CN102908612A - TRAIL截短型变异体活化NF-κB和在炎症反应中的应用 - Google Patents
TRAIL截短型变异体活化NF-κB和在炎症反应中的应用 Download PDFInfo
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Abstract
本发明属于肿瘤坏死因子-(TNF-)相关的细胞凋亡诱导配体(tumor necrosis factor-(TNF-)-related apoptosis-inducing ligand(TRAIL))领域。特别是，本发明涉及TRAIL的7个截短型TRAIL变异体(AK，E2，E3，E4，DA，BX424和BX439)，其中DA与TRAILβ编码的蛋白质序列完全相同，而BX424则和TRAILδ蛋白质序列完全相同。实验发现在这些截短型变异体中，除了BX439之外，都有不同程度的活化NF-κB的能力。进一步研究发现，过表达DA，BX424和E4能够显著活化巨噬细胞炎性蛋白-1β(MIP-1β/CCL4)、巨噬细胞炎性蛋白-3α(MIP-3α/CCL20)以及白介素8(IL8/CXCL8)启动子报告基因。本发明TRAIL的变异体在治疗炎症反应中具有广泛的应用前景。
Description
技术领域
本发明涉及7个截短型TRAIL变异体作为在治疗炎症反应药物制备中的应用，特别涉及在细胞中活化NF-κB和治疗炎症中的作用。
背景技术
肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand，TRAIL)属于肿瘤坏死因子超家族成员，其编码基因定位于3号染色体(3q26)，共由5个外显子组成，编码281个氨基酸，其中第18位～38位氨基酸为跨膜区，第39位～281位为胞外区，属于II型跨膜蛋白。TRAIL可在许多正常组织表达，对大多数正常细胞没有明显的毒性，但能够选择性的杀伤肿瘤细胞，因此在肿瘤治疗中具有巨大潜力(例如参见Stegehuis JH.TRAIL receptor targeting therapies for non-small cell lung cancer：current status and perspectives.Drug Resist Updat 2010，13(1-2)：2-15；Holoch PA.TNF-related apoptosis-inducing ligand(TRAIL)：a new path to anti-cancer therapies.Eur J Pharmacol 2009，625(1-3)：63-72.)。目前，用于临床应用研究的最常用的TRAIL重组蛋白(recombinant soluble human TRAIL，rsTRAIL)有两种形式，分别为第95～281位、第114～281位氨基酸的TRAIL分子。目前，TRAIL共有4个转录变异体(TRAILα/β/γ/δ)报道(例如参见Krieg A.TRAIL-beta and TRAIL-gamma：two novelsplice variants of the human TNF-related apoptosis-inducing ligand(TRAIL)without apoptoticpotential.Br J Cancer 2003，88(6)：918-927.；Woods DC.Cisplatin-mediated sensitivity toTRAIL-induced cell death in human granulosa tumor cells.Gynecol Oncol 2008，108(3)：632-640.)，但其功能尚缺乏系统性的研究。
TRAIL同其他TNF家族成员一样，形成同源三聚体经由死亡受体途径介导肿瘤细胞凋亡(例如参见Lavrik I.Death receptor signaling.J Cell Sci 2005，118(Pt 2)：265-267.；Tansey MG.The TNF superfamily in 2009：new pathways，new indications，and new drugs.Drug Discov Today2009，14(23-24)：1082-1088.)。目前已知的TRAIL受体共有5种：DR4，DR5，DcR1，DcR2和OPG。DR4(death receptor 4，又称TRAIL-R1)和DR5(death receptor 4，又称TRAIL-R2、TRICK-2、KILLER)属于可以介导肿瘤细胞凋亡的功能性受体，具有胞浆内死亡结构域，通过接头分子FADD(Fas associated death domain)招募CASPASE-8/10，形成死亡受体信号复合体(death-inducing signaling complex，DISC)，进一步激活CASPASE蛋白酶解级联反应来诱导细胞死亡(例如参见Holoch PA.TNF-related apoptosis-inducing ligand(TRAIL)：a new pathto anti-cancer therapies.Eur J Pharmacol 2009，625(1-3)：63-72.