谢胜男 侯文睿 鲁憬莉 奚炜 向明

　　基金项目：国家自然科学基金资助项目(30772580)

　　作者单位：430030武汉，华中科技大学同济医学院药学院药理教研室(谢胜男、侯文睿、鲁憬莉、向明);宜昌市第一人民医院药剂科(奚炜)

　　通讯作者：向明，E-mail: xiangming@mails.tjmu.edu.cn

　　【摘要】目的 探讨PBudCE4.1/Reg基因联合胰岛素自身抗原共同防治1型糖尿病的作用及机制。方法  构建PBudCE4.1/Reg基因;采用40 mg/kg链脲佐菌素(STZ)连续5 d腹腔注射建立1型糖尿病小鼠模型，小鼠每周肌肉注射Reg基因，皮下注射胰岛素不完全弗氏佐剂(IFA)混合乳剂各1次，连续4周。每周测定小鼠血糖，6周后处死小鼠。采用四唑盐比色法(MTT)检测小鼠脾淋巴细胞增殖反应;采用流式细胞术(FACS)分析脾淋巴细胞CD4+CD25+ Foxp3+调节性T细胞阳性率;采用ELISA法检测小鼠血清胰岛素分泌水平;采用HE染色法进行胰腺组织病理组织学检查。  结果  Reg/胰岛素联合给药可有效减少胰岛细胞损伤及炎性细胞浸润，提高糖尿病小鼠胰岛素分泌水平，从而显著降低糖尿病小鼠血糖值(P<0.01)，且停药2周后，降糖作用仍持续存在;同时可降低脾淋巴细胞增殖能力(P<0.01)，促进CD4+CD25+ Foxp3+调节性T细胞分化(P<0.05)。  结论  Reg基因联合胰岛素自身抗原对STZ诱导的T1DM模型有明显的抗糖尿病作用及协同效应，作用机制可能与免疫耐受的诱导及β细胞修复再生有关。

　　【关键词】PBudCE4.1/Reg基因;胰岛素;糖尿病，1型

　　doi:12.3969/j.issn.1006-6187.2011.02.014

　　Antidiabetic effects of PBudCE4.1/Reg gene combined with insulin on type 1 diabetic mice

　　XIE Sheng-nan, HOU Wen-rui, LU Jing-li, et al. School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

　　Corresponding author: XIANG Ming, E-mail: xiangming@mails.tjmu.edu.cn

　　【Abstract】Objective　　To investigate the antidiabetic effects of PBudCE4.1/Reg gene combined with insulin on streptozotocin (STZ)-induced type 1 diabetic mice and explore a new way for diabetes gene therapy.  Methods  The recombinant vector carrying Reg gene was constructed (PBudCE4.1/Reg). The T1DM model was established in BALB/c mice by a daily intraperitoneal injection of 40 mg/kg of STZ per mouse for 5 consecutive days, PBudCE4.1/Reg gene and/or insulin were i.m. and s.c. injected, respectively, into the diabetic mice once a week for consecutive 4 weeks. At the end of experiment, pancreas tissues were taken for histologic examination with HE staining. Splenocytes were harvested for the assay of lymphocyte proliferation with MTT method. Serum insulin was determined according to the protocol described by the manufacturer of the mouse insulin ELISA kit. Besides, splenocytes were also subjected to the determination of the ratio of the CD4+CD25+ Foxp3+ T cell subpopulations to FACS.ResultsPBudCE4.1/Reg gene was constructed successfully. The level of blood glucose in mice treated with Reg combined with insulin was significantly lower than diabetic model control (P<0.01), which lasted stably after the treatment withdrawal. No lymphocyte infiltration in the islets of the pancreas and no obvious β cell destruction could be found in the pancreatic tissues from mice receiving the combination treatment. And administration of Reg combined with insulin tended to bring serum insulin to normal values as compared with the diabetic control mice. A depressed lymphocyte proliferation and higher ratio of the CD4+CD25+ Foxp3+ T cell subpopulations to FACS were demonstrated in the Reg/insulin treated mice as compared with those in the diabetic control ones (P<0.01 and P<0.05, respectively).ConclusionsReg gene combined with insulin prevents mice from developing hyperglycemia, possibly by inducing immunotolerance and accelerating the growth of β cell in diabetic mice.

　　【Key words】PBudCE4.1/Reg; Insulin; Diabetes mellitus, type 1