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

胰岛素依赖型糖尿病基因治疗的发展，解决胰腺β细胞死亡的消退。我们使用小鼠胰腺β细胞系Min-6和β-TC作为材料。我们检测了抗氧化活性、胰岛素分泌、前胰岛素原基因的表达，并对前胰岛素原基因启动子进行了分析。β-细胞损伤估计为用TNF-α和IL-1 β处理这些细胞所诱导的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（gamma-glutamylcysteine synthetase，gamma-GCS）基因，并分析了gamma-GCS基因的5 '侧翼区。为抑制γ-GCS基因的表达，将核酶构建的γ-GCS反义密码子转染β细胞。Min-6和beta-TC细胞中GSH水平和GSH相关酶活性较其他小鼠组织细胞降低了90%，表明beta-TC细胞具有较低的氧化还原活性，更易受到氧化应激的影响. GSH下调胰岛素分泌.前胰岛素原的表达不受GSH的调节. GSH保护细胞因子对β细胞的损伤。5.抗γ-GCS核酶转染引起胰岛素分泌增加。这些数据表明，β细胞中低浓度的GSH对胰岛素分泌是必需的，同时对氧化剂的细胞损伤敏感。

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.