；Lavrik I.Death receptor signaling.J Cell Sci 2005，118(Pt 2)：265-267.)；DcR1(decoy receptor 1，又称LIT、TRID、TRAIL-R3)和DcR2(decoy receptor 2，又称TRUNDD、TRAIL-R4)属于诱骗受体，前者无胞内区，后者胞内死亡结构域(death domain)不完整，因此与TRAIL结合后无法传递凋亡信号；OPG(osteoprotegerin)是一种分泌型糖蛋白，属于可溶性受体，可通过竞争结合从而抑制TRAIL的功能。正常细胞一般广泛表达4种膜受体DR4、DR5、DcR1、DcR2，而肿瘤细胞往往高表达DR4和DR5，因此在正常细胞中诱骗受体DcR1/2可以干预TRA IL与死亡受体DR4/5的结合而免受攻击，肿瘤细胞由于缺乏诱骗受体保护而易于被TRAIL诱导而发生凋亡。
由于TRAIL对大多数正常细胞没有明显的毒性，但能够选择性的杀伤肿瘤细胞，因此在肿瘤治疗中具有巨大潜力(例如参见Stegehuis JH.TRAIL receptor targeting therapies fornon-small cell lung cancer：current status and perspectives.Drug Resist Updat 2010，13(1-2)：2-15；Holoch PA.TNF-related apoptosis-inducing ligand(TRAIL)：a new path to anti-cancer therapies.Eur J Pharmacol 2009，625(1-3)：63-72.)。但研究表明有近半数的肿瘤细胞系不同程度的对TRAIL产生抵抗(TRAIL resistance)，这成为限制TRAIL临床应用的重要原因之一。肿瘤细胞对TRAIL产生抵抗的机制有多方面，如细胞膜上TRAIL死亡受体与诱骗受体表达量的不同会影响到对TRAIL的抵抗(例如参见Horak P.Contribution of epigenetic silencing of tumornecrosis factor-related apoptosis inducing ligand receptor 1(DR4)to TRAIL resistance and ovariancancer.Mol Cancer Res 2005，3(6)：335-343.；Elias A.Epigenetic silencing of death receptor 4mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in gliomas.Clin CancerRes 2009，15(17)：5457-5465.；Sanlioglu AD.Surface TRAIL decoy receptor-4 expression iscorrelated with TRAIL resistance in MCF7 breast cancer cells.BMC Cancer 2005，5：54.)；TRAIL受体突变(例如参见Park WS.Inactivating mutations of KILLER/DR5 gene in gastric cancers.Gastroenterology 2001，121(5)：1219-1225.；Lee SH.Somatic mutations of TRAIL-receptor 1 andTRAIL-receptor 2 genes in non-Hodgkin′s lymphoma.Oncogene 2001，20(3)：399-403.)；细胞内凋亡-抗凋亡分子之间的平衡(例如参见Wang X.Akt-mediated eminent expression of c-FLIP andMcl-1 confers acquired resistance to TRAIL-induced cytotoxicity to lung cancer cells.Mol CancerTher 2008，7(5)：1156-1163.；Morioka S.TAK1 kinase determines TRAIL sensitivity by modulatingreactive oxygen species and cIAP.Oncogene 2009，28(23)：2257-2265.；Thakkar H.Pro-survivalfunction of Akt/protein kinase B in prostate cancer cells.Relationship with TRAIL resistance.J BiolChem 2001，276(42)：38361-38369.；Khanbolooki S.Nuclear factor-kappaB maintains TRAILresistance in human pancreatic cancer cells.Mol Cancer Ther 2006，5(9)：2251-2260.