STZ诱导的糖尿病小鼠,观察移植前后的血糖、体重、糖刺激的胰岛素释放及生存时间,结果表明,转染了NIT-GRP78细胞的糖尿病小鼠的体重及血糖逐渐恢复正常,生存时间延长且在移植后7天检测糖刺激的胰岛素释放量也显著增高。【结论】以上结果表明,GRP78与胰岛β细胞内质网应激有密切关联;GRP78可以保护胰岛β细胞抵抗免疫损伤诱导的凋亡;且可诱导对Th1细胞具有负性调节作用的IL-4的分泌,有可能在胰岛细胞移植促进重建胰岛细胞功能中发挥重要作用。更多还原

【Abstract】 Objective: Endoplasmic reticulum(ER) stress-mediated apoptosis plays an important role in the destruction of pancreatic beta-cells, and contributes to the development of type 1 diabetes. The chaperone molecule, glucose regulated proteins 78(GRP78), is required to maintain ER function during toxic insults. In this study, we investigated the changes of GRP78 expression in different phase of streptozotocin(STZ)-affected beta-cells, to explore the relationship between GRP78 and the response of beta-cell to ER stress; we investigated the effect of GRP78 on the beta-cell apoptosis; and we investigated the effect of GRP78 on the development of STZ-induced type I diabetes mice.Methods:①The expression of GRP78 in different phase of STZ-affected beta-cell A insulinoma cell line(NIT-1) treated with STZ for different time course, and STZ-induced diabetic Balb/C mices at different time points were used as the model system. The level of GRP78 and C/EBP homologous protein (CHOP) mRNA were detected by real-time PCR and their protein by immunoblot, apoptosis and necrosis were measured by flow cytometry, and morphological changes of apoptotic cells by fluorescence microscope. In addition, the changes of GRP78 protein in STZ-treated non-diabetic mices was also detected by immunoblot;②The effect of GRP78 on the beta-cell apoptosis induced by STZ, cytokines and CTLNIT-1 cells transfected with GRP78 was established, named NIT-GRP78, and used to study apoptosis, which was induced by streptozotocin, inflammatory cytokines or cytotoxic T lymphocyte(CTL). Apoptosis of NIT-1 or NIT-GRP78 cells was detected by flow cytometry, by monitoring the transcription of CHOP with real-time PCR, the concentration of nitric oxide and the activity of superoxide dismutase with colorimetric method; ③The effect of GRP78 on the development of STZ-induced type I diabetes mice NIT-1 cells transfected with GRP78 were used to study the immunosuppressive and protective ability of GRP78, which was evaluated by extended CTL killing assay measured with flow cytometry through target cells dyed with CFSE cultured with effector cells and finally stained with PI, by measurement of concentrations of IL-4 and IFN-γdetected with ELISA, by transplantation of these cells into STZ-induced diabetic Balb/c mices.Results:①The expression of GRP78 in different phase of STZ-affected beta-cell GRP78 expression significantly increased in early phase but decreased in later phase of affected beta-cell, while along with the decrease of GRP78, CHOP was induced and apoptosis occured. Interestingly, the GRP78 protein of STZ-treated non-diabetic mices increased stably compared with control;②The effect of GRP78 on the development of STZ-induced type I diabetes mice In comparison to NIT-1 cells, NIT-GRP78 cells responded to the streptozotocin or cytokines treatments with decreased concentration of nitric oxide, but increased activity of superoxide dismutase. In addition, the level of CHOP was also decreased in the NIT-GRP78 cells, which may mediate the resistance of the GRP78 overexpressed NIT-1 cells from apoptosis. NIT-GRP78 cells were also more resistant than NIT-1 cells to CTL specific killing;③The effect of GRP78 on the beta-cell apoptosis induced by STZ, cytokines and CTLCultured with NIT-GFP-primed lymphocytes, NIT-GRP78 cells had moderate necrosis(“+ +”, P<0.05) compared with the severe necrosis of NIT-GFP cells(“+ + +”). While cultured with NIT-GRP78-primed and expanded lymphocytes, NIT-GRP78 cells had mild necrosis(“+”, P<0.05), NIT-GFP cells had moderate necrosis(“+ +”, P<0.05). A increase of IL-4(P<0.01) secretion from beta-cell-primed splenocytes when GRP78 presences was observed. Diabetic mice reached normoglycemia promptly and gained weight after transplantation of NIT-GRP78. More importantly, the survival time for recipients transplanted with NIT-GRP78 cells was significantly longer than that with NIT-GFP cells(P < 0.01). In addition, we observed a significant increase of insulin concentration after glucose stimulation for diabetic mice received NIT-GRP78 cells at day 7 of post-transplantation.Conclusions: From the results we can conclude that there is a close relationship between GRP78 and the response of beta-cells to ER stress; Modulating GRP78 expression could be useful in preventing pancreatic beta-cell from the immunological destruction in type 1 diabetes individuals; GRP78 could have a dual functions of protecting NIT-1 cells from CTL mediated lysis and of stimulating a population of T cells with cytokine profile of IL-4 which may be part of a mechanism to down-regulate an immune response, should be paid more attention to improvement of immunorejection in beta-cell transplantation.更多还原