摘要
凋亡和坏死对胚胎发育及维持淋巴系统动态平衡起关键作用。既往研究表明敲除小鼠外源性凋亡途径中的Fas基因可导致淋巴增殖性疾病，而其下游基因FADD、Caspase8和cFLIP基因敲除的纯合子小鼠则不能得以出生，常在孕10.5天时死胎。这种孕中期死亡由另一种类似于坏死的细胞死亡引起。进一步研究发现这种坏死与RIP1有关，即在FADD基因敲除基础上同时敲除RIP1基因，能使胚胎小鼠存活至出生，且大小、外观与野生型小鼠无差别。而Caspase8可阻止RIP3介导的坏死，同时敲除Caspase8、RIP3可使小鼠出生,且随着年龄的增长出现淋巴增殖性疾病，表现为全身的淋巴结及脾脏较野生型对照小鼠明显增大。FADD对RIP3介导的坏死的抑制作用也已经在小肠上皮细胞上得到证实。在T淋巴细胞功能方面，条件性FADD基因敲除的小鼠的T淋巴细胞对TCR介导的增殖反应不良，但在同时敲除RIP1基因后T淋巴细胞增殖能力得到纠正。而RIP3的敲除也使Caspase8敲除的T淋巴细胞功能得到纠正。有研究表明RIP3能够与RIP1相互作用，表明两者具有相似的作用。
既然RIP1的缺失能够纠正FADD缺失导致的胚胎死亡及T淋巴细胞增殖功能障碍，RIP3与RIP1有相似的作用，我们推测RIP3的缺失也能够纠正FADD缺失导致的胚胎死亡及T淋巴细胞增殖功能障碍，为验证上述假说，进一步证实上述凋亡和坏死途径的FADD、RIP3蛋白相互作用关系是否成立，为临床上淋巴增殖性疾病及肿瘤的治疗提供新的探索方向，本文进行了以下研究：
一、将FADD+/-RIP3+/-双重杂合子小鼠杂交获得FADD/RIP3双重基因敲除小鼠DKO
将6周龄大小FADD+/-RIP3+/-双重杂合子异性小鼠置于同一笼内，每天早晨检查阴道栓，结果发现小鼠能够正常受孕，小鼠出生后3周断奶并剪尾，提取DNA，PCR确定基因型，多次传代得到DKO小鼠。Western blot法确定小鼠的淋巴器官已达到FADD、RIP3的双重敲除。将基因型检测结果进行统计分析，结果表明该双重基因敲除DKO小鼠的出生符合孟德尔规律，能够正常出生、成长，监测该种小鼠的体重、进食、活动、生殖行为与对照小鼠无明显差别。该结果说明基因敲除RIP3，能够完全逆转FADD缺失导致的小鼠胎儿的组织坏死，保证胚胎顺利发育。不同于FADD/RIP1双重基因敲除小鼠出生后即死亡于围生期，FADD/RIP3双重基因敲除小鼠出生后可存活且顺利生长。说明在生理情况下，FADD蛋白对RIP3的引起坏死功能有制约作用。该结果未见FADD-/-RIP3+/+和FADD-/-RIP3+/-小鼠，再次证实FADD-/-小鼠不能出生，与既往的研究相符。另外，该实验三种基因型的交配模式中不论FADD为何种基因型均可见到RIP3-/-子代小鼠的出现，也符合既往的研究，即RIP3基因敲除的小鼠能够正常出生并成长，其T淋巴细胞也无增殖缺陷。总结该部分实验， RIP3的敲除纠正了FADD敲除小鼠在孕10.5天时的死胎，首次获得了FADD/RIP3双重基因敲除小鼠，该双重基因敲除小鼠的子代能顺利按孟德尔规律出生及成活，为深入研究该两种蛋白功能奠定了基础。
二、小鼠的淋巴细胞亚群分析
随着小鼠年龄的增长，我们观察到双重基因敲除小鼠出现淋巴增殖性疾病，与同窝的对照小鼠相比较，表现为明显增大的淋巴结和脾脏。脾的重量一般为对照小鼠的5-6倍。不但淋巴器官体积大、重量大，其含有的淋巴细胞数亦较对照小鼠明显增多，脾脏细胞数一般在对照小鼠的6倍左右，淋巴结细胞计数最多可达对照小鼠的10-12倍。
为明确分析该种小鼠中淋巴细胞亚群情况，我们分别将小鼠的淋巴结、脾脏、胸腺细胞行CD3-PE及B220-TC染色，流式细胞仪分析。结果显示该种DKO小鼠的淋巴结、脾脏中出现一个CD3及B220双阳性的异常淋巴细胞群，与对照组比较差异非常明显。胸腺中该群细胞仅轻度增多。
为进一步明确该种细胞的CD4CD8表达情况，将其T细胞再次进行CD4-PE、CD8-TC染色并流式细胞仪分析，从结果中可以看到DKO小鼠单阳性的CD4、CD8细胞比例少于对照小鼠，而CD4、CD8双阴性细胞较对照小鼠增多。明确了DKO小鼠的异常增多的细胞群表型为：CD3、B220双阳性，CD4、CD8双阴性。进一步对该群细胞进行淋巴细胞激活标志的染色，结果表明，不论在淋巴结或是脾脏， CD69表达增高，而CD25、 CD28和CTL-A4的表达两组之间无明显差异。用免疫荧光法检测小鼠血清中抗核抗体情况，1:40稀释小鼠血清，100倍荧光共轭显微镜观察结果，可以看到DKO小鼠荧光强度明显强于对照组小鼠。且随着年龄的增加，DKO小鼠的荧光强度逐渐增强，在6个月时达到最强。
该部分实验结论对于淋巴增殖性疾病的发病机制研究提供新的方向，并为治疗提供新的靶点。
三、小鼠的T淋巴细胞功能分析
通过磁珠法将小鼠T淋巴细胞分离，3H掺入法和CellTrace法检测T淋巴细胞增殖功能。分析表明T淋巴细胞的TCR介导的增殖功能在年轻双重基因敲除小鼠较对照组增强，在相对年老的双重基因敲除小鼠有所减弱。
进一步通过研究DKO小鼠胸腺细胞的杀伤情况及外周T淋巴细胞的AICD来明确T细胞死亡情况。通过anti-Fas杀伤胸腺细胞后PI染色，流式细胞仪分析T淋巴细胞的死亡情况。结果表明年轻、年老DKO小鼠胸腺细胞均表现为对抗Fas抗体诱导的杀伤的抵抗；将外周T淋巴细胞经抗CD3抗体活化后再用抗Fas抗体、TNFR1、抗CD3抗体诱导激活诱导的细胞死亡（AICD）情况，结果表明DKO小鼠的T淋巴细胞死亡情况与野生对照及RIP3-/-对照无差别，但低于FADD-/-的小鼠的T细胞死亡情况。
该部分实验说明RIP3的缺失成功纠正了小鼠FADD缺失导致的T淋巴细胞增殖缺陷，且该DKO小鼠的胸腺细胞对抗Fas抗体诱导的杀伤有抵抗作用。