；Franco AV.The role of NF-kappa B in TNF-related apoptosis-inducing ligand(TRAIL)-induced apoptosis ofmelanoma cells.J Immunol 2001，166(9)：5337-5345.；Lee TJ.Acquired TRAIL resistance in humanbreast cancer cells are caused by the sustained cFLIP(L)and XIAP protein levels and ERKactivation.Biochem Biophys Res Commun 2006，351(4)：1024-1030.)；TRAIL死亡受体的翻译后修饰(例如参见Wagner KW.Death-receptor O-glycosylation controls tumor-cell sensitivity to theproapoptotic ligand Apo2L/TRAIL.Nat Med 2007，13(9)：1070-1077.)，等。为克服这些TRAIL抵抗机制，目前的策略是根据肿瘤细胞的抵抗机制有针对性的采取用药策略，尤其联合用药以提高肿瘤细胞对TRAIL的敏感性，如重组TRAIL蛋白或TRAIL死亡受体特异性的单克隆抗体(HGS-ETR1/mapatumumab和HGS-ETR2/lexatumumab等)，联合化学药物一起使用，可以达到更理想的效果。这些化学药物通过各自机制，如以P53非依赖方式上调死亡受体的表达(大多数抑制剂都可以上调TRAIL死亡受体表达)，抑制NF-κB的活性，抑制PI3K、AKT激酶活性、抑制Bcl-2家族成员抗凋亡活性，抑制组织蛋白去乙酰化酶(histone deacetylase，HDAC)活性(可上调DR5的表达)等，增强肿瘤细胞对TRAIL的促凋亡效应(例如参见Stegehuis JH.TRAIL receptor targeting therapies for non-small cell lung cancer：current status andperspectives.Drug Resist Updat 2010，13(1-2)：2-15；Holoch PA.TNF-related apoptosis-inducingligand(TRAIL)：a new path to anti-cancer therapies.Eur J Pharmacol 2009，625(1-3)：63-72.；BevisKS.Overcoming TRAIL resistance in ovarian carcinoma.Gynecol Oncol，119(1)：157-163.)。例如，rhTRAIL联合Paclitaxel，Carboplatin和Bevacizumab用于治疗NSCLC(已进入临床II期)(例如参见Stegehuis JH.TRAIL receptor targeting therapies for non-small cell lung cancer：currentstatus and perspectives.Drug Resist Updat 2010，13(1-2)：2-15)；此外，rhTRAIL联合抗CD20的单克隆抗体rituximab用于治疗非霍奇金淋巴瘤(non-Hodgkin′s lymphoma，NHL)(例如参见Daniel D.Cooperation of the proapoptotic receptor agonist rhApo2L/TRAIL with the CD20antibody rituximab against non-Hodgkin lymphoma xenografts.Blood 2007，110(12)：4037-4046.)。
另一策略是从TRAIL自身出发提高重组TRAIL蛋白的促凋亡效应，如在氨基端添加适当的标签，如亮氨酸拉链(LZ-TRAIL)，可提高TRAIL的稳定性和聚集度从而增加TRAIL的效应(例如参见Rozanov DV.Engineering a leucine zipper-TRAIL homotrimer with improvedcytotoxicity in tumor cells.Mol Cancer Ther 2009，8(6)：1515-1525.)，而且标签也有利于重组蛋白质的表达和纯化，但是增加标签可能会导致安全隐患，例如，LZ-TRAIL可诱导体内分离的星形胶质细胞(astrocytes)死亡(例如参见Walczak H.Tumoricidal activity of tumor necrosisfactor-related apoptosis-inducing ligand in vivo.Nat Med 1999，5(2)：157-163.)，His-TRAIL可引起肝毒性(例如参见Lawrence D.Differential hepatocyte toxicity of recombinant Apo2L/TRAILversions.Nat Med 2001，7(4)：383-385.