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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,降糖作用仍持续存存;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。
Objective To investigate the role and mechanism of PBudCE4.1 / Reg gene combined with insulin autoantigen in prevention and treatment of type 1 diabetes mellitus. Methods PBudCE4.1 / Reg gene was constructed. A mouse model of type 1 diabetes mellitus was established by intraperitoneal injection of streptozotocin (STZ) 40 mg / kg for 5 days. The mice were intramuscularly injected with Reg gene and subcutaneous injection of insulin (IFA) mixed emulsion once each for 4 weeks. Mouse blood glucose was measured weekly and mice were sacrificed after 6 weeks. The spleen lymphocyte proliferation reaction was detected by tetrazolium salt (MTT) assay. The positive rate of CD4 ~ + CD25 ~ + Foxp3 ~ + regulatory T cells in splenic lymphocytes was analyzed by flow cytometry (FACS) The level of insulin secretion was detected in mice. Pathological examination of pancreatic tissue was performed by HE staining. Results The combination of Reg / insulin could effectively reduce islet cell injury and inflammatory cell infiltration, increase the level of insulin secretion in diabetic mice and significantly reduce the blood glucose level in diabetic mice (P <0.01) (P <0.01), and promote the differentiation of CD4 ~ + CD25 ~ + Foxp3 ~ + regulatory T cells (P <0.05). Conclusion Reg gene combined with insulin autoantigen has obvious anti-diabetic and synergistic effects on STZ-induced T1DM model. The mechanism may be related to the induction of immune tolerance and β-cell repair and regeneration.