总结该实验，RIP3的敲除纠正了FADD缺失小鼠在孕10.5天时的死胎，使双重基因敲除小鼠的子代能顺利按孟德尔规律出生及成活，RIP3的缺失也纠正了FADD缺失所致的T淋巴细胞增殖不良。双重基因敲除小鼠的胸腺T细胞对抗Fas抗体诱导的死亡表现为抵抗，对抗Fas抗体、TNFR1、抗CD3抗体诱导的AICD与野生对照无差别。通过将DKO小鼠T细胞增殖及死亡功能分析，表明DKO小鼠发生淋巴细胞堆积的主要原因是细胞死亡减少，推测死亡减少的原因为FADD、RIP3的缺失同时阻断了凋亡和坏死两条死亡信号传导通路，导致细胞永生化生长。该研究为阐明FADD、RIP3在死亡通路上的作用及相互关系提供了新的思路。
Apoptosis and necrosis play a key role in keep embryos development andhomeostasis of the immune syetem. Deletion of the Fas gene leads todevelopment of autoimmune-lymphoproliferative disease.However,thedeletion of FADD,Caspase8or cFLIP resulted in the embryonic lethalityat the day of10.5th of the pregnency.A kind of cell death was found inthe middle pregnancy death of the fetals that is similar to the necrosis.It was exhibitted by the further studies that FADD can suppress RIP1mediated necrosis in embryo development and T cell function. At the sametime it was confirmed that Caspase8is important in keeping away thenecrosis mediated by RIP3. FADD can prevent epithelial cell fromRIP3-mediated necrosis has been demonstrated in previous intestinalepithelial cell related study.T cells proliferation defect in FADDconditional knockout mice were correctted meanwhile by the deletion ofRIP1. Deletion of RIP3restore the proliferation dysfunction of Caspase8deleted T cells.Some studies have indicated that RIP3can interactionwith RIP1.Since RIP3has the similary function with RIP1and RIP1deletioncan rescue the embryos development defect and T cell proliferationdysfunction in FADD knockout mice,we assume that RIP3deletion can alsorescue the embryos development defect and T cell proliferationdysfunction in FADD knockout mouse.To confirm this hypothesis,we do someexperiments as below:
At first,we set up mating of FADD+/-RIP3+/-heterozygous6weeks miceto get FADD RIP3double knockout mice.As expecting, FADD and RIP3doubleknockout mice were generated with predicted Mendelian frequencies in ourlab and the DKO mice can not be distinguished from its littermates inweight,food intake,activity and reproduction at the beginning.Actuallyit shows that RIP3deletion can rescue the embryos development defect inFADD knockout mice. It is different from the RIP1FADD double knockoutmice died right after born,the FADD/RIP3DKO mice can survive as thewild type control mice.It implys that the necrosis caused by RIP3wascontrolled by FADD.It consistent with other research that there is noFADD-/-RIP3+/+and FADD-/-RIP3+/-genetype mouse was born at birth andafter that within the offsprings of the all3differtent genetype mating.No matter what is the FADD genetype,the RIP3-/-mice can be born andsurvival the whole life as the wildtype control.In general,The FADD RIP3double knockout mice is visible and can survival like its littermatecontrol.