；Jo M.Apoptosis induced in normal human hepatocytes bytumor necrosis factor-related apoptosis-inducing ligand.Nat Med 2000，6(5)：564-567.)，Flag-TRAIL可诱导体外培养的脑细胞死亡(例如参见Nitsch R.Human brain-cell death inducedby tumour-necrosis-factor-related apoptosis-inducing ligand(TRAIL).Lancet 2000，356(9232)：827-828.)，但是无标签的重组TRAIL蛋白则无毒性(例如参见Lawrence D.Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions.Nat Med 2001，7(4)：383-385.；Hao C.Induction and intracellular regulation of tumor necrosis factor-relatedapoptosis-inducing ligand(TRAIL)mediated apotosis in human malignant glioma cells.CancerRes 2001，61(3)：1162-1170.；Hao C.TRAIL inhibits tumor growth but is nontoxic to humanhepatocytes in chimeric mice.Cancer Res 2004，64(23)：8502-8506.)。
此外，从TRAIL自身与受体复合物结构出发，通过定点突变对TRAIL分子胞外区进行改造，在保障其凋亡活性的基础上，寻找与DR4或DR5选择性结合的TRAIL突变体(TRAILvariant)，在选择性诱导肿瘤细胞凋亡方面较为理想(例如参见Kelley RF.Receptor-selectivemutants of apoptosis-inducing ligand 2/tumor necrosis factor-related apoptosis-inducing ligandreveal a greater contribution of death receptor(DR)5 than DR4 to apoptosis signaling.J Biol Chem2005，280(3)：2205-2212.；Tur V.DR4-selective tumor necrosis factor-related apoptosis-inducingligand(TRAIL)variants obtained by structure-based design.J Biol Chem 2008，283(29)：20560-20568.；van der Sloot AM.Designed tumor necrosis factor-relatedapoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor.Proc NatlAcad Sci U S A 2006，103(23)：8634-8639.；Duiker EW.Enhanced antitumor efficacy of aDR5-specific TRAIL variant over recombinant human TRAIL in a bioluminescent ovarian cancerxenograft model.Clin Cancer Res 2009，15(6)：2048-2057.
)。例如，将TRAIL的第269位天冬氨酸(Asp269，D)突变为组氨酸(His，H)的单点突变体D269H，以及双位点突变体D269H/T214R及D269H/E195R可选择性的结合DR5，而与其他TRAIL受体的结合力大大降低；结肠癌细胞系Colo205和ML-1(myeloid leukaemia cell)细胞系表面的四种TRAIL受体(DR4/5及DcR1/2)表达模式近似，但是前者主要由DR5、后者主要由DR4介导TRAIL的促凋亡效应；上述DR5选择性TRAIL突变体能够诱导Colo205细胞凋亡而无法诱导ML-1细胞凋亡，且能够选择性诱导卵巢癌细胞系A2780(表达DR5，不表达DR4)细胞凋亡，而无法诱导EM-2(chronic myeloid leukemia cell，表达DR4，不表达DR5)细胞死亡；DR5选择性TRAIL突变体对正常细胞HUVEC(人脐静脉内皮细胞)无促凋亡效应(例如参见van der Sloot AM.Designed tumor necrosis factor-relatedapoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor.Proc NatlAcad Sci U S A 2006，103(23)：8634-8639.)。