GAD抗原肽抗体对IDDM糖尿病基因治疗研究

Gene therapy of IDDM diabetics by antibody to GAD Ag peptide

目的 探讨GAD抗原肽抗体诱导特异性免疫耐受对IDDM糖尿病的基因治疗。方法 构建Ig MBAE、GAD Ig MBAE融合蛋白表达载体 ,制备产重组病毒的病毒包装细胞 ,经逆转录病毒载体介导基因转染B细胞 ,对非胖型糖尿病小鼠 (NOD)进行基因治疗 ,以血糖指标和病理组织学分析评价疗效。结果 1.酶切鉴定证实Ig MBAE、GAD Ig MBAE表达载体构建正确。 2 .RT PCR可检测到转染 4 8hGAD Ig B细胞中的GAD Ig融合基因表达 ;Westernblot和双夹心ELISA可检测到转染细胞胞浆和细胞上清中的GAD Ig融合蛋白 ;3.在治疗第 36周 ,GAD Ig B细胞治疗组的糖尿病发病率由B细胞和Ig B细胞组的10 0 %降低至 5 5 .5 % ,胰腺组织病理分析提示 ,GAD Ig B细胞治疗组的胰岛组织形态接近正常 ,胰岛组织中淋巴单核细胞的浸润得到有效控制。结论 GAD抗原肽抗体通过逆转录病毒介导的基因治疗 ,可有效地延迟NOD小鼠的糖尿病发病及明显降低发病率。

Objective Gene therapy with GAD-IgG tolerogens that induces specific tolerance to IDDM. Methods Identification of GAD-IgG were expressed in frame on a soluble IgG fusion protein scaffold and delivered via retroviral gene therapy in LPS-stimulated B cells in vitro by restriction enzyme,RT-PCR,ELISA and Western blot;its role is estimated for the treatment of NOD by in vivo. Results GAD-IgG fusion protein could detected by ELISA and Western blot in cytoplasm of GAD-IgG/B cells and cultured supernatant,respectively. After treatment of 36 weeks, IDDM morbidity of GAD-IgG/B cells treating group were decreased from 100% of IgG/B cells and B cells treating groups to 55.5%. Analysis pathology were showed that pancreatic islet cell might be protected from GAD-IgG/B cell treating NOD mice. Conclusion GAD-IgG could be used to interfere with the diabetic disease and delay the diabetic disease progress. It is important protocols for treatment of developing IDDM or for patients receiving islet cells transplantation.