【作者】 冯皓；

【导师】 郭葆玉；

【作者基本信息】 第二军医大学 ， 微生物和生化药学， 2004， 硕士

【摘要】 1型糖尿病旧称胰岛素依赖性糖尿病(IDDM),系T淋巴细胞介导的器官特异性自身免疫性疾病,以胰腺β细胞浸润、胰岛炎症、β细胞凋亡、胰岛素分泌不足为特点。其发病机理目前尚不完全明了。根据氨基酸序列中两个半胱氨酸的位置,趋化因子可分为四个亚家族:CXC、CC、C和CX3C。近年来的研究显示CC趋化因子及其受体家族在诸如实验性过敏反应性脑脊膜炎、多发性硬化症、类风湿性关节炎和1型糖尿病等自身免疫性疾病的发病过程中有重要作用。目 的:本研究拟用我实验室保存的抗小鼠CCR5膜外第一襻多克隆抗体,在1型糖尿病模型动物体内封闭CCR5分子,观察动物发病的变化,并对其中可能的机制作初步的研究。方 法:NOD.Scid(severe combined immunodeficiency)小鼠系先天性缺乏T淋巴细胞、B淋巴细胞和NK细胞的严重联合免疫缺陷鼠,利用自身免疫疾病可以通过过继致敏淋巴细胞被动转移的原理,我们将发病的1型糖尿病模型动物-NOD(Nonobese diabetic)鼠的脾细胞过继至上述NOD.Scid小鼠体内,建立过继性1型糖尿病小鼠模型。将20接受过继的NOD.Scid小鼠随机分成3组(A组6、B组7、C组7)。A组腹腔注射上述抗CCR5膜外第一襻抗体,一周3次,共12次;B组用相同方法注射PBS;C组在细胞过继后4～5周开始注射抗体,一周3次,共12次。测定三组小鼠血糖和体重。细胞过继10周或小鼠出现重度糖尿病临床表现时处死。取小鼠胰腺组织作H&E染色,观察其病理学变化,作胰岛炎性积分;取胰腺组织作免疫组织化学染色,检测CCR5在发病小鼠胰腺中的表达。取小鼠脾细胞制成单细胞悬液,用尼龙毛分离得到T淋巴细胞作为反应细胞,以NOD.Scid小鼠胰腺组织作为刺激物,在二氧化碳孵箱中培养,于24小时和48小时分别收集细胞上清作IL-2和IL-10、INF-γ的ELISA分析。<WP=6>结 果:(1)早期注射抗CCR5抗体可部分抑制1型糖尿病。在细胞过继后70天,A组中有2只小鼠出现血糖升高(1只死亡),而B组中所有小鼠均出现血糖升高、多尿、体重减轻的糖尿病临床表现,其中2只死亡。(2)晚期注射抗CCR5抗体未对1型糖尿病的发展产生影响。(3)早期注射抗CCR5抗体可抑制胰岛炎的发生。从胰腺的H&E染色切片上可见,A组未发病小鼠呈正常状态,B组小鼠胰岛单核细胞浸润明显,且数量减少,面积减小。炎性积分A组与B组差异显著。(A组:0.3±0.1,n=5;B组:2.5±0.2,n=4;p<0.001)(4)免疫组织化学检测显示发病的小鼠胰腺CCR5较正常NOD.Scid小鼠有明显提高。(5)抗CCR5抗体对T淋巴细胞产生细胞因子的影响。A组小鼠T细胞上清中所含的IL-2、IL-10、INF-γ水平分别为25.2pg/ml、27.3pg/ml、0.24ng/ml,而B组小鼠T细胞上清中含三种细胞因子水平为57.7pg/ml、31.6pg/ml、18.03ng/ml,统计学分析,两组小鼠的IL-2和INF-γ水平间有显著差异,而IL-10间无显著差异。结 论:本实验进行了趋化因子蛋白受体5(CCR5)同1型糖尿病发生关系的研究。结果表明早期运用体内注射抗CCR5膜外第一襻多克隆抗体封闭过继性1型糖尿病模型小鼠体内CCR5分子,可部分的抑制疾病的发生。起潜在机制,我们认为可能同封闭CCR5分子后影响小鼠的单核细胞向胰腺聚积、浸润,以及影响Th1类细胞因子的分泌有密切的关系。更多还原