体内实验进一步证明了D269H/E195R突变体抗肿瘤效果比野生型重组TRAIL更理想(例如参见Duiker EW.Enhanced antitumor efficacy of aDR5-specific TRAIL variant over recombinant human TRAIL in a bioluminescent ovarian cancerxenograft model.Clin Cancer Res 2009，15(6)：2048-2057.)。TRAIL的D218H及D218Y突变体则可选择性结合DR4，诱导DR4介导的细胞凋亡效应(例如参见Tur V.DR4-selective tumornecrosis factor-related apoptosis-inducing ligand(TRAIL)variants obtained by structure-baseddesign.J Biol Chem 2008，283(29)：20560-20568.)。
除了促凋亡效应外，TRAIL还能通过死亡受体DR4、DR5并借助接头分子TRADD和TRAF2等够激活NF-κB等信号通路，由此介导细胞存活、增殖、迁移、侵袭以及促进炎性细胞因子释放等非凋亡效应(例如参见Belyanskaya LL.TRAIL-induced survival andproliferation of SCLC cells is mediated by ERK and dependent on TRAIL-R2/DR5 expression inthe absence of caspase-8.Lung Cancer 2008，60(3)：355-365.；Secchiero P.TRAIL promotes thesurvival，migration and proliferation of vascular smooth muscle cells.Cell Mol Life Sci 2004，61(15)：1965-1974.；Ehrhardt H.TRAIL induced survival and proliferation in cancer cells resistanttowards TRAIL-induced apoptosis mediated by NF-kappaB.Oncogene 2003，22(25)：3842-3852.；Ishimura N.Trail induces cell migration and invasion in apoptosis-resistant cholangiocarcinomacells.Am J Physiol Gastrointest Liver Physiol 2006，290(1)：G129-136.；Tang W.TRAIL receptormediates inflammatory cytokine release in an NF-kappaB-dependent manner.Cell Res 2009，19(6)：758-767.；Tang W.Tumour necrosis factor-related apoptosis-inducing ligand(TRAIL)-induced chemokine release in both TRAIL-resistant and TRAIL-sensitive cells via nuclearfactor kappa B.Febs J 2009，276(2)：581-593.)，是一种具有多方面效应的细胞因子(例如参见Schaefer U.TRAIL：a multifunctional cytokine.Front Biosci 2007，12：3813-3824.)。NF-κB是个同源或异源二聚体转录因子，亚基主要包括p50(由p105加工而来，又称NF-κB1)，p52(由p100加工而来，又称NF-κB2)，p65(RelA)，c-Rel和RelB等。在静息状态下，NF-κB以无活性形式存在细胞质，在细胞因子、LPS等刺激因子作用下，可通过经典途径和非经典途径活化后进入细胞核并结合靶序列5’-GGGRNYYYCC-3’(R为嘌呤，Y为嘧啶，N为任意核苷酸)，从而激活靶基因转录。NF-κB的活化程度与肿瘤细胞系对TRAIL的敏感程度密切相关(例如参见Plantivaux A.Is there a role for nuclear factor kappaB in tumor necrosis factor-relatedapoptosis-inducing ligand resistance？Ann N Y Acad Sci 2009，1171：38-49.)。TRAIL在免疫系统发挥重要的功能，参与了固有免疫和适应性免疫应答过程(例如参见Schaefer U.TRAIL：amultifunctional cytokine.Front Biosci 2007，12：3813-3824.；Anel A.Apo2L/TRAIL and immuneregulation.Front Biosci 2007，12：2074-2084.；Lee HO.TRAIL：a mechanism of tumor surveillancein an immune privileged site.J Immunol 2002，169(9)：4739-4744.)