Secondly, we analysed the T cell subgroup by flowcytometry. Enlargetedlymph nodes and spleen was found in these older mice though it is notdifferent with control at the birth.It is the FADD-/-/RIP3-/-DKO micethat develop age-dependent LPD (lymphoproliferative disease) resemblingthat of Fas-/-mice. There are distinct spleen cell count differencebetween DKO and control mice, A CD3、B220double positive and CD4、CD8double negative cell population was found in lymph node and spleen of theseDKO mice.To confirm whether the cell is activated, CD25, CD28, CD69and CTL-A4were stained as the T cell activation marker and analysed byflowcytometry.It shows that the CD69expression is higer in DKO mice,andother markers are negative.A anti-ANA kit was used to test the anti-ANAantigen in DKO mice serum by immunofluorescent techniques.It indicatedthat the DKO mice have unanimous positive results comparing with the control.So wo got the conclusion that the DKO mice occur the LPD and itcan provide a new target for investigating the therapy of LPD.
At last, T cell proliferation and cell death were detected. T cellproliferation induced by TCR is a little bit higer than the control inyoung mice,but there is a contray outcome in old DKO mice.Not only youngDKO mice but also old DKO mice have decreased anti-Fas induced thymocytekilling contrasted with their control respectively.It can not bedistinguished of anti-Fas,TNFR1and anti-CD3relatived activationinduced cell death(AICD) from DKO mice and their wild type control thoughthere is a big difference between DKO mice and FADD-/-mice in AICD.
In total,our data indicate that RIP3knockout correct the FADD-/-embryonic dysplasia and the DKO mice gave birth to viable thendevelopment with LPD in adult. The CD69expression is higer in DKO micethan the control though there is no difference in others activationmarker.The DKO mice have unanimous positive results contrasted with thecontrol in serum anti-ANA analysed by immunofluorescent.T cellproliferation defect in FADD knockout mice was restored by RIP3deletion.DKO mice thymocyte was resistent to anti-Fas inducedkilling.There is no differencd in DKO mice and wild type control inanti-Fas,TNFR1and anti-CD3induced AICD.It is supposed the reason leadto LPD is the decreased cell death which is caused by blocking twopathway of apoptosis and necrosis at the same time by double knockout ofFADD and RIP3.
引文
[1] Gluecksmann, A. Cell death in normal vertebrate ontogeny. Biological Reviews,1951,26:59-86.
