谷氨酸脱羧酶特异调节的T细胞抑制NOD小鼠的自身免疫性糖尿病

目的 探讨抗原特异调节的T细胞在非肥胖型糖尿病(NOD)小鼠自发性糖尿病中的作用。方法 应用流式细胞计数仪辅以荧光标记组织相容性抗原Ⅱ类分子四聚体染色，从自发性糖尿病的NOD小鼠和非糖尿病的BALB／c小鼠中分离出谷氨酸脱羧酶(GAD)多肽特异性T细胞。分别应用抗原刺激试验、酶联免疫反应、细胞内细胞因子染色检测上述T细胞的细胞因子表达。最后将上述T细胞给NOD／scid小鼠静脉注射，检测这些T细胞对糖尿病发生的影响。结果 当用不同多肽诱导同一品系时，NOD小鼠T细胞分泌不等量的干扰素-γ，相似量的白介素(IL)-4和IL-10；BALB／c小鼠T细胞分泌类似量的细胞因子。当同一多肽诱导不同品系时，NOD小鼠T细胞较BALB／c小鼠T细胞分泌较少量的IL-2但较大量的IL-4和IL-10。这些NOD小鼠T细胞均可以有效地抑制NOD／scid小鼠的糖尿病发生。结论 两种毗邻的GAD多肽诱导NOD小鼠产生的T细胞表达不同的细胞因子分泌类型，但这些NOD小鼠T细胞均可以有效地抑制NOD／scid小鼠的糖尿病，因此推论这些细胞为分泌独特细胞因子且抑制糖尿病的T调节细胞。     

白细胞介素17介导致糖尿病性T细胞诱导的NOD小鼠糖尿病的发生

目的 研究白细胞介素17(IL-17)在致糖尿病性BDC2.5 T细胞转移性糖尿病发病中的作用.方法 通过注射转基因IL-17 BDC2.5 T细胞(IL-17组,n=6)、IL-17 siRNA BDC2.5 T细胞(silL-17组,n=5),观察高表达和低/无表达IL-17转基因BDC2.5 T细胞对NOD.scid小鼠转移性糖尿病的影响,进行小鼠胰岛组织学鉴定、脾脏和胰腺淋巴结染色和血浆细胞因子测定.非转基因BDC2.5 T细胞注射为对照组(n=8).结果 在观察期内,IL-17组小鼠全部发生糖尿病,而silL-17组和对照组则无一例发病.胰腺病理HE染色显示IL-17组小鼠出现严重的胰岛炎,胰岛受到破坏,结构模糊不清;而silL-17组小鼠胰腺仅表现为轻、中度胰岛炎,可见胰岛内散在的细胞浸润,胰岛组织结构基本完整,细胞形态正常.小鼠脾脏和淋巴结染色显示,IL-17组小鼠脾脏和胰腺淋巴结中CD11c+/CD11b+的树突细胞增殖显著高于silL-17组和对照组(均P＜0.01),且胰腺淋巴结中该细胞增殖显著高于脾脏.IL-17组小鼠血浆IL-17、肿瘤坏死因子α、γ-干扰素和白细胞介素6水平均显著高于siIL-17组和对照组(均P＜0.01),后两组间差异无统计学意义(P＞0.05).结论 BDC2.5 T细胞的IL-17高表达可显著加速NOD.scid小鼠发生转移性糖尿病,且与宿主树突细胞增殖和在胰腺淋巴结局部的聚集以及多种细胞因子作用有密切关系.     

Th1细胞在NOD小鼠糖尿病早期的应用

目的评价Th1细胞对NOD小鼠糖尿病早期变化的作用。方法选择4 w、8 w和16 w的雌性NOD小鼠,采用流式细胞术测定脾Th1细胞,采用免疫组化法检测胰腺内Th1细胞。结果随年龄增加,雌性NOD小鼠脾和胰腺内Th1细胞逐渐增加。结论 Th1细胞参与NOD小鼠糖尿病早期的病理过程。     

雷公藤多甙对NOD小鼠胰岛β细胞半胱天冬蛋白酶表达的影响

用环磷酰胺促进NOD小鼠1型糖尿病的发病，腹腔注射雷公藤多甙（TWP）4周，监测糖尿病发病率，TUNEL法检测胰岛细胞凋亡，免疫组化法观察胰岛β细胞半胱天冬蛋白酶（caspase）3的表达，RT-PCR方法检测胰腺caspase-8 mRNA的表达。结果提示TWP组糖尿病发病率下降，胰岛β细胞凋亡减少，胰岛β细胞caspase-3蛋白及胰腺caspase-8 mRNA的表达均减少。     

