【作者】 方妮；

【导师】 娄晋宁；

【作者基本信息】 北京协和医学院 ， 病理学与病理生理学， 2014， 博士

【摘要】 高糖、高脂和细胞因子等刺激所导致的内质网应激,在2型糖尿病的胰岛素抵抗和胰岛p细胞功能衰竭中发挥了重要作用。胰岛p细胞对内质网应激极为敏感,由内质网应激导致的p细胞凋亡是2型糖尿病发病的重要环节。研究内质网应激诱导胰岛p细胞凋亡的分子机制对阐明2型糖尿病的发病机制以及糖尿病的防治都具有极其重要的意义。Tribble同源蛋白3(tribbles homolog3, TRIB3)是近年发现的一种重要的应激相关蛋白,可被多种刺激因素诱导,具有广泛的生物学功能。TRIB3在胰岛素靶组织中的上调表达,参与了胰岛素抵抗的发生及2型糖尿病的进展。内质网应激反应可通过转录激活因子4(Activating transcription factor4, ATF4)-C/EBP同源蛋白(C/EBP homology protein, CHOP)诱导TRIB3表达,但TRIB3在内质网应激诱导的胰岛β细胞凋亡中的作用及机制尚不清楚。目的：通过检测糖尿病动物模型胰岛组织,以及内质网应激条件下胰岛p细胞系(INS.1)中TRIB3和CHOP的表达情况,分析TRIB3在糖尿病胰岛β细胞功能衰竭中的作用；并通过上调或敲降TRIB3,研究其对内质网应激诱导的INS.1细胞凋亡的影响,阐明TRIB3在糖尿病胰岛β细胞功能衰竭中的作用及机制。方法：为明确TRIB3是否参与糖尿病状态下胰岛的内质网应激反应,采用免疫蛋白印迹(western blots)和实时荧光定量多聚酶链反应(real-time PCR)方法,分别检测发生糖尿病的GK大鼠胰岛和db/db小鼠胰腺中TRIB3和CHOP的表达水平；并利用毒胡萝卜素(Thapsigargin, Tg)于腹腔给予db/m小鼠在体内诱导胰岛p细胞内质网应激和细胞凋亡,检测p细胞caspase-3/7活性,western blots检测β细胞TRIB3和CHOP表达水平。为分析TRIB3在内质网应激诱导胰岛β细胞凋亡中的作用,利用Tet-on系统构建可诱导表达TRIB3的大鼠胰岛β细胞系(TRIB3细胞系),采用real-time PCR、 western blots和细胞免疫荧光等方法检验所构建的p细胞Trib3基因的可诱导性。然后利用Tg或衣霉素(Tunicamycin, Tm)诱导内质网应激反应,采用DNA ladder和脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)方法观察Tg或Tm诱导的INS-1细胞凋亡；在我们构建的可诱导表达TRIB3的β细胞系中,分析TRIB3过表达或敲降(RNA干扰)后对Tg、Tm诱导p细胞凋亡的影响。进一步,为分析TRIB3在体内对内质网应激诱导胰岛p细胞凋亡的影响,将TRIB3细胞系移植到经链脲霉素(streptozotocin,STZ)造模的糖尿病小鼠肾包膜下,分别于腹腔单独给予Dox诱导TRIB3过表达,或单独给予Tg诱导内质网应激反应,或两者联合应用,尾静脉监测动物空腹血糖水平和ELISA检测胰岛素分泌水平,并应用免疫组化染色检测移植瘤细胞TRIB3的表达和TUNEL染色检测移植瘤细胞的凋亡为研究TRIB3介导内质网应激诱导胰岛β细胞凋亡的机制,首先在体外对TRIB3细胞系给予Dox诱导TRIB3表达,另给予Tg诱导内质网应激。然后采用western blots检测核转录因子(NF-KB)的活性,real-time PCR检测诱导型一氧化氮合成酶(inducible nitric oxide synthase,iNOS)的表达,以及Griess法检测一氧化氮(nitric oxide, NO)的生成。并利用吡咯烷二硫代氨基甲酸盐(pyrrolidine dithiocarbamate, PDTC)来抑制NF-κB的活性,通过TUNEL染色检测PDTC的干预对Dox单独应用或Dox与Tg联合应用对β细胞凋亡的影响。最后进行体内实验来验证TRIB3的作用机制：免疫组化染色分别检测GK大鼠和db/db小鼠糖尿病发病前后胰岛p细胞NF-KB活性的改变,为体内研究提供实验依据。将TRIB3细胞系移植至STZ造模小鼠肾包膜下,在单独应用Dox或Dox与Tg联合应用的基础上,给予PDTC预处理2h进行干预,检测动物空腹血糖水平和胰岛素分泌水平。免疫组化染色观察PDTC干预对移植瘤细胞NF-κB活性的影响。TUNEL染色检测移植瘤细胞凋亡的情况。结果：TRIB3与糖尿病动物胰岛的内质网应激反应有关：糖尿病GK大鼠和db/db小鼠p细胞CHOP和TRIB3的表达明显上调。应用内质网应激药物诱导db/m小鼠体内胰岛p细胞凋亡,伴随有CHOP和TRIB3的表达明显上调,说明TRIB3与糖尿病状态下的内质网应激反应有关,并可能参与了胰岛β细胞凋亡TRIB3促进内质网应激诱导的胰岛p细胞凋亡：采用Tet-on系统,我们建立了Trib3基因可诱导表达的胰岛p细胞系。Dox对Trib3基因表达的诱导作用具有明显的时-效和量-效关系。利用该细胞系的分组研究发现,单独给予Dox组使TRIB3过表达以及单独给予Tg组诱导的内质网应激均可一定程度地诱导p细胞凋亡,两者联合组(Dox+Tg+)p细胞凋亡比单独给药组显著增加,组间统计学差异显著(P<0.001)。而敲降TRIB3的表达可显著减少内质网应激诱导的β细胞凋亡。说明TRIB3介导了内质网应激诱导的胰岛β细胞凋亡。体内验证实验发现,TRIB3细胞系在体内成瘤后,未给药组糖尿病动物空腹血糖下降急剧,单独给予Dox或Tg可减缓血糖下降幅度,但两者联合给药(Dox+Tg+)可使动物空腹血糖水平升高、胰岛素分泌水平降低。病理学检查发现其机制是在Tg诱导的内质网应激条件下,TRIB3过表达诱导了大量移植瘤细胞凋亡。TRIB3通过活化NF-KB促进内质网应激诱导的胰岛β细胞凋亡：Tg诱导内质网应激后,TRIB3过表达明显增强NF--κB活化,iNOS表达上调,NO和活性氧(Reactive oxygen species,ROS)生成增多,以及caspase-3活化。应用PDTC抑制NF-κB活化后,TRIB3过表达所诱导的细胞凋亡明显减少；而且在Tg诱导内质网应激条件下,TRIB3过表达所诱导的细胞凋亡减少得更明显。说明TRIB3通过激活NF-κB通路介导内质网应激诱导的胰岛p细胞凋亡。体内研究发现NF-κB在糖尿病GK大鼠和db/db小鼠胰岛内均活化的证据。进一步在我们以TRIB细胞系建立的移植瘤动物模型中,发现经PDTC干预后,在Tg诱导内质网应激条件下,TRIB3使NF-KB活化的作用明显受抑制。不仅如此,NF-κB活化受抑制后,在Tg诱导内质网应激条件下,TRIB3过表达不再使动物空腹血糖水平升高,反而出现了动物空腹血糖水平下降,血清胰岛素水平升高,p细胞凋亡减少的结果。这些结果进一步提示,TRIB3通过活化NF-KB通路发挥其促进内质网应激诱导胰岛p细胞凋亡的作用。结论：糖尿病状态下胰岛p细胞TRIB3表达上调,参与内质网应激诱导的胰岛β细胞凋亡,其机制是TRIB3使NF-κB通路活化并启动其下游的氧化应激和细胞凋亡途径促进β细胞凋亡更多还原

