【作者】 赵燕英；

【导师】 陈正望；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 1型糖尿病是一种T细胞介导、以胰腺beta细胞特异性损伤为特征的自身性免疫疾病，目前临床上主要靠终身注射胰岛素维持患者生命。1型糖尿病的早期病理特征是胰岛炎，主要表现为活化的单核免疫细胞(巨噬细胞、树突状细胞和T细胞等)浸润胰岛，这些细胞释放的介质(细胞因子、一氧化氮和氧自由基等)，直接损伤胰腺beta细胞。虽然1型糖尿病发病的分子机理还不清楚，但积累的证据显示与机体免疫相关的细胞因子在这一过程中发挥了关键作用。上世纪90年代，Dr Chen从猪小肠中分离并鉴定出一个多肽，命名为daintain。与此同时，Utans等从鼠心脏移植排斥物中克隆出一个巨噬细胞因子—异体移植炎症因子1 (allograft inflammatory factor-1，AIF-1)，两者氨基酸序列高度同源，故称daintain／AIF-1。作为一个与炎症相关的巨噬细胞因子，daintain／AIF-1引起了许多学者的关注。本实验室前期研究显示在发生胰腺炎的1型糖尿病模型鼠胰腺中存在大量的daintain／AIF-1，而正常鼠胰腺中很少，提示了daintain／AIF-1参与了1型糖尿病的发生，为了进一步探讨daintain／AIF-1在1型糖尿病发生中的作用及机理，本论文主要开展了以下工作：1．分离纯化daintain／AIF-1以猪小肠为原料，通过Sephadex G-25(fine)凝胶过滤和DEAE纤维素弱阴离子交换层析以及两次高效液相色谱等方法分离蛋白组分，纯化出了daintain／AIF-1。2．制备daintain／AIF-1和胰岛素的单克隆抗体。以纯化的daintain／AIF-1和购买的胰岛素为免疫原免疫Balb／c小鼠，利用杂交瘤技术，采用间接酶联免疫吸附(enzyme linked immunosorbent assay，ELISA)的方法筛选分别针对daintain／AIF-1和胰岛素的单克隆细胞株，并鉴定了抗体的效价、亚类以及亲和常数。3．研究daintain／AIF-1在1型糖尿病的发生发展中的可能作用。利用制备的daintain／AIF-1及其单克隆抗体，通过免疫共沉淀和western blotting，发现小鼠血清中存在daintain／AIF-1，证明daintain／AIF-1是一个分泌的胞外蛋白。间接ELISA显示1型糖尿病模型NOD(non obese diabetic)小鼠血清中daintain／AIF-1的含量显著高于同周龄的Balb／c小鼠。用组织免疫化学法，发现daintain／AIF-1在发生1型糖尿病模型BB(biobreeding)大鼠的胰腺中密集，且胰腺beta细胞逐渐消失，说明daintain／AIF-1参与了损害beta细胞的作用，从而在1型糖尿病的发生发展中起一定作用。4．进而在细胞以及机体水平上研究了daintain／AIF-1促进并导致1型糖尿病发生发展的分子机理。给NOD鼠静脉注射daintain／AIF-1后，其白细胞升高，血中胰岛素减少，伴随血糖升高，导致NOD鼠提前发生糖尿病。免疫组化结果显示其胰腺中胰岛萎缩，beta细胞量很少。同时，体外实验观察到daintain／AIF-1可抑制胰岛素分泌，直接破坏胰岛细胞。将daintain／AIF-1标记上异硫氰酸荧光素(fluorescinisothiocyate，FITC)，利用全内反射荧光显微镜和流式细胞技术，检测胰岛细胞的荧光强度，显示daintain／AIF-1可与胰岛细胞结合。利用亲和层析，寻找并提取daintain／AIF-1在胰腺中的相互作用蛋白，经过胶内酶切，肽指纹图谱测定，以及肽段测序，鉴定出一个相互作用蛋白为胱硫醚beta合成酶。5．daintain／AIF-1对血液生物学特性的影响。将外源daintain／AIF-1静脉注射入昆明鼠可引发炎症反应，例如激发氧化物产生，降低超氧化物岐化酶的活性，引起游离血红素释放，增高血浆c反应蛋白和纤维蛋白原的含量，伴随着血液粘度的提高，造成血液在空气中更快速的凝固，提高在体内促进血液凝固的功能。daintain／AIF-1处理后的小鼠血清可以消耗更多的还原剂二巯基苏糖醇。镍-组氨酸pull down以及western blotting鉴定daintain／AIF-1在血液中与血红蛋白beta亚基1结合，促进红细胞裂解，血红素释放。炎症、氧化应激以及游离的血红素作为糖尿病血管并发症主要诱因的事实提示daintain／AIF-1与糖尿病的血管并发症相关。以上研究表明daintain／AIF-1是1型糖尿病的致病因子之一，参与并促进了1型糖尿病的发生发展，与糖尿病血管并发症相关，可以作为1型糖尿病治疗的新的靶点。更多还原

【Abstract】 Type 1 diabetes is a multifactorial autoimmune disease, which is characterised by Tcell mediated destruction of the beta cells in the pancreas. The early stages of the diseaseprocess are termed insulitis, the infiltration of the pancreatic islets by mononuclearimmune cells, including macrophages, dendritic cells, and T cells. Beta-cell death in thecourse of insulitis is probably caused by direct contact with activated macrophages andT-cells, and/or exposure to soluble mediators secreted by these cells, including cytokines,nitric oxide (NO), and oxygen free radicals. Although the molecular pathways for theinitiation, perpetuation, and eventual destruction of the beta cell in the disease areunknown, accumulating experimental evidence implicates cytokines as key mediators.In the mid 1990s, Dr Chen isolated and characterized a polypeptide from porcineintestines and named it as "daintain". Contemporaneously, Utans et al identified a novelmacrophage factor, allograft inflammatory factor-1(AIF-1), in rat cardiac allografls withchronic rejection. Daintain is identical to AIF-1 in amino acid sequence, therefore we callthe polypeptide as daintain/AIF-1. As an inflammation maker in macrophages, thiscytokine has rapidly gained interest from a fast growing group of scientists. Our perviousstudy demonstated there was an