己酮可可碱对NOD小鼠胰腺、脾脏Fas和FasL表达的影响

环磷酰胺加速NOD小鼠发病,腹腔注射己酮可可碱(pentoxifylline, PTX)后监测糖尿病发病率, 检测胰岛细胞凋亡、胰岛素表达以及胰腺、脾脏Fas和FasL mRNA的表达.结果 提示PTX组糖尿病发病率下降(P<0.05),胰岛细胞凋亡减少,而胰岛细胞胰岛素的表达增加,胰腺FasL mRNA的表达下降(P<0.05),Fas有下降趋势但差异无统计学意义,脾脏Fas和FasL mRNA表达明显增强(P<0.05或P<0.01).     

大黄酸对NOD小鼠糖尿病肾病的治疗作用观察

目的 :观察大黄酸对NOD小鼠糖尿病肾病的防治作用。　 方法 :在NOD小鼠发生糖尿病后 ,以大黄酸 15 0mg/ (kg·d)灌胃 ,连续 15周 ,进行有关生化和形态学检查。　 结果 :随着观察时间的延长 ,糖尿病NOD小鼠的血糖持续升高 ;而治疗组小鼠则随着大黄酸治疗时间的延长 ,血糖水平进行性下降。 15周时 ,治疗组与模型组血糖水平比较 ,差异非常显著 [(7 8± 3 80 )mmol/Lvs(13 9± 4 80 )mmol/L ,P <0 0 1]。大黄酸明显降低糖尿病NOD小鼠血甘油三酯 [(0 74± 0 13)mmol/Lvs (2 16± 0 73)mmol/L ,P <0 0 1]和胆固醇 [(1 84± 0 5 5 )mmol/Lvs (6 5 3±5 2 7)mmol/L ,P <0 0 5 ]水平 ,预防血肌酐的升高 [(4 5 6± 9 0 ) μmol/Lvs (6 1 8± 6 6 ) μmol/L ,P <0 0 1],也减少 2 4h尿蛋白的排泄 [(0 37± 0 17)mg/ 2 4hvs (3 32± 0 6 8)mg/ 2 4h ,P <0 0 1]。组织学显示 ,大黄酸治疗后的糖尿病NOD小鼠肾小球和丝球体面积明显减少 ,系膜增宽及细胞外基质积聚明显改善。双侧肾脏重量和肾重指数也明显降低。免疫荧光染色结果显示 ,经大黄酸治疗后的糖尿病NOD小鼠其肾小球FN荧光染色明显减弱 ,免疫球蛋白沉积减少。电镜下可见 ,糖尿病NOD小鼠系膜区增宽 ,系膜基质增多 ,肾小球毛细血管袢基膜增厚 ,上     

茶多糖对NOD小鼠1型糖尿病的预防作用

目的探讨茶多糖（TPS）对非肥胖糖尿病（NOD）小鼠1型糖尿病（DM）的预防作用。方法比较TPS预免疫组和生理盐水（NS）对照组NOD小鼠1型DM的发病率、血清C肽和谷氨酸脱羧酶抗体水平、胰岛组织病理学和免疫组化、脾脏T细胞亚群比例。结果TPS预免疫组与NS组比较，DM发病率显著降低，血清C肽水平显著增高，胰岛炎症程度减轻，CD8T细胞亚群比例显著增高，CD4／CD8比例显著降低。结论早期应用茶多糖预免疫可以预防或延缓NOD小鼠1型糖尿病的发生。     

胰岛素样生长因子Ⅰ基因转移对NOD鼠胰岛炎及胰岛β细胞凋亡的保护作用

探讨腺病毒介导的胰岛素样生长因子Ⅰ(Ad-IGF-Ⅰ)对NOD鼠胰岛炎及胰岛β细胞凋亡的影响.结果 发现Ad-IGF-Ⅰ可减少糖尿病的累积发病率,降低胰岛炎积分、促进胰岛β细胞增殖,减少β细胞凋亡率,使Fas、半胱氨酸天冬氨酸蛋白酶3(caspase-3)的蛋白低表达,Bcl-xl蛋白高表达.提示Ad-IGFⅠ可减少NOD鼠胰岛炎,通过Fas和Bcl-xl途径发挥抗凋亡作用.