[2] Lockshin, RA and Zakeri, Z. Programmed cell death and apoptosis: origins of thetheory. Nat Rev Mol Cell Res,2001,256(1):19-26.
[3] Lockshin, RA and Williams, CM. Programmed cell death. II. Endocrinepotentiation of the breakdown of the intersegmental muscles of silkmoths. J InsectPhysiol,1964,10:643-649.
[4] Kerr, JF, Wyllie, AH and Currie, AR. Apoptosis. a basic biological phenomenonwith wide-ranging implications in tissue kinethcs. Br J Cancer,1972,26(4):239-57.
[5] Arul M. Chinnaiyan，Clifford G. Tepper Michael F. Seldin，Karen O'Rourke, et al.FADD/MORT1is a common mediator of CD95(Fas/APO-1) and tumor necrosisfactor receptor–induced apoptosis. J Biol Chem,1996,271:4961-5.
[6] Frank C.Kischkel, Stefan Hellbardt, Iris Behrmann, et al. Cytotoxicity-dependentAPO-1[Fas/CD95]-associated proteins form a death-inducing signalingcomplex (DISC) with the receptor. EMBOJ,1995,14:5579-88.
[7] Teresa Fernandes-Alnemri, Robert C. Armstrongt, Joseph Krebst, et al. In vitroactivation of CPP32and Mch3by mch4, a novel human apoptotic cysteineprotease containing two FADD–like domains. Proceedings of the NationalAcademy of Sciences of the United States of America,1996,93:7464-9.
[8] Srinivasa M. Srinivasula, Manzoor Ahmad, Teresa Fernandes-Alnemre, et al.Molecular ordering of the Fas–apoptotic pathway: the Fas/APO-1Protease Mch5is a CrmA–inhibitable protease that activates multiple Ced-3/ICE–like cysteineproteases. Proceedings of the National Academy of Sciences of the United Statesof America,1996,93:14486-91.
[9] Xu Luo, Imawati Budihardjo, Hua Zou, et al. Bid, a Bcl2interacting protein,mediates cytochrome c release from mitochondria in response to activation of cellsurface death receptors. Cell,1998,94:481-90.
[10] Acehan, D, Jiang, X, Morgan, DG,Heuser, JE, et al. Three-dimensional structureof the apoptosome: implications for assembly, procaspase-9binding, andactivation.Mol Cell,2002,9(2):423-32.
[11] Slee, EA, Harte, MT, Kluck, RM, et al. Ordering the cytochrome c-initiatedcaspase cascade:hierarchical activation of Caspases-2,-3,-6,-7,-8, and-10in aCaspase-9-dependent manner.J Cell Biol,1999,144(2):281-92.
[12] Kaufmann,SH and Earnshaw,WC. Induction of apoptosis by cancerchemotherapy.Exp Cell Res,2000,256(1):42-9.
[13] Wang, X. The expanding role of mitochondria in apoptosis.Genes Dev,2001,15(22):2922-33.
[14] Philip L. Cohen and Robert A. Eisenberg.lpr and gld: single gene models ofsystemic autoimmunity and lymphoproliferative disease.Annu. Rev. Immunol,2001,9:243-269.
[15] Jianke Zhang, Dragana Cado, Ann Chen, et al. Fas-mediated apoptosis andactivation-induced T-cell proliferation are defective in mice lacking FADD/Mort1.Nature,1998Mar19;392:296-300.
[16] Eugene E Varfolomeev, Marcus Schuchmann, Victor Luria, et al. Targeteddisruption of the mouse Caspase8gene ablates cell death induction by the TNFreceptors,Fas/Apo1.and DR3and is lethal prenatally.Immunity,2002,9:267-276.
[17] Wen-Chen Yeh, Annick Itie, Andrew J Elia, et al. Requirement for Casper(c-FLIP) in regulation of death receptor-induced apoptosis and embryonicdevelopment.Immunity,2002,12:633-642.
[18] Haibing Zhang, Xiaohui Zhou, Thomas McQuade, Jinghe Li, Francis Ka-MingChan and Jianke Zhang.Functional complementation between FADD and RIP1in embryos and lymphocytes Nature,2011, Mar17,471(7338):373-6.
