Study on the development of therapy for insulin-dependent diabetes melitus addressing the regulation of pancreatic beta-cells death.

针对胰岛β细胞死亡调节的IDDM基因治疗进展。以小鼠胰岛β细胞系Min-6和β-TC为材料。测定抗氧化活性、胰岛素分泌、胰岛素原基因表达，并对胰岛素原基因启动子进行分析。β细胞损伤是指用肿瘤坏死因子-α和白介素1-β处理这些细胞所引起的DNA损伤。为了调节抗氧化活性和氧化还原，我们克隆了谷胱甘肽(GSH)合成的限速酶--γ-谷氨酰半胱氨酸合成酶(GCS)基因，并对其5‘侧翼区进行了分析。为抑制γ-GCS的表达，将核酶构建的伽马-GCS反义密码子导入β细胞。与其他小鼠组织细胞相比，Min-6和β-TC细胞的GSH水平和GSH相关酶活性降低了90%，提示β细胞具有较低的氧化还原活性和氧化应激能力。GSH.3可下调胰岛素的分泌。胰岛素原的表达不受GSH.4的调节。GSH对细胞因子诱导的胰岛β细胞损伤有保护作用。反义γ-GCS-核酶的表达增加了胰岛素的分泌。这些数据表明，β细胞中低浓度的GSH对胰岛素的分泌是必不可少的，但对氧化剂对细胞的损伤很敏感。综上所述，针对抑制GSH合成的新基因疗法被认为对IDDM有效。

Development of gene therapy for IDDM addressing regration of pancreatic beta-cell death. We used mouse pancreatic beta-cell lines, Min-6 and beta-TC as materials. We estimated antioxidant activities, insulin secretion, expression of preproinsulin gene, and analyzed the preproinsulin gene promoter. beta-cell damage was estimated as DNA damage induced by the treatment of these cells with TNF-alpha and IL- 1beta. To regulate the antioxidant activity and redox, we cloned the gene of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme for the glutathione (GSH) synthesis, and analyzed the 5'-flanking region of gamma-GCS gene. To suppress the expression of gamma-GCS,beta-cells were transfected with antisense codon of gamma-GCS constructed with ribozyme.Results1. Levels of GSH and activities of GSH related enzymes decreased by 90% in Min-6 and beta-TC cells compared to other mouse tissues cells, suggesting beta-cells are feasible to oxidative stress and possess less redox activity.2. The insulin secretion was down-regulated by GSH.3. The expression of preproinsulin was not regulated by GSH.4. GSH protected beta-cell damage by cytokines.5. Transfection of anti-gamma-GCS-ribozyme caused of increase in insulin-secretion.6. These data suggest low concentration of GSH in beta-cells is essential for the insulin secretion while sensitive to cellular damage by oxidants.Collectively, new gene therapy targeting the suppression of GSH synthesis thought to be effective to IDDM.