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目的探讨肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)诱导L929-A细胞发生受体相互作用蛋白激酶1(receptor-interacting protein 1,RIP1)依赖性凋亡的分子机制。方法通过胰蛋白酶浓度梯度消化及蛋白质印迹法检测RIP1、胱天蛋白酶8(caspase-8)和Bid蛋白的表达和线粒体定位;利用荧光探针标记法检测TNF-α处理后L929-A细胞内的活性氧(reactive oxygen species,ROS)水平、胞内钙离子浓度、线粒体膜电位(mitochondrial membrane potential,MMP)及三磷酸腺苷(adenosine triphosphate,ATP)浓度,应用试剂盒检测线粒体呼吸链复合体Ⅰ、Ⅲ的活性变化;采用RIP1激酶特异性抑制剂坏死抑素1(necrostatin-1,Nec-1)和Bid基因敲除的L929-A细胞评估RIP1激酶活性和Bid蛋白在介导细胞死亡中的作用。结果 RIP1、caspase-8和Bid蛋白均定位在线粒体外膜上;TNF-α处理后3 h即可诱导Bid剪切,伴随Bid剪切,线粒体呼吸链复合体功能检测显示复合体Ⅲ的活性受到抑制,MMP下降。TNF-α处理后6~12 h细胞内ROS升高、钙离子浓度上升、ATP浓度降低;抑制RIP1激酶活性或敲低Bid蛋白可完全拮抗TNF-α诱导的细胞毒性。结论 TNF-α通过诱导RIP1激酶活性依赖的Bid剪切,继而抑制线粒体呼吸链和细胞能量代谢,诱导细胞死亡。
Objective To investigate the molecular mechanism of tumor necrosis factor alpha (TNF-α) -induced receptor-interacting protein 1 (RIP1) -dependent apoptosis in L929-A cells. Methods The expression of RIP1, caspase-8 and Bid protein and the mitochondrial localization of mitochondria were detected by trypsin concentration gradient digestion and Western blotting. Fluorescent probe labeling was used to detect the expression of RIP1, The levels of reactive oxygen species (ROS), intracellular Ca2 +, mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) were measured by enzyme linked immunosorbent assay The activity of RIP1 kinase and the effect of Bid protein in mediating cell death were evaluated by using RIP1 kinase-specific inhibitor necrostatin-1 (Nec-1) and Bid gene-knockdown L929-A cells. Results Both RIP1, caspase-8 and Bid proteins were located on the mitochondrial outer membrane. Bid-cutting was induced at 3 h after treatment with TNF-α, and with Bid cleavage, the activity of complex III was detected Inhibition, MMP decline. Intracellular ROS increased, calcium concentration increased and ATP concentration decreased 6 ~ 12 h after TNF-α treatment. Inhibition of RIP1 kinase activity or knockdown of Bid protein completely antagonized TNF-α-induced cytotoxicity. Conclusion TNF-α can inhibit the mitochondrial respiratory chain and cell energy metabolism and induce cell death by inducing RIP-dependent kinase bidirectional cleavage.