【Abstract】 Endoplasmic reticulum (ER) stress induced by hyperglycemia, palmitate and pro-inflammatory cytokines, contributes to insulin resistance and pancreatic β cell dysfunction in type2diabetes. Pancreatic β-cells are vulnerable to ER stress. ER stress-mediated pancreatic β cell apoptosis plays a crucial role in the development of type2diabetes. Elucidating the molecular mechanisms underlying ER stress-induced β cell apoptosis would facilitate the understanding of the causes of diabetes mellitus, and open avenues for the development of new therapies.Tribbles homolog3(TRIB3) is an important stress-related protein. It is widely expressed in many tissue and organism with multiple biological functions and can be induced by various stimulis. TRIB3ovexpression has been observed in many insulin target tissues, shaping the risk of insulin resistance and type2diabete. TRIB3can be induced via ATF4-CHOP pathway under ER stress condition, however, whether TRIB3involved in ER stress-induced β cell apoptosis and its mechanism are unclear.Objectives:The TRIB3and CHOP expression in the diabetic animal pancreas, and the TRIB3and CHOP expression in INS-1cells under conditions of ER stress were studied, to analyse the potential influence of TRIB3on β cell dysfunction; upregulated or knockdown of TRIB3expression to investigate the behavior of TRIB3in ER stress induced β-cell apoptosis and it mechanism.Methods:To analyse whether TRIB3is involved in ER stressed islets in a diabetic environment, the expression of TRIB3and CHOP were detected by western blots and real-time PCR in diabetic animal islets;db/m mice were intraperitoneally injected with thapsigargin(Tg) to induce β cell apoptosis, caspase-3/7activity was studied, then TRIB3and CHOP expression were measured by western blots. The effect of TRIB3on ER stress induced β cell apoptosis were studied both in vitro and in vivo. By use of tetracycline tet-on system, the Dox inducible TRIB3stable cell line (named TRIB3cells) was established. The induction of TRIB3expression in β cell line was confirmed by real-time PCR and immunofluorescence staining. The time-response and dose-response relationship between Dox and TRIB3were analyzed by western blots. We used RNA interference to knockdown Trib3. Tg or Tunicamycin(Tm) was used to induce ER stress, and INS-1cell apoptosis was analyzed by DNA fragementation and TUNEL staining. The apoptosis of βcells with TRIB3overexpression or Trib3knockdown under ER stress condition was analyzed by TUNEL staining. To study the effect of TRIB3on ER stress induced β cell apoptosis in vivo, TRIB3cells were used for sub-renal capsule transplantation in streptozotocin (STZ)-diabetic mice. The mice were intraperitoneally injected once daily with Dox to induce TRIB3expression, and with Tg to induce ER stress. We studied the levels of blood glucose and insulin in mice. TRIB3expression in the xenografts was studied by western blots, insulin and C-peptide secreted in the xenografts were measured by immunohistochemical staining. The apoptosis of TRIB3cells with or without TRIB3overexpression, treated with Tg or not, were analyzed by TUNEL staining.To study the mechanism involved in TRIB3mediated ER stress-induced β cell apoptosis in vitro. TRIB3cells were treated with Dox to induce TRIB3expression and Tg to induce ER stress. NF-κB activation was analyzed by Western blots, inducible nitric oxide synthase (iNOS) expression was analyzed by real-time PCR, and inducible nitric oxide synthase (NO) production was analyzed by Griess method. We then used the pyrrolidine dithiocarbamate (PDTC) to inhibit NF-κB activity, and studied its effect on β cell apoptosis induced by single use of Dox or combined use of Dox with Tg.We further studied the mechanism in vivo.The activation of