Abstract：

To explore the protective effect of adenovirus mediated IGF-Ⅰgene(Ad-IGF-Ⅰ)transfer on non-obese diabetic(NOD)mice. The results showed that the incidence of diabetes and degree of insulitis were significantly reduced in mice receiving Ad-IGF- Ⅰ . Treatment with Ad-IGF- Ⅰ significantly decreased apoptosis rate,expression of Fas and caspase-3, and increased expression of Bcl-xl. This study indicates the potential of IGF- Ⅰ gene therapy in protecting NOD mice from insulitis and apoptosis.     

非肥胖糖尿病鼠的致糖尿病T细胞与年龄的相关性

目的研究非肥胖糖尿病(NOD)鼠是否带有致糖尿病的T细胞及其致糖尿病能力是否随着NOD鼠年龄增长而增加。方法用NOD鼠和NOD背景的转基因鼠为动物模型,通过过继转移将不同周龄的NOD鼠T细胞转移到T细胞缺陷的NOD鼠(即NOD.scid鼠)和在β细胞上表达共刺激分子CD80的NOD.scid鼠(即NOD.scid.Rip.B7鼠)。观察过继转移后诱导受鼠糖尿病的发病率及各受鼠组发病率。结果 (1)80%的NOD.scid.Rip.B7鼠在转移脾细胞11 w后发生糖尿病,而NOD.scid受鼠在转移后20 w尚没有1例受鼠发生糖尿病;(2)在转移后同一时间,不同周龄的NOD供鼠在相同受鼠中诱导的糖尿病发病率差异显著(P0.001);不同周龄的受鼠糖尿病发病率无统计学差异(P0.05)。结论 (1)幼年NOD鼠的胰岛没有淋巴细胞浸润是因为β细胞特异性的T细胞数有限,其致糖尿病能力随着NOD供体鼠年龄的增长而增加;(2)在胰岛炎启动前,新生的和幼年的NOD.scid鼠就已经表达与疾病有关的β细胞自身抗原。     

口服胰岛素、谷氨酸脱羧酶对NOD小鼠糖尿病发生率的影响

目的 观察雌性非肥胖糖尿病（ＮＯＤ）小鼠口服胰饥素和氨酸脱羧酶（ＧＡＤ）对糖尿病发生的影响。以及对白细胞介素４（ＩＬ－４）的血甭水平和胰腺ＩＬ－４ｍＲＮＡ转录的影响。方法 雌性ＮＯＤ小鼠分为３组：第一组给予ＰＢＳ作为对照组第二组给予胰岛素,第三组予以ＧＡＤ。ＥＬＩＳＡ法检测血清ＩＬ－４水平,Ｎｏｒｔｈｅｒｎ杂交检测胰腺Ｌ－４ｍＲＮＡ的表达。结果 胰岛素或ＧＡＤ均能抑制ＮＯＤ小鼠糖尿病的发生,ＰＢＳ     

谷氨酸脱羧酶通过调节T细胞亚群预防NOD小鼠发生1型糖尿病

目的 了解谷氨酸脱羧酶（ＧＡＤ）对非肥胖糖尿病（ＮＯＤ）小鼠的１型糖尿病是否具有预防作用并探讨其免疫作用机制。方法 用猪脑ＧＡＤ和不完全弗氏佐剂（ＦＩＡ）混合后给４周龄雌性ＮＯＤ小鼠腹腔注射（３２例）,同时单独射等量ＦＩＡ作为对照组。８周龄时腹腔注射环磷酰胺以加速糖尿病。每周测定体重、血糖,糖尿病形成２周或２０周龄处死小鼠后血清Ｃ－肽、ＧＡＤ抗体（ＧＡＤ－Ａｄ）和脾组织Ｔ细胞亚群,观察胰腺病理、免     

