【摘要】：目的探討腫瘤壞死因子α(tumor necrosis factor alpha,TNF-α)誘導L929-A細胞發(fā)生受體相互作用蛋白激酶1(receptor-interacting protein 1,RIP1)依賴性凋亡的分子機制。方法通過胰蛋白酶濃度梯度消化及蛋白質(zhì)印跡法檢測RIP1、胱天蛋白酶8(caspase-8)和Bid蛋白的表達和線粒體定位;利用熒光探針標記法檢測TNF-α處理后L929-A細胞內(nèi)的活性氧(reactive oxygen species,ROS)水平、胞內(nèi)鈣離子濃度、線粒體膜電位(mitochondrial membrane potential,MMP)及三磷酸腺苷(adenosine triphosphate,ATP)濃度,應用試劑盒檢測線粒體呼吸鏈復合體Ⅰ、Ⅲ的活性變化;采用RIP1激酶特異性抑制劑壞死抑素1(necrostatin-1,Nec-1)和Bid基因敲除的L929-A細胞評估RIP1激酶活性和Bid蛋白在介導細胞死亡中的作用。結果 RIP1、caspase-8和Bid蛋白均定位在線粒體外膜上;TNF-α處理后3 h即可誘導Bid剪切,伴隨Bid剪切,線粒體呼吸鏈復合體功能檢測顯示復合體Ⅲ的活性受到抑制,MMP下降。TNF-α處理后6~12 h細胞內(nèi)ROS升高、鈣離子濃度上升、ATP濃度降低;抑制RIP1激酶活性或敲低Bid蛋白可完全拮抗TNF-α誘導的細胞毒性。結論 TNF-α通過誘導RIP1激酶活性依賴的Bid剪切,繼而抑制線粒體呼吸鏈和細胞能量代謝,誘導細胞死亡。
[Abstract]:Objective to investigate the molecular mechanism of receptor interacting protein kinase 1 (receptor-interacting protein 1) -dependent apoptosis in L929-A cells induced by tumor necrosis factor 偽 (tumor necrosis factor alpha-TNF- 偽. Methods the expression of RIP1, cystatin 8 (caspase-8) and Bid protein and mitochondrial localization were detected by trypsin concentration gradient digestion and Western blotting, and the levels of reactive oxygen species (reactive oxygen speciesRos in L929-A cells treated with TNF- 偽 were detected by fluorescence probe labeling. Intracellular calcium concentration, mitochondrial membrane potential (mitochondrial membrane potentialMMP and adenosine triphosphate ATP concentration were used to detect the activity of mitochondrial respiratory chain complex 鈪，

本文編號：2141340