【作者】 何苏丹；

【导师】 王晓东；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 细胞死亡对于多细胞生物体的发育和自稳平衡起着至关重要的作用。细胞凋亡是生理情况下最主要的程序性细胞死亡方式,而细胞坏死曾被认为是一种被动的意外死亡,但是,最近很多证据表明,细胞坏死也是受到调控的,并参与缺血性心脑血管疾病和神经退行性等疾病的发生。因此,阐明程序性坏死的分子机制和研发有效的抑制剂将为制定有效的治疗策略提供强有力的科学依据。肿瘤坏死因子TNFα是一个多效性细胞因子,参与调节炎症反应、细胞凋亡和坏死等。我们实验室已经发现Smac/Diablo蛋白的类似物Smac mimetic能诱导TNFα-依赖性细胞凋亡,此途径需要激酶RIPK1的参与。有趣的是,当细胞凋亡受到caspase抑制剂zVAD阻止时,Smac mimetic能诱导一些细胞发生依赖于TNFα和RIPK1的细胞坏死。我们利用高通量RNAi筛选技术鉴定了RIPK1的同家族蛋白RIP3是调控TNFα-诱导性细胞坏死的关键蛋白,其激酶活性是必不可少的。在受到坏死信号刺激时,一个包含有RIP3和RIPK1的蛋白复合体会被诱导形成。过量表达的激酶死亡突变体会与内源性RIPK1相结合从而抑制细胞坏死途径。我们还发现RIP3只在一些细胞中选择性表达,其表达跟TNFα-诱导性细胞坏死密切相关,但不参与TNFα诱导的细胞凋亡途径。而且,异位表达RIP3使得对TNFα-诱导性细胞坏死有抗性的细胞转变成敏感型细胞。我们还建立了RIP3基因敲除小鼠用于RIP3-依赖性细胞坏死的生理学意义。同样的,RIP3-/-MEF也表现出对TNFα-诱导性细胞坏死的抗性。在雨蛙素诱导的小鼠急性胰腺炎模型中,RIP3基因的敲除显著地减少了胰腺细胞的坏死,有效降低了胰腺组织的损伤。这些发现表明RIP3是TNFα-诱导性细胞坏死途径的关键调控蛋白,并可成为新的药物靶点,为相关疾病的治疗提供新的思路。此外,我们对20万个化合物进行高通量筛选寻找细胞坏死途径的抑制剂,由此发现超过20个化合物可通过干扰信号途径的不同阶段从而有效的抑制TNFα-诱导性细胞坏死。这些化合物应用前景广泛,对其作用靶点的鉴别将有利于揭示细胞坏死途径中新的调控蛋白,而且在动物疾病模型上的深入研究还将促进有效药物的开发,为相关疾病的治疗提供新的手段。更多还原

【Abstract】 Cell death is a crucial process for development and tissue homeostasis in multicellular organisms by eliminating unwanted and harmful cells.Apoptosis,a form of programmed cell death,is the principal mechanism by which cells are physiologically removed.In contrast to apoptosis,necrosis was originally described as an accidental and passive form of cell death. However,recent evidences suggest necrosis is also well controlled and programmed,and plays a prominent role in pathological conditions including ischemia and neurodegeneration. Understanding the molecular mechanisms of programmed necrosis and exploring potent inhibitors could lead to powerful therapeutic strategies for these devastating diseases. Tumor necrosis factor(TNF)αis a pleiotropic factor involved in diverse cellular responses, including inflammation,apoptosis and necrosis.Previously,our lab has shown that Smac mimetic,a small molecule mimic of Smac/Diablo protein,induces TNFα-dependent apoptosis in a receptor interacting protein kinase 1(RIPK1)-dependent manner.Interestingly,when apoptosis was bloCked by a pan- caspase inhibitor,z-VAD,cell death phenotype was switched from apoptosis to necrosis in certain cell lines.This Smac mimetic induced necrosis is also TNFαand RIPK1-dependent.Using the high-throughput RNA interference(RNAi) screening assay,we identified RIP3,a serine-threonine kinase and homolog of RIPK1,as an essential regulator in the TNFα-induced necrosis.Stable expression of shRNA targeting RIP3 blocked necrotic cell death while the cell death phenotype was rescued by introducing a shRNA-resistant wild type form of RIP3,but not a kinase deficient RIP3.Upon necrotic stimuli,a protein complex containing both RIPKI and RIP3 was formed.Overexpression of a kinase-dead form of RIP3 blocked necrosis by sequestering endogenous RIPK1.We also noted that RIP3 was selectively expressed in certain cell lines and the endogenous expression of RIP3 correlates very well with the cellular response to TNFα-induced necrosis.Furthermore,ectopic expression of RIP3 rendered the resistant cells to TNFα-induced necrosis.RIP3 knockout mice were generated to further investigate the physiological functions of RIP3.Consistently,RIP3-/- MEF was resistant to TNFα-induced necrosis.RIP3 knockout mice are devoid from inflammation inflicted tissue damage in an acute pancreatitis model.These findings demonstrate that RIP3 is a key determinant for TNFα-induced necrotic cell death and therefore offers a new drug target for evading necrotic damage.In addition,we screened a chemical library of 200,000 compounds for the chemical inhibitor of the TNFα-induced necrosis.More than 20 compounds exerted profound inhibition on the TNFα-induced necrosis by interfering with necrosis at different stages of the signaling pathway and provide novel opportunities to develop successful therapies.Further insight into the specific targets of the compounds will uncover novel molecules in the regulation of necrosis.更多还原