，例如TRAIL在NK细胞介导的IFN-γ依赖的抑制肿瘤细胞生长和转移过程起重要作用；重组TRAIL蛋白能够诱导TRAIL敏感性和抗性细胞系以NF-κB依赖的方式释放趋化因子CXCL2、CXCL8(IL8)、CCL2、CCL4(MIP-1β)以及CCL20(MIP-3α)等；重组TRAIL刺激支气管内皮细胞分泌CCL20，诱导髓性DC及CC6+的CD4+T细胞归巢(homing)，在过敏性哮喘的发生过程中起重要作用(例如参见Weckmann M.Critical link between TRAIL and CCL20 for the activation of TH2 cellsand the expression of allergic airway disease.Nat Med 2007，13(11)：1308-1315.)。
目前，关于TRAIL转录变异体，除了全长形式的TRAIL，即α变异体(或称RefSeq)之外，还有3个被报道，分别是TRAILβ、γ(例如参见Krieg A.TRAIL-beta and TRAIL-gamma：two novel splice variants of the human TNF-related apoptosis-inducing ligand(TRAIL)withoutapoptotic potential.Br J Cancer 2003，88(6)：918-927.)和δ变异体(例如参见Woods DC.Cisplatin-mediated sensitivity to TRAIL-induced cell death in human granulosa tumor cells.Gynecol Oncol 2008，108(3)：632-640.)。与TRAILα相比，TRAILβ缺少第3个外显子、TRAILγ缺少第2个和第3个外显子，而TRAILδ则是缺少第3个和第4个外显子。定位分析发现TRAILγ还可定位于细胞核膜(例如参见Krieg A.TRAIL-beta and TRAIL-gamma：two novelsplice variants of the human TNF-related apoptosis-inducing ligand(TRAIL)without apoptoticpotential.Br J Cancer 2003，88(6)：918-927.)。TRAILβ和γ无法诱导TRAIL敏感性细胞系HeLa凋亡，因此这种截短(truncated)形式的变异体被认为可能是为了减少有活性的TRAIL全长而出现的一种被动调节机制(例如参见Krieg A.TRAIL-beta and TRAIL-gamma：two novelsplice variants of the human TNF-related apoptosis-inducing ligand(TRAIL)without apoptoticpotential.Br J Cancer 2003，88(6)：918-927.)。关于TRAIL变异体，后来仅有的一篇研究表明在炎症细胞因子TNFα刺激下，通过前提mRNA的剪接导致TRAILα会向无凋亡活性的TRAILβ方向变化，说明在细胞因子的刺激下，可以转换TRAIL的促凋亡潜能(proapoptotic potential)(例如参见Kamachi M.Activation of protein phosphatase causes alternative splicing of tumornecrosis factor-related apoptosis-inducing ligand(TRAIL)：potential effect on immune surveillance.Biochem Biophys Res Commun 2007，360(1)：280-285.)。除此之外，关于TRAILβ、γ和δ的功能无其他文献报道，也无报道这些截短形式的TRAIL是否具有抑制TRAIL的促凋亡作用。
本研究通过生物信息学分析，共克隆到了7个截短型TRAIL变异体(AK，E2，E3，E4，DA，BX424和BX439)，其中DA与TRAILβ编码的蛋白质序列完全相同，而BX424则和TRAILδ蛋白质序列完全相同。凋亡实验结果表明，所有截短形式的TRAIL变异体都无法诱导细胞凋亡，且E4和AK的胞外区也无法抑制TRAIL促凋亡效应。但是，这些截短型变异体中，除了BX439之外，都有不同程度的活化NF-κB的能力，尤其是DA，BX424和E4，它们活化NF-κB的能力甚至强于RefSeq。鉴于TRAILβ在胞内有分布(例如参见Krieg A.TRAIL-beta and TRAIL-gamma：two novel splice variants of the human TNF-relatedapoptosis-inducing ligand(TRAIL)without apoptotic potential.Br J Cancer 2003，88(6)：918-927.)，而最新的一篇报道提示人肥大细胞在细胞因子IFN-γ、LPS刺激下上调TRAIL的表达，但TRAIL分布仅局限在胞内，缺乏膜表面形式TRAIL(例如参见Berent-MaozB.Human mast cells express intracellular TRAIL.Cell Immunol，262(2)：80-83.)。这些提示TRAIL及其变异体存在胞内活化NF-κB的途径。进一步研究发现，过表达DA，BX424和E4能够显著活化巨噬细胞炎性蛋白-1β(MIP-1β/CCL4)、巨噬细胞炎性蛋白-3α(MIP-3α/CCL20)以及白介素8(IL8/CXCL8)启动子报告基因，而过表达RefSeq以及其他TRAIL变异体的活化能力相对较弱，说明TRAIL变异体在炎症反应过程可能扮演着重要角色。
