【作者】 范国权；

【导师】 陈向伟；

【作者基本信息】 山西医科大学 ， 免疫学， 2003， 硕士

【摘要】 目的：1 谷氨酸脱羧酶的免疫干预NOD小鼠糖尿病的确定 2 谷氨酸脱羧酶对NOD小鼠的量效观察及其机理的探查 3 谷氨酸脱羧酶对NOD小鼠不同周龄发病的影响 方法： 实验一 将3周龄20只体重10±09g NOD小鼠分为4组分别腹腔注射谷氨酸脱羧酶(GAD)3mu、GAD3mu+IFA50μl、IFA50μl、PBS50μl，定期做糖尿病检测，动态观察各组糖尿病发生发展的过程。根据发病率来确定免疫干预药物，经过实验后GAD3mu+IFA50μl发病率最低，效果最好，所以GAD3mu+IFA50μl为下一步实验研究对象。 实验二 将确定免疫干预药物GAD3mu+IFA50μl作为观察对象，探测GAD与NOD小鼠糖尿病的量效关系，将3周龄体重10±0.9g糖尿病小鼠40只随机分为4组，第一组为GAD0.03+IFA50μl，第二组为GAD3mu+IFA50μl，第三组为GAD0.3mu+IFA50μl，第四组PBS对照组。定期检测血糖，眼内眦取血分离血清。到28周时，全部处死，取脾、胸腺作淋巴细胞亚群测定，胰腺作病理组织学观察。 实验三 由于NOD小鼠3周龄时，开始出现自身抗原GAD，8周龄时达高峰，随后降低。为探讨GAD3mu+IFA50μl对不同周龄NOD小鼠糖尿病的影响，将3周龄的糖尿病小鼠40只随机分为4组，第1组在3周龄时腹腔注射GAD3mu+IFA50μl，第2组在8周龄时腹腔注射山厄叮医科大学布皿创匕学位卡仑文GAD3mu+IFAS印1，第3组在12周龄时腹腔注射GAD3mu+正A50州，第4组PBS对照组。动态观察血糖、尿糖，并且眼内毗取血分离血清。28周龄时全部处死，取脾、胸腺检测淋巴细胞亚群的变化及胰腺作病理组织学检测。 采用双抗夹心法测定血清中细胞因子的变化，应用流式细胞仪测定淋巴细胞亚群的变化，并采用普通HE染色观察胰腺病理组织学改变。实验血糖、血清中细胞因子含量资料采用SAS软件作等时间距重复测量处理。T淋巴细胞亚群和胰岛病理学检测采用单因素方差分析，另外胰岛炎评分采用SAS非参数检验。结果: 1.通过对实验一血糖资料的分析，发现GAD3mu十IFA50川对NOD小鼠有稳定血糖的作用，各个时间点血糖无差别，并且与其他处理组有显著差异。3周龄时对NOD小鼠腹腔注射GAD3mu十丁A50川，对其糖尿病的发病有明显干预作用，可以作为本实验的研究对象。 2.实验二GAD各剂量组的血糖研究发现第2组GAD3mu+IFAS卯l，具有维持NOD小鼠血糖稳定，并且第2组在12周龄时与其他剂量组有差别。第2组NOD小鼠血清中细胞因子水平，IL一4在13周龄时比其他剂量组显著升高(P<0.05)，最后到28周时各剂量组无差别。血清中Y一IFN水平，2组增加比其他剂量组缓和，28周时2组均值比其他剂量组值显著降低(P<0.05)。2组胸腺淋巴细胞亚群分析CD4+/CDS+比例为2.34士O，21，相对的CD4+细胞所占比例最小(P<0.05);脾淋巴细胞亚群分析，2组在各剂量组中CD矿/c DS十比例为1.91士0.犯，比值最小(P<0.05)。胰腺病理切片观察，2组胰岛个数最多7.88士1.22个/张，而且胰岛炎评分最低。从中说明2组通过调节淋巴亚群变化以至血清中细胞因子变化，来维持NOD小鼠血糖的稳定性。 3.实验三探讨GAD3mu+IFA50川对各周龄的NOD小鼠糖尿病的影响，发现第1组胰岛个数最多，胰岛炎评分最低。GAD在NOD小鼠抗原未出现前诱导T细胞耐受，可能产生TS抑制性淋巴细胞。在胸腺中，第1组CD4+阳性细胞所占比例最小，于第2组有显著差异，而于第3组无差异;CDS+细胞在各组无显著差异;CD4+/C DS+比率在各组有显著差异。在脾脏中，CD4+细胞各组间无差别，CDS+细胞各组间有显著性（P＜0．05人血清中IL＊水平，总趋势增加，各组之间有差异。YJFN水平第1组比其他组显著降低，到28周时第1组于第2组YJFN水平无差别。从以上说明GAD对NOD小鼠糖尿病的作用受自身GAD表达水平的影响，通过调节淋巴细胞亚群变化以及分泌 Thl和 ThZ类细胞因子来调节血糖。结论： 谷氨酸脱校酶（GAD）早期可以对NOD小鼠糖尿病的发生进行调节。GAD3mu＋IFA50…剂量组对NOD小鼠糖尿病的免疫干预效果最好，主要是通过调节胸腺，脾淋巴细胞亚群分类，并且通过淋巴细胞分泌Th．1类因子和 Th－2类因子来调节Tho细胞的转化，从而抑制NOD小鼠胰岛炎，胰岛p细胞破坏减少，最终GAD免疫干预NOD小鼠糖尿病的发生。GAD3mu＋IFA50…对各周龄组的影响。由于GAD自身抗原在3周龄时开始出现，8周龄时达最高峰，随后降低。本实验三采用3周龄，8周龄，12周龄，3个周龄组观察，发现单体 GAD在 3周龄组主要是通过诱导ThZ类团于表达增加，抑制Th类因子表达，促进CDS”阳性TS细胞比率增加。8周龄组是通过单体GAD封闭自身反应性淋巴细胞，导致 Thl类因子分泌减低，Th－2类因子分泌相对增加。12周龄组是自身抗原GAD经过8周时表达高峰，再次注射GAD对已经活化的自身反应性淋巴细胞作用不大，对其分泌细胞因于的调节作用不明显。但比对照组ThZ类细胞因子增加。总的看来，谷氨酸脱校酶在早期适宜的剂量腹腔注射可以免疫干预NOD小鼠糖尿病的发生。更多还原