[19] William J. Kaiser, Jason W. Upton, Alyssa B. Long, Devon Livingston-Rosanoff,Lisa P. Daley-Bauer, Razqallah Hakem, Tamara Caspary&Edward S.Mocarski.RIP3mediates the embryonic lethality of Caspase-8-deficient mice.Nature,2011Mar17,471(7338):368-72.
[20] Andrew Oberst, Christopher P. Dillon, Ricardo Weinlich, Laura L. McCormick,Patrick Fitzgerald, Cristina Pop, Razq Hakem, Guy S. Salvesen&Douglas R.Green. Catalytic activity of the Caspase-8-FLIP(L) complex inhibitsRIPK3-dependent necrosis. Nature,2011Mar17,471(7338):363-7.
[21] Ch’en, I. L., Tsau, J.S., Molkentin, J.D., Komatsu, M.&Hedrick, S.M.Mechanisms of necroptosis in T cells. The Journal of experimental medicine,2011,208:633-41.
[22] Edward S. Mocarski Jason W. Upton&William J. Kaiser。Viral infection and theevolution of Caspase8-regulated apoptotic and necrotic death pathways. TureReviews Immunology aop,2011,10:1038-1041.
[23] Vandenabeele,P,et al.Molecular mechanisms of necroptosis: an ordered cellularexplosion.Nature Rev.Mol.cell Biol,2010,11:700-714.
[24] He, S, et,al. Receptor interacting protein kinase-3determines cellular necroticresponse to TNFa. Cell,2009,138:229-232.
[25] Zhang, D, W, et al. RIP3, an energy metabolism regulator that switchesTNF-induced cell death from apoptosis to necrosis.science,2009,325:332-336.
[26] Stanger BZ, Leder P, Lee T-H, Kim E, Seed B. RIP: a novel protein containing adeath domain that interacts with Fas/APO-1(CD95) in yeast and causes celldeath. Cell,1995,81:513-23.
[27] Hsu H, Huang J, Shu H-B, Baichwal V, Goeddel DV. TNF-dependent recruitmentof the protein kinase RIP to the TNF receptor-1signaling complex. Immunity,1996,4:387-96.
[28] Itoh N, Nagata S. A novel protein domain required for apoptosis. J Biol Chem,1993,268:10932-7.
[29] Tartaglia LA, Ayres TM, Wong GHW, Goeddel DV. A novel domain within the55kd TNF receptor signals cell death. Cell,1993,74:845-53.
[30] Sun X, Yin J, Starovasnik MA, Fairbrother WJ, Dixit VM. Identification of anovel homotypic interaction motif required for the phosphorylation ofreceptor-interacting protein (RIP) by RIP3. J Biol Chem,2002,277:9505-11.
[31] Lin Y, Devin A, Rodriguez Y, Liu ZG. Cleavage of the death domain kinase RIPby Caspase-8prompts TNF-induced apoptosis. Genes Dev,.1999,13:2514-26.
[32] Martinon F, Holler N, Richard C, Tschopp J. Activation of a proapoptoticamplification loop through inhibition of NF-kappaB-dependent survival signalsby Caspase-mediated inactivation of RIP. FEBS Lett,2000,468:134-6.
[33] Kim JW, Choi EJ, Joe CO. Activation of death-inducing signaling complex(DISC) by pro-apoptotic C-terminal fragment of RIP. Oncogene,2000,19:4491-9.
[34] Barcia RN, Valle NS, Mcleod JD. Caspase involvement in RIP-associatedCD95-induced T cell apoptosis. Cell Immunol,2003,226:78-85.
[35] Ea CK, Deng L, Xia ZP, Pineda G, Chen ZJ. Activation of IKK by TNF alpharequires site-specific ubiquitination of RIP1and polyubiquitin binding byNEMO. Mol Cell,2006,22:245-57.
[36] Li H, Kobayashi M, Blonska M, You Y, Lin X. Ubiquitination of RIP is requiredfor tumor necrosis factor alpha-induced NF-kappaB activation. J Biol Chem,2006,281:13636-43.
[37] O’Donnell MA, Legarda-Addison D, Skountzos P, Yeh WC, Ting AT.Ubiquitination of RIP1regulates an NF-kappaB-independent cell-death switch inTNF signaling. Curr Biol,2007,17:418-24.