accumulative daintain/AIF-1 immunostraining in thepancreas of BB rats when they were suffering from insulitis. To further investigate theroles of daintain/AIF-1 in type 1 diabetes, the following work has been done, including:1. daintain/AIF-1 purification and identification. Daintain/AIF-1 was purified fromporcine intestine with a sephadex G-25 (fine) column chromatography, DEAE anionexchange column chromatography and reverse-phase HPLC purification.2. monoclonal antibodies (mabs) against daintain/AIF-1 and insulin production. Thepurified daintain/AIF-1 and insulin were then used to immunize Balb/c mice as theimmunogen. Both hybridization technique and enzyme linked immunosorbent assay(ELISA) were performed to generate cell lines which secreted mabs against the twopeptides. Isotypes, titer and affinity of the mabs were identified.3. daintain/AIF-1 participated in the pathogenesis of type 1 diabetes. Using the mabagainst daintain/AIF-1, daintain/AIF-1 was found as a circulating protein in the blood ofBalb/c and NOD mice with immunoprecipitation followed by western blotting. Theconcentration of daintain/AIF-1 in the plasma of NOD mice was distinctly higher compared with the same aged Balb/c mice, which suggested daintain/AIF-1 wasassociated with type 1 diabetes. Furthermore there was still daintain/AIF-1immunostraining in the pancreas of BB rats when they were suffering from type 1 diabetes,but no beta cells in pancreas. These data implicated that daintain/AIF-1 was involved inbeta cell death in type 1 diabetes.4. the roles and action mechanism of daintain/AIF-1 in type 1 diabetes. Whendaintain/AIF-1 was intravenously injected into NOD mice, it promoted white blood cellproliferation, increased the concentration of blood glucose, and decreased theconcentration of insulin in the blood and pancreas, aggravated diabetes in NOD mice. Invitro, daintain/AIF-1 inhibited insulin secretion from islets of Langerhans, and damagedthe primary pancreas islet cells. Daintain/AIF-1 was labeled with FITC, and fluorescencewas detected on pancreas islet cells by total internal reflection fluorescence microscope(TIRFM) and flow cytometer. Daintain/AIF-1-interacting protein was identified ascyatathionine-beta-synthase (CBS) in the mice pancreas by peptide mass fingerprinting.5. As a circulating protein in the blood, daintain/AIF-1 impaired plasma superoxidedismutase activity, enhanced concentrations of C-reactive protein and fibrinogen,accompany with higher blood viscosity and faster blood coagulation. The serum from themice administrated with daintain/AIF-1 oxidized more dithiothreitol (DTT). Furthermore,AIF-1 interacting protein was separated by an AIF-1/histidine fusion protein nickelaffinity assay. MALDI-TOF mass analysis identified hemoglobin subunit beta-1 as anAIF-1 interacting protein. This interaction was verified by westem blotting. This was afunctional interaction, because AIF-1 induced cytolysis of erythrocytes, and further boundwith hemoglobin subunit beta-1 resulting in heme release in vitro. Oxidative stress,inflammation, free heme and blood coagulation contribute to the vesicular complicationsin diabetes. These data suggested that daintain/AIF-1 associated with the vesicularcomplications in diabetes.In conclusion, dainain/AIF-1 as an inflammatory factor taken critical roles in theinitiation and progress of type 1 diabetes.更多还原