【Abstract】 Type 1 diabetes is an autoimmune disease characterized by the destruction of pancreatic islet β-cells by invading leukocytes and the consequent deterioration of the insulin-dependent glucose homeostasis. Although the pathway of the disease has not yet clarified, many studies show that chemokines and chemokine receptors are critically involved in this autoimmune disease by activating migration monocytes and recruitment of T cells into islets. Depending on the juxtaposition of the first two cysteine residues in amino acid sequence, chemokines are divided into four classes: the CXC, CC, C and CX3C families. Chemokines act through specific receptors that belong to the 7-transmenbrane spanning, G-protein-coupled receptor family. Recent observations suggested CC chemokines might play an important role in autoimmune diseases such as experimental allergic encephalomyelitis, active multiple sclerosis and type 1 diabetes.Objective: We used a specific polyclonal antibody targeting the first extracellular loop of CCR5, in the well-established NOD mouse transfer model, which has higher incidence of diabetes than the spontaneous one, to explore: 1) whether the development of diabetes could be affected by blocking part of CCR5; 2) whether they can serve as the potential therapeutic targets? Methods: Two diabetic female NOD mice, with blood glucose of 27.2mmol/L and 29.4mmol/L respectively, were killed and suspensions of splenocytes were washed twice in RPMI1640 medium containing 10% fetal calf serum. Each NOD.Scid mouse was injected i.p. with 1×107 splenocytes. 20 NOD.Scid mice were randomly divided into three groups. One group was injected i.p. with 200 μl PBS. The other two were injected with 10μg of anti-CCR5 Ab (diluted in 200 μl PBS) per mouse each time. PBS, <WP=8>anti-CCR5Ab were injected i.p. into NOD.Scid mice respectively 1 h before cell transfer and then the injection continued for 4 wks three times a week after transfer (a total of 12 injections of 120 μg of Ab per mouse). Blood glucose levels were measured to observe the anti-diabetogenic effect of the antibody. Histological examination, immunohistochemical analysis and ELISA analysis were performed to find the underlying mechanism. Results:(1) Early administration of anti-CCR5Ab prevents diabetes. Within 70 days, all of the control mice developed diabetes while 4 of the 6 mice treated with anti-CCR5 Ab were diabetes-free. (2) Late administration of anti-CCR5Ab does not affect the development of diabetes and insulitis. (3) Early administration of anti-CCR5Ab inhibits insulitis. The results of H&E staining and histological examination showed that the mice treated with PBS (7 wks after transfer) had extensive insulitis, whereas anti-CCR5-treated mice revealed no insulitis). The difference of inflammation scores between two groups was significent. (mean±SD: PBS: 2.5±0.2 n=4 mice/group; anti-CCR5: 0.3±0.1 n=5 mice/group; p<0.001 anti-CCR5 vs PBS, Student’s-t test)(4) Immunohistochemical analysis of pancreatic sections prepared from diabetic recipient mice showed that the islet cells were intensively stained after incubation with anti-CCR5 Ab. The pancreatic islet of normal NOD.Scid mice failed to be stained with the same concentration of anti-CCR5 Ab.(5) Effects of Anti-CCR5 on the secretion of islet Ag-reactive Th1 cells. The supernatant of cultured T cells derived from anti-CCR5-treated mice contained little amounts of IFN-γ (0.24ng/ml) and IL-2 (25.2pg/ml) whereas that from control mice contained much more amounts of IFN-γ (18.03ng/ml) and IL-2 (57.7pg/ml). There was no significant difference in IL-10 (anti-CCR5 vs control Ab 27.3pg/ml vs 31.6pg/ml) produced by islet Ag-specific T cells.Conclusion:Early administration of polyclonal antibody, targeting the first extracellular <WP=9>loop of CCR5, could induce the resistance to type 1 diabetes in the adoptive transfer model. The mechanisms by which anti-CCR5 Ab prevents the diabetes may be included the remission in insulitis by direct inhibition of migration of monocytes int更多还原