NF-κB was detected by immunohistochemical staining in the islets of diabetic GK rats and db/db mice.In order to investigate the role of NF-κB in β cell apoptosis, TRIB3cells were again used for sub-renal capsule transplantation in STZ-diabetic mice. The mice were pretreated with PDTC for2h, then treated with single use of Dox or combined use of Dox with Tg. We examined the levels of blood glucose and insulin in mice. We applied immunohistochemical staining to study the effect of PDTC on NF-κB activation influenced by TRIB3overexpression under ER stress condition. The apoptosis of TRIB3cells in the xenografts were analyzed by TUNEL staining. Results:TRIB3upregulation was correlated with ER stressed pancreatic islets. TRIB3and CHOP expression were upregulated in islets of diabetic GK rat and db/db mice.β cell apoptosis induced by thapsigargin(Tg) in db/m mice, was accompanied by upregulation of TRIB3and CHOP. These results indicated that TRIB3was involved in ER stress-induced β cell apoptosis.TRIB3overexpression enhanced ER stress induced β cell apoptosis. The Dox inducible TRIB3cell line was established, and TRIB3expression was induced by Dox in a dose-dependent and time-dependent manner. Treatment with Dox or Tg increased cell apoptosis in the inducible TRIB3cell line, while treatment with both Dox and Tg significantly increased cell apoptosis than the single use of Dox or Tg(P<0.001). Under conditions of ER stress, Trib3knockdown significantly inhibited β cell apoptosis. Studies in the sub-renal capsular transplantation animal model discovered that, after TRIB3cells formed xenografts in vivo, the levels of blood glucose decreased. However, the decreased blood glucose improved after administration of Dox or Tg. Besides, administration both of Dox and Tg increased blood glucose levels and decreased insulin levels. TUNEL studied in the xenografts uncovered that, TRIB3overexpression enhanced ER stress induced β cell apoptosis.TRIB3enhanced ER stress induced β cell apoptosis via NF-κB activation. Mechanism studied in vitro discovered that NF-κB pathway plays an important role in TRIB3-mediated β cell apoptosis. Under the condition of ER stress, NF-κB activation, iNOS mRNA upregulation, NO and Reactive oxygen species(ROS) overproduction, as well as caspase-3activation, were enhanced significantly after TRIB3induction; however, after exposed to PDTC for2h, TRIB3induction failed to increase apoptosis in β cells under the condition of ER stress. These results indicated that TRIB3mediated ER stress induced β cell apoptosis via NF-κB activation and the downstream target molecular of NF-κB. Studies in vivo discovered that, NF-κB activated in the islets of diabetic GK rats and db/db mice. Again in our established transplantation animal model, we discovered that, after treated with PDTC, NF-κB activation by TRIB3induction under ER stress was inhibited obviously. As NF-κB activation was inhibited, TRIB3induction failed to increase blood glucose, but to increase the levels of insulin and inhibit β cell apoptosis, under the condition of ER stress. These results indicated that TRIB3mediated ER stress induced β cell apoptosis via NF-κB pathway. Conclusions:TRIB3is upregulated in the ER stressed pancreatic islets in a diabetic environment. TRIB3overexpression enhanced ER stress induced β cell apoptosis and the mechanism is refered to NF-κB activation and the oxidative stress.更多还原