[38] Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS. NF-kappa Bantipoptosis: induction of TRAF1and TRAF2and c-IAP1and c-IAP2tosuppress Caspase-8activation. Science,1998,281:1680-3.
[39] Beg AA, Baltimore D. An essential role for NF-κB in preventing TNF-α-inducedcell death. Science,1996,274:782-4.
[40] Zhang H, Zhou X, McQuade T, Li J, Chan FK, Zhang J. Functionalcomplementation between FADD and RIP1in embryos and lymphocytes. Nature,2011,471:373-6.
[41] Cusson N, Oikemus S, Kilpatric ED, Cunningham L, Kelliher M. The deathdomain kinase RIP protects thymocytes from tumor necrosis factor receptor Type2-induced cell death. J Exp Med,2002,196:15-26.
[42] A. Degterev, Z. Huang, M. Boyce, et al. Chemical inhibitor of nonapoptotic celldeath with therapeutic potential for ischemic brain injury. Nat. Chem. Biol,2005,1:112-119.
[43] C. C. Smith, S. M. Davidson, S. Y. Lim, et al. A potentially novelcardioprotective agent? Cardiovasc. Drugs Ther,2007,21:227-233.
[44] W. Han, L. Li, S. Qiu, et al. Shikonin circumvents cancer drug resistance byinduction of a necroptotic death. Mol. Cancer Ther,2007,6:1641-1649.
[45] Newton, K. et al. Kinase RIP3is dispensable for normal NF-κBs, signaling bythe B-cell and T-cell receptors, tumor necrosis factor receptor1, and Toll-likereceptors2and4. Mol. Cell. Biol,2004,24:1464-9.
[46] Sun, X. et al. RIP3, a novel apoptosis-inducing kinase. J. Biol. Chem,1999,274:16871-5.
[47] Yu, P.W. et al. Identification of RIP3, a RIP-like kinase that activates apoptosisand NF-κB. Curr. Biol,1999,9:539-42.
[48] Kim Newton, Xiaoqing Sun, and Vishva M. Dixit.Kinase RIP3is dispensable fornormal NF-kappa Bs,signaling by the B-cell and T-cell receptors,tumor necrosisfactor receptor1,and Toll-like receptors2and4. Mol. Cell. Biol,2004,24:1464-1469.
[49] Y. S. Cho, S. Challa, D. Moquin, et al. Phosphorylation-driven assembly of theRIP1-RIP3complex regulates programmed necrosis and virus-inducedinflammation. Cell,2009,137:1112-1123.
[50] Alexei Degterev, Junichi Hitomi, Megan Germscheid, Irene L Ch'en, OlgaKorkina, Xin Teng, Derek Abbott, Gregory D Cuny, Chengye Yuan, GerhardWagner, Stephen M Hedrick, Scott A Gerber, Alexey Lugovskoy&JunyingYuan.Idendification of RIP1kinase as a specific cellular target ofnesrostatins.Nat.Chem.Biol,2008,4:313-321.
[51] Sudan He, Lai Wang, Lin Miao, Tao Wang, Fenghe Du, Liping Zhao, XiaodongWang. Receptor interacting protein kinase-3determines cellular necroticresponse to TNF-alpha.Cell,2009,137:1100-1111.
[52] V.Goossens et al:Regulation of tumor necrosis factor-induced,mitochondria-andreactive oxygen species-dependent cell death by the electron flux through theelectron transport chain complexI.Antioxid. Redox Signal,1999,1:285-295.
[53] VERA GOOSSENS, JOHAN GROOTEN, KURT DE VOS, AND WALTERFIERS. Direct evidence for tumor necrosis factor-induced mitochondrial reactiveoxygen intermediates and their involvement in cytotoxicity. Proc. Natl. Acad. Sci.U.S.A.,1995,92:8115-8119.
[54] Duan-Wu Zhang, Jing Shao, Juan Lin, Na Zhang, Bao-Ju Lu, Sheng-Cai Lin,Meng-Qiu Dong and Jiahuai Han. RIP3,an energy metabolism regulator thatswitches TNF-induced cell death from apoptosis to necrosis.Science,2009,325:332-336.
