自身抗原胰岛素皮下注射诱导1型糖尿病模型小鼠的免疫耐受作用

A Study of Immune Tolerance Induced by Subcutaneous Administration of Autoantigen Insulin in Mice with IDDM

目的探讨自身抗原诱导免疫耐受防治1型糖尿病(IDDM)的方法及机制。方法将32只8周龄BALB/c小鼠随机分为4组:空白对照组、模型对照组、皮下注射给药组和灌胃给药组,各8只。各组小鼠每日腹腔注射链脲佐菌素(STZ)40 mg.kg-1,连续5 d,建立IDDM模型。皮下注射给药组于造模前1 d每只皮下注射胰岛素100μg,以后每周给药1次,连续4周。灌胃给药组于造模前1 d每只灌胃给予胰岛素150μg,以后每周给药2次,连续4周。每周测定血糖值。6周后处死小鼠,取胰腺组织HE染色观察胰岛组织变化,四唑盐比色法(MTT法)测定小鼠脾淋巴细胞增殖反应。另取脾细胞采用荧光激活细胞分类技术(FACS)分析CD4+CD2+5T细胞亚群比例。结果与模型对照组比较,皮下注射给药组血糖值明显降低(P<0.05),而灌胃给药组则差异无显著性。胰腺HE染色发现模型对照组胰腺有明显的炎性细胞浸润,而皮下注射给药组胰岛基本无浸润。与空白对照组和模型对照组比较,皮下注射给药组脾淋巴细胞增殖能力降低(P<0.05)。FACS显示皮下注射给药组CD4+CD2+5T细胞亚群比例明显高于空白对照组和模型对照组。结论皮下注射自身抗原胰岛素可以诱导免疫耐受防治糖尿病,其机制与促进体内CD4+CD2+5T细胞亚群产生相关。

Objective To probe into the method and mechanism of the prevention and treatment of IDDM in mice by induction of immune tolerance with the autoantigen insulin, Methods 32 male BALB/c mice 8 weeks of age were randomly divided into 4 equal groups： blank control, model control, insulin s, c. （subcutaneous injection） and insulin gg （gastrogavage）, The IDDM model was established in mice of the 4 groups by a daily intraperitoneal injection of 40 mg·kg^-1 of streptozotocin （STZ） per mouse for 5 consecutive days, Mice of the insulin s. c, group were given each a subcutaneous injection of 100μg of bovine insulin one day before the establishment of the IDDM model and once weekly thereafter for 4 consecutive weeks, Animals of the insulin gg group were given each 150μg of bovine insulin administered by gastrogavage one day before the establishment of the IDDM and the same treatment was thereafter repeated twice weekly for 4 consecutive weeks. Blood glucose was then determined weekly and the animals of all 4 groups were sacrificed 6 weeks later, Pancreas tissues were taken for histologic examination with HE staining and splenocytes harvested for the assay of lymphocyte proliferation with the MTr method. Besides, splenocytes were also subjected to the determination of the ratio of the CD^＋ CD25^＋ T cell sulpopulations with FACS. Results An IDDM model was successfully established in BALB/c mice by repeated intrapritoneal injection of low dose STZ as evidenced in mice of the model control group by beta cell destruction as a result of lymphocyte infiltration. The level of blood glucose in mice of the insulin s.c. group, but not that in animals of the insulin gg group, was significantly lower than that in mice of the model control group （ P 〈 0.05 ）. By and large no lymphocyte infiltration in the islets of the pancreas and no obvious 13 cell destruction could be found in the pancreatic tissues from mice of the insulin s. c. group. A depressed lymphocyte proliferation and higher ratio of the CD^＋ CD25^＋ T cell subpopulations were demonstrated in mice of the insulin s.c. group as compared with.those in mice of the blank and model control groups（P 〈 0.05 ）. Conclusion Subcutaneous administration of bovine insulin in mice with IDDM was shown to induce immunotolerance resulting in protection against the disease, possibly by the up-regulation of the CD^＋ CD25^＋T cell subpopulations.