人胰岛素原DNA疫苗预防非肥胖糖尿病鼠胰岛β细胞凋亡的机制探讨

目的探讨人胰岛素原DNA疫苗预防非肥胖糖尿病（NOD）小鼠胰岛β细胞凋亡的机制。方法（1）4周龄NOD雌鼠随机分为磷酸盐缓冲液（PBS）、质粒载体PcDNA3．1（PcDNA）、胰岛素原（Pins）3组，由胫前肌分别注射PBS、质粒PcDNA3．1、人胰岛素原DNA疫苗50pg，1周后重复1次。（2）各组取12周龄未发病的NOD鼠10只，分离胰腺，HE染色观察胰岛炎；TUNEL＋SABC法检测胰岛β细胞凋亡；取脾制成细胞悬液培养72h，ELISA法测定血清白介素4（IL-4）和γ干扰素（IFN-γ）水平；MTT法检测脾细胞增殖反应。结果（1）12周龄时Pins组正常胰岛比例高于PBS组和PcDNA组（均P〈0．01），胰岛周围炎比例低于PcDNA组（P=0．02），胰岛内炎比例则低于PBS组（P〈0．01）和PcDNA组（P=0．006）；（2）Pins组胰岛β细胞凋亡率低于PBS组和PcD—NA组（均P〈0．01）；（3）PIns组血清IL-4／IFN-γ比值显著高于PBS组（P=0．014）和PcDNA组（P=0．036）；（4）PIns组脾细胞对人胰岛素增殖反应的刺激指数（SI）低于PcDNA组（P=0．021）。结论人胰岛素原DNA疫苗通过上调IL-4／IFN-γ比值，使免疫平衡向Th2偏移，致NOD鼠胰岛炎减轻，胰岛β细胞凋亡减少。     

Lymphocyte vaccination protects prediabetic non-obese diabetic mice from developing diabetes mellitus

Summary In the therapeutic manoeuvre termed “lymphocyte vaccination”, activated lymphocytes capable of transferring an autoimmune disease are instead attenuated and given in vaccine form. We have previously shown that such a therapy administered to non-obese diabetic (NOD) mice at 6 weeks of age prevents diabetes mellitus. To assess whether this therapy has potential clinical relevance, in the present study lymphocyte vaccination was applied in NOD mice in 3 weekly doses commencing in the immediate prediabetic period (age 12 weeks), when insulitis is advanced and diabetes incipient. Of 30 NOD mice receiving active vaccine (composed of attenuated lymphocytes from diabetic NOD mice) 13 (43.3 %) remained non-diabetic to the age of 30 weeks, in comparison with 2 of 30 (6.7 %; p < 0.01) mice receiving a control vaccine (composed of attenuated lymphocytes from non-diabetic NOD/B10 mice) and 5 of 26 (19.2 %; p < 0.01) mice receiving saline carrier alone. Moreover, in an additional group of 10 NOD mice receiving active vaccine weekly between 12 and 30 weeks, 8 remained diabetes free at the end of the treatment. The most notable effect of the vaccine was that the delay in diabetes onset was accompanied by a reduction in insulitis and in some cases a complete absence of infiltrating lymphocytes at 30 weeks of age. Immunocytochemistry indicated that when present, islet infiltrating lymphocytes in non-diabetic mice that received active vaccine showed significantly reduced staining for interferon-γ, compared with the infiltrate seen in diabetic mice receiving the control vaccine or saline. This study demonstrates that the rapid progression to diabetes typically seen in 12-week-old NOD mice can be delayed by lymphocyte vaccination, supporting the possibility that a vaccine composed of attenuated autologous peripheral blood lymphocytes could be effective in high risk first degree relatives of patients with insulin dependent diabetes mellitus. [Diabetologia (1997) 40: 1388–1395] Received: 6 May 1997 and in revised form: 24 July 1997     

Changing patterns of cell surface mono (ADP-ribosyl) transferase antigen ART2.2 on resting versus cytopathically-activated T cells in NOD/Lt mice

Aims/hypothesis. ART2.2 is a mouse T-cell surface ectoenzyme [mono (ADP-ribosyl) transferase] shed upon strong activation. We analysed temporal changes in ART2.2 expression in unmanipulated and cyclophosphamide-treated NOD/Lt mice compared with diabetes-resistant control strains. We used NAD, the ART2.2 substrate, to test whether ART-mediated ADP-ribosylation could retard diabetogenic activation of islet-reactive T cells in vitro. Methods. ART2.2 and CD38, another NAD-utilizing enzyme, were measured by flow cytometry. ADP-ribosylation from ethano-NAD was followed by flow cytometry using a reagent specific for etheno-ADP ribose. Results. Although mature NOD CD4 + and C D8 + T cells expressed ART2.2, this expression was delayed in young NOD mice when compared with control strains. This ontological delay at 3 weeks of age correlated with an early burst of CD25 expression unique to NOD splenic T cells. This pattern was reproduced in cyclophosphamide-accelerated diabetes in young NOD/Lt males, wherein a retarded repopulation of ART2.2 T cells in spleen and islets correlated with development of heavy insulitis and diabetes. NAD inhibited anti-CD3 induced activation of splenic T cells in vitro and also retarded killing of beta-cell targets by NOD islet-reactive CD8 effectors in vitro at concentrations equal to or greater than 1 μmol/l. Evidence suggested that CD38 on B lymphocytes competes with ART2.2 for substrate needed by B lymphocytes for ADP ribosylation. Conclusions. ART2.2 on T cells may not simply mark the resting state, but could also contribute to it via ADP-ribosylation. [Diabetologia (2001) 44: 848–858] Received: 27 November 2000 and in revised form: 1 March 2001     

