【摘要】 目的:研究重组热休克蛋白65通过鼻黏膜免疫途径对NOD小鼠胰腺炎和糖尿病发生的影响。方法:通过PCR方法克隆出结核分枝杆菌的热休克蛋白65的基因序列,构建了pET28a-HSP65高效表达载体,在大肠杆菌中高效可溶性表达,利用阴离子交换柱层析分离纯化。在不添加佐剂的情况下以纯化的HSP65通过3次鼻黏膜免疫非肥胖型1型糖尿病模型动物(NOD)小鼠,每月采集小鼠的血清,通过酶联免疫吸附试验(ELISA)和Western blot检测血清中抗体的产生,利用自动生化分析仪检测血糖浓度。结果:通过鼻黏膜免疫途径可成功诱导小鼠产生特异的抗HSP65抗体,组织病理学分析也显示HSP65通过鼻黏膜免疫能降低NOD小鼠胰腺病变的产生。结论:HSP65通过鼻黏膜免疫能预防NOD小鼠糖尿病的发生,证实了HSP65经鼻黏膜免疫能降低和自身免疫性糖尿病有关的炎症过程的严重性,对1型糖尿病的治疗提供了一个新的途径。更多还原

【Abstract】 AIM: To study the efficacy of heat shock protein 65 kDa(HSP65) of Mybobacterium tuberculosis var.bovis in prevention of autoimmune diabetes by intranasal.METHODS: The HSP65 gene was derived from Mybobacterium tuberculosis var.bovis genome by PCR and successfully expressed as soluble protein in Escherichia coli.The recombinant protein HSP65 was purified by anion exchange column chromatography,then used to immunize prediabetic NOD(non-obese diabetic) mice via three intranasal(i.n.) delivery in absence of adjuvants.Serum samples from the immunized mice were collected at monthly intervals.The anti-HSP65 antibody was detected by enzyme-linked immunosorbent assay(ELISA) and verified by Western blot analysis.The concentration of blood glucose was measured by automatic analyzer.RESULTS: Specific anti-HSP65 antibodies were successfully induced in mice immunized via intranasal routes.Histochemical analysis of mice pancreas tissue showed that HSP65 intranasal vaccination could decrease pathological changes in NOD mice.CONCLUSION: Intranasal vaccination with HSP65 in NOD mice could prevent the development of diabetes.Our results demonstrate that intranasal vaccination with HSP65 reduces significantly the inflammatory process associated with auto-immune diabetes.This approach may offer novel therapeutic avenues for the treatment for of type 1 diabetes mellitus.更多还原