[55] Franky Van Herreweghe, Jianqiang Mao, Frank W. R. Chaplen, Johan Grooten,Kris Gevaert, Jo l Vandekerckhove, and Katia Vancompernolle.Tumor necrosisfactor-induced modulation of glyoxalase I activities through phosphorylation byPKA results in cell death and is accompanied by the formation of a specificmethylglyoxal-derived AGE. Proc. Natl. Acad. Sci.U.S.A.,2002,99:949-954.
[56] P.Boya,G.Kroemer,Lysosomal membrane permeabilization in cell death.Oncogene,2008,27:6434-6451.
[57] Koh Ono, Xiaofei Wang, and Jiahuai Han.Resistence to tumor necrosisfactor-induced cell death mediated by PMCA4deficiency. Mol. Cell. Biol.,2001,21:8276-8288.
[58] Cho,Y, et al. Phosphorylation-driven assembly of the RIP1-RIP3complexregulates programmed necrosis and virus-induced inflammation. Cell,2009,137:1112-1123.
[59] He,S, et al.Receptor interracting protein kinase-3determines cellular necroticresponse to TNFa. Cell,2009,137:1100-1111.
[60] Patrick-Simon Welz, Andy Wullaert, Katerina Vlantis, Vangelis Kondylis, VanesaFernandez-Majada1, Maria Ermolaeva, Petra Kirsch, Anja Sterner-Kock, Geertvan Loo&Manolis Pasparakis. FADD prevents RIP3-mediated epithelial cellnecrosis and chronic intestinal inflammation. Nature,2011,477(7364):330-334.
[61] Marion C, et al.The adaptor protein FADD protects epidermal keratinocytes fromnecroptosis in vivo and prevents skin inflammation.Immunity,2011,35:1-11.
[62] Zhang, J. et al. Fas-mediated apoptosis and activation-induced T-cellproliferation are defective in mice lacking FADD/Mort1. Nature,1998,67:1567-1579.
[63] Hua, Z. C. et al. A function of Fas-associated death domain protein in cell cycleprogression localized to a single amino acid at its C-terminal region. Immunity,2003,18:513-521.
[64] Falk, M. et al. Caspase inhibition blocks human T cell and cell cycle-associatedproteins. J. Immunol,2004,173:5077-5085.
[65] Walsh, C. M. et al. A role for FADD in T cell activation and development.Immunity,1998,8:439-449.
[66] Newton, K. et al. A dominant interfering mutant of FADD/MORT1enhancesdeletion of autoreactive thymocytes and inhibits proliferation of mature Tlymphocytes. EMBO J,1998,17:706-718.
[67] Kabra, N. H. et al. T cell-specific FADD-deficient mice: FADD is required forearly T cell development. Proc. Natl. Acad. Sci. U.S.A.,2001,98:6307-6312.
[68] Salmena, L. et al. Essential role for caspase8in T-cell homeostasis and T-cellmediated immunity. Genes,2003,17:883-895.
[69] Beisner, D. R. et al. The requirements for Fas-associated death domain signalingin mature T cell activation and survival. J. Immunol,2003,171:247-256.
[70] N. Festjens, T. Vanden Berghe, P. Vandenabeele. Necrosis, a well-orchestratedform of cell demise: Signalling cascades, important mediators and concomitantimmune response. Biochim. Biophys. Acta,2006,1757:1371-1387.
[71] A. Degterev, J. Hitomi, M. Germscheid, et al. Identification of RIP1kinase as aspecific cellular target of necrostatins. Nat. Chem. Biol,2008,4:313-321.
[72] Kim Newton, Xiaoqing Sun, and Vishva M. Dixit. Kinase RIP3is dispensablefor normal NF-κBs, signaling by the B-cell and T-cell receptors, tumor necrosisfactor receptor1, and toll-like receptors2and4. Molecular and cellular biology,2004, Feb,1464-1469.
[73] William J. Kaiser, Jason W. Upton, Alyssa B. Long, ect. RIP3mediates theembryonic lethality of caspase-8-deficient mice. Nature,2011Mar,17,471:368-372.
[74] Laouar, Y.&Ezine, S. In vivo CD4+lymph node T cells from lpr mice generateCD4–CD8-B220+TCR-beta low cells. J. Immunol,1994,153:3948-3955.