1型糖尿病小鼠模型构建及胰岛B细胞中胰岛素表达

Establishment of Type 1 Diabetic Mouse Models and Study on the Expression Changes of Insulin in Islet B Cells

目的:构建1型糖尿病(T1DM)小鼠模型,研究T1DM小鼠胰岛B细胞表达胰岛素(Ins)及血清Ins水平的变化。方法:正常雄性C57BL/6J小鼠104只,随机分为实验组、盐水对照组和正常对照组;模型的诱导采用连续多次小剂量链脲佐菌素给药法(MLDSTZ)制作T1DM小鼠模型;分别于注射后第3、7、10、14、21及28天检测小鼠的空腹血糖、体重,取血清及胰腺组织,应用免疫组织化学SABC单染法观察胰岛B细胞Ins表达,图像分析、形态计量法检测Ins阳性细胞平均灰度值、计算面数密度(NA),酶联免疫吸附法(ELISA)检测血清Ins水平。结果:与正常及盐水对照组比较,实验组小鼠第10天以后出现明显的多饮、多尿、活动减少等典型糖尿病(DM)表现,空腹血糖水平从实验第14天开始明显升高,以第28天组最高,差异有统计学意义(P<0.01);实验组小鼠胰岛面积有所减小。胰岛Ins阳性细胞在实验第10、14及21天组染色加深,免疫反应强度增强,平均灰度值降低,差异有统计学意义(P<0.01);NA从第7天开始减小,呈逐渐下降趋势,与正常及盐水对照组比较,差异有统计学意义(P<0.01);实验组小鼠与对照组相比,血清Ins浓度从实验第7天后均处于较低水平。结论:T1DM模型小鼠胰岛面积减小,胰岛B细胞数量减少,合成和分泌Ins总量减少且表达下调,血清Ins水平下降。

Objective： To establish mouse models of type 1 diabetes mellitus （T1DM）, and to inves- tigate the expression changes of insulin in islet B cells and serum insulin levels of mice with T1DM. Methods： A total of 104 normal male C57BL/6J mice were randomly divided into three groups： experiment group （EG）,saline control group （SCG） and normal control group （NCG）. Mouse models of T1 DM were induced through injecting low dose of streptozotocin （MLDSTZ） continuously and repeat- edly. Fasting blood-glucose and weights of the mice were measured subsequently on the 3th, 7th ,10th, 14th, 21st and 28th days after the first injection, and the serum and pancreatic tissues of the mice were taken at the same time. The expressions of Ins in islets were detected by immunohistochemical SABC staining method. Islet area, numerical density on area（ Ns ）, and Ins positive cell number in islet were detected with image analysis method. Serum Ins levels were determined with enzyme linked immunosorbent assay （ELISA） method. Results： Compared with NCG and SCG, mice in EG appeared typical diabetes mellitus manifestations such as polydipsia, polyuria and less activity on the 10th day. Fasting blood-glucose level was increased from the 14th day onwards, and with the highest value on the 28th day（P 〈 0. 01）; The area of islets in EG was reduced. Compared with NCG and SCG,the immune staining intensity of islet Ins immunoreaction （Ins-IR） cells was enhanced on the 10th, 14th ,21st days in EG,and the mean gray value was decreased at the same time （P 〈 0.01 ） ; NA of Ins-IR cells was apparently decreased in EG from the 7th day onwards （P 〈 0.01 ） ; The ELISA results showed that the concentration of serum insulin in EG was at a low level from the 7th day onwards. Conclusions： The ar- ea of pancreatic islet of T1DM mice is reduced, the number of B ceils in islet is decreased, the synthesis and secretion of insulin is down-regulated, the serum insulin level is low.