Mechanism of Fas-mediated apoptosis of pancreatic β-cells.

我们将人Fas基因导入小鼠β细胞系(βTc1)，建立了表达人Fas基因的β细胞克隆。该克隆命名为Hfas/βTc1，在抗Fas抗体作用下发生了凋亡，表现为染色质凝集、核仁解体、核小体间DNA片段化和Annexin V染色。Fas被抗Fas抗体交联后，Caspase-3样蛋白水解酶活性升高，但Caspase-1样蛋白水解酶活性无明显变化。Caspase-3抑制剂可抑制Fas介导的β-细胞凋亡。此外，反义caspase-3构建阻断了caspase-3的激活并显著抑制了Fas诱导的Hfas/βTc1细胞的凋亡。这些观察结果提示，Caspase-3在Fas介导的β细胞株的凋亡中起重要作用。油酸可促进Fas介导的β细胞凋亡，提示脂肪酸可能参与了酮症酸中毒引起的β细胞的凋亡。为了评估caspase抑制剂对NOD小鼠胰岛素炎的影响，我们在注射环磷酰胺后开始注射z-VAD-fmk，每天0.2 mg。15只对照小鼠中有8只发生糖尿病，15只接受治疗的小鼠中有5只发生糖尿病。在为期21天的实验中，所有接受治疗的小鼠都存活了下来，而对照组有3只小鼠死亡。15只对照小鼠中有3只和15只接受治疗的小鼠中有9只仍有轻微的胰岛素炎。因此，自身免疫性胰岛素炎可以通过抑制半胱氨酸天冬氨酸氨基转移酶来部分减轻。

We transfected human Fas cDNA into a mouse β-cell line (βTC1) and established a β-cell clone expressing human Fas. This clone, designated hFas/βTC1, underwent apoptosis when exposed to anti-Fas showing hallmarks of apoptosis, i.e. , chromatin condensation, nucleolar disintegration, internucleosomal DNA fragmentation, and Annexin V staining. The crosslinking of Fas by anti-Fas resulted in the elevation of caspase-3-like, but not caspase-1-like, protease activity. A caspase-3 inhibitor attenuated the Fas-mediated β-cell apoptosis. Furthermore, an antisense caspase-3 construct blocked caspase-3 activation and substantially suppressed Fas-triggered apoptosis of hFas/βTC1 cells. These observations suggest the essential role of caspase-3 in Fas-mediated apoptosis of the β-cell line. Fas-mediated apoptosis of hFas/βTC1 cells was augmented by the addition of oleate, suggesting that fatty acids may be involved in β cell apoptosis associated with ketoacidosis. To assess the effects of caspase inhibitors on insulitis of NOD mice, we started injections of z-VAD-fmk of 0.2 mg/day following a cyclophosphamide injection. Diabetes developed in 8 of 15 control mice and in 5 of 15 treated mice. All treated mice survived during the 21-day experiment while 3 mice died in the control group. Insulitis remained mild in 3 of 15 control mice and in 9 of 15 treated mice. Thus autoimmune insulitis may be partially attenuated by inhibition of caspases.