Preventive effects of Cyclosporin on diabetes in NOD mice

Summary Non-obese diabetic mice aged 30 to 60 days were treated orally with Cyclosporin at doses of 25,15 and 2.5 mg/kg every 2 days until 160 days of age. Diabetes developed in 12 out of 18 oil-treated mice (67%), with partial to complete Langerhans' islet destruction associated with lymphocytic infiltration. The non-obese diabetic mice showed a plasma glucose concentration of 6.62 ± 0.92 mmol/l (mean ± SD) at 50 days of age. The plasma glucose level of oil-treated non-obese diabetic mice gradually increased after 130 days of age and reached 14.0 to 19.0 mmol/l at 160 days of age, while Cyclosporin-treated non-obese diabetic mice showed neither clear increase of plasma glucose levels nor development of insulitis. The cumulative incidence of diabetes in Cyclosporin-treated mice was significantly lower than that in oil-treated mice (p < 0.01). Subsequently, Cyclosporin treatment was started after development of glucose intolerance. Twenty-five mg/kg of Cyclosporin was administered every 2 days for 35 days. Cyclosporin appeared to have little therapeutic effect on diabetes in non-obese diabetic mice.     

成年发病狼疮鼠的T细胞受体Vβ基因测序分析

目的分析MRL/lpr和(NZB×NZW)F1两种狼疮鼠成年发病时,广泛浸润于多脏器的T细胞克隆T细胞受体(TCR)Vβ基因编码的氨基酸基序特点,揭示该T细胞克隆的抗原特异性。方法用反转录聚合酶链反应(RT-PCR)分别扩增不同成年发病狼疮鼠个体的肾脏、大脑中的TCRVβ6基因,对扩增产物进行基因克隆和DNA测序分析;单链构象多态性分析法(SSCP)鉴定广泛浸润的T细胞克隆TCRVβ6基因编码的氨基酸基序。结果两种成年发病狼疮鼠的不同个体之间,广泛浸润于多脏器的T细胞克隆TCRVβ6基因的互补决定区3(CDR3)中出现相同的氨基酸基序。结论不同个体成年发病狼疮鼠的多脏器中广泛浸润的T细胞克隆TCRVβ基因的CDR3中出现相同的氨基酸,表明该克隆针对特定的自身抗原。     

Apoptosis and beta-cell destruction in pancreatic islets of NOD mice with spontaneous and cyclophosphamide-accelerated diabetes

Summary Autoimmune-mediated destruction of pancreatic islet beta cells leads to insulin-dependent diabetes in non-obese diabetic (NOD) mice. Although both direct cytotoxic T cell- and indirect cytokine-, nitric oxide- or free radical-mediated mechanisms induce beta-cell apoptosis in vitro, beta-cell death in vivo in spontaneous autoimmune diabetes is not well-characterized. Furthermore, whether beta cells die gradually, or rapidly in the late pre-clinical stage, is a question of current interest. To investigate beta-cell death in vivo, we measured the frequency and intra-islet localisation of apoptosis, defined as DNA strand breaks by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) technique, during spontaneous and cyclophosphamide-accelerated diabetes in NOD mice. In spontaneous diabetes, the frequency of apoptosis in islets correlated with the progression of beta-cell destruction with age. Although apoptosis was detected at low frequency within the reduced insulin-positive islet area of pre-diabetic mice at 90 days of age, it was rarely co-localised to beta cells. After acceleration of beta-cell destruction with cyclophosphamide, the frequency of apoptosis reached maximum at 12 days, at which time 3.2 % of apoptotic cells were beta cells. Apoptosis was most frequent in the insulin-negative islet area comprised of mononuclear cell infiltrate and was localized to CD8⁺ T cells. The rarity of detectable apoptotic beta cells in spontaneous pre-diabetic mice with pronounced insulitis and reduced insulin-positive islet areas most likely reflects the rapid clearance of apoptotic beta cells. Our findings are more consistent with gradual destruction of non-renewable beta-cells in spontaneous diabetes, than with their rapid, accelerated destruction (as after cyclophosphamide) in the late pre-clinical stage. [Diabetologia (1998) 41: 1381–1388] Received: 6 May 1998 and in revised form: 14 July 1998