【Abstract】 Objective1. Immune medicament can be chosen.2. Different dose of GAD effects the incidence of diabetes in NOD mice. And the function can be discovered.3. GAD effects the incidence of diabetes in different week old NOD mice. And mechanism can be found.Methods1. Twenty NOD mice in three-week old were devided into 4 groups at random . Group 1 was injected GAD3mu ; group 2 was injected GAD3mu+IFA50ul ;group 3 was injected EFA50ul ;group 4 was injected PBSSOul as control group. Blood glucose were measured in every two weeks. At last group 2 was the lowest in the incidence of diabetes. Therefore GAD3mu+IFA50 l is as the observation in this experiment.2. Based on experiment 1 result ,GAD3mu+IFA50 l is the observation. Being found different dose of GAD effection the incidence of diabetes in NOD mice,40 mice were devided into 4 groups at random. In group 1 mice were injected GAD0.03mu+IFA50 l; in group 2 mice were injected GAD3mu+IFA50 l; in group 3 mice were injected GAD0.3mu+IFA50 l; group 4 is control group.Blood glucose and the serum leveles of IL-4 and y-IFN were measured in regulation time . At the age of 28 weeks , mice were killed and the subsets of T cell in spleen and in thymus were determined by two-clolor FACS analysis . Parcreatic histopathology studies were performed by H-E.3. Injection GAD3mu+IFA50 l in belley effect the incidence ofdiabetes in different week old NOD mice.40 mice that were three week old are randomly devided into 4groups.In group l,mice were injected in three week old ;in group 2, mice were injected in eight week old;in group3 ,mice were injected in twelve week; group 4 was control group.Blood glucose and the serum leveles of IL-4 and y-IFN were measured in regulation time . At the age of 28 weeks , mice were killed and the subsets of T cell in spleen and in thymus were determined by two-clolor FACS analysis . Parcreatic histopathology studies were performed by H-E. Results1. In GAD3mu+IFA50 l group,mice blood glucose balance . Therefore GAD3mu+IFA50ul is observation in thise experiment.2. In group 2 , mice that were injected GAD3mu+IFA50ul had kept balancein blood glucose. The serum levels of IL-4 were increased significantly ,and y-IFN was on the contray (p<0.01). The lymphocytic inflammation of pancreatic islets in group 2,and the insulitis score decreased markedly(p<0.01). A strong proliferative response of CD8+ subsets in splenocytes occurred in 2group, and CD4+/CD8+ ratio was lowest in the groups (p<0.01) .3. In group 1 blood glucose are normal level ,on the contrary other groups increased very rapidly .In group 1 mice IL-4 level in surms kept balance, and y-IFN level increased lowest in all groups(p<0.001). The insulitis score was the smallest(p<0.01). CD4+/CD8+ratio is lowest in all groups(p<0.01).ConclusionGAD3mu+IFA50ul prevents the onset of type 1 diabetes and reduces the risk of insulitis in female NOD mice at the age of three weeks.Its mechanisms seem to be related to the regulation ThO cell switch to the Th2 eel by cytokine such as IL-4 .更多还原