在TRAIL变异体中，BX439和BX424的区别仅在于BX439缺乏外显子2(138个核苷酸)，但过表达时BX439活化NF-κB的能力大大降低，说明外显子2编码产物对于活化NF-κB至关重要。DA缺乏外显子3(43个核苷酸)，但过表达时能显著活化NF-κB，说明该外显子编码产物对于NF-κB的活化并不必需。
综上所述，尽管截短型TRAIL变异体已经丧失了促凋亡或抑制凋亡的能力，但是TRAIL变异体，尤其是DA，BX424和E4能够显著活化NF-κB，且上调CCL4，CCL20及CXCL8(IL-8)等趋化因子基因表达，说明TRAIL变异体能够发挥重要的非凋亡作用。
发明内容
本发明人利用生物信息学分析，克隆到了7个截短型TRAIL变异体(AK，E2，E3，E4，DA，BX424和BX439)，并且通过实验验证这些截短型变异体具有同时活化NF-κB和某些炎性蛋白的功能。
这些截短型变异体具有很好的EST支持，目前还没有相关的功能报道。本实验证明这些截短型变异体在8种人源细胞中有不同程度的表达。并且首次证实了7个截短型TRAIL变异体在293T和HCT116细胞中能够同时活化NF-κB和某些炎性蛋白，并且这些作用不是通过诱导细胞凋亡引起的。
因此通过这些截短型变异体的功能，可以达到影响炎症反应的目的，同时为研制治疗炎症反应药物奠定了基础。
附图说明
图1A为TRAIL转录变异体基因结构
图1B为TRAIL变异体蛋白质序列比对结果
图2为TRAIL变异体在细胞系中的表达情况
图3为TRAIL各变异体在细胞膜表面的表达情况
图4为TRAIL各变异体在293T细胞中激活NF-κB的情况
图5为荧光显微镜下TRAIL各变异体在293T细胞激活NF-κB的情况
图6为在共培养293T细胞的条件下TRAIL各变异体激活NF-κB的情况
图7为TRAIL各变异体在293T细胞中活化炎性蛋白IL8，CCL4和CCL20的情况
图8A为过表达时TRAIL各变异体对293T细胞的凋亡的影响
图8B为过表达时TRAIL各变异体对HCT116细胞的凋亡的影响
图8C为TRAIL各变异体对293T和HCT116细胞凋亡的影响
具体实施方式
实施例1、7个截短型TRAIL变异体的结构分析和蛋白序列比对
利用EST数据库分析，并结合UCSC基因组浏览器查看，我们绘制出7个截短型TRAIL变异体的基因结构。利用DNAstar和DNAMAN等生物信息学软件将7个截短型TRAIL变异体的蛋白序列与RefSeq的蛋白序列进行比对。
结果参见图1，其中1A显示所有这些变异体序列跨越的内含子边界均符合GT/AG规则，其中AK(编码65个氨基酸)和DA(编码98个氨基酸)分别在TRAIL的第1个和第2个，以及第4个和第5个外显子之间包含有一个新的外显子，DA缺少外显子3。E2(编码88个氨基酸)，E3(编码123个氨基酸)和E4(编码145个氨基酸)分别是在原有的外显子2、3、4上进行了延伸。BX424(编码101个氨基酸)缺少外显子3和4。BX439则缺少外显子2、3、4。黑色方块表示已知的外显子，而灰色方块表示新外显子及已知的外显子延伸部分。全长形式的TRAIL即RefSeq，或称TRAILα；DA转录本和TRAILβ不一样，但编码蛋白质的氨基酸序列完全相同；BX424和TRAILδ一致。其中1B可以看出所有TRAIL变异体氨基端序列相同，包含有完整的跨膜结构域，但羧基端各有不同。由于TRAIL属于II型跨膜蛋白，羧基端位于胞外区并参与受体的结合，所以各变异体羧基端不同提示有可能丧失了与受体的结合能力或具有不同的受体结合特异性。
实施例2、7个截短型TRAIL变异体在不同细胞系中的表达情况
将8种细胞(Raji，Jurkat，THP-1，HCT116，BT-325，HepG2，PC12，293T，均购自美国ATCC)以30万/孔铺于六孔板(NEST Biotecl，703001)中常规培养24小时。按Invitrogen逆转录试剂盒说明书提RNA，逆转录为cDNA，PCR扩增条件94℃5分钟，94℃变性30s，58℃退火30s，72℃延伸40s，30个循环。
结果如图2所示，通过RT-PCR检测7个截短型TRAIL变异体在不同细胞系中mRNA水平，除了DA仅在THP-1中检测到表达外，其它变异体均在多种细胞系中有表达。
实施例3、7个截短型TRAIL变异体在细胞膜上的表达情况
将HEK293T细胞(购自美国ATCC)以30万/孔铺于六孔板(NEST Biotecl，703001)中常规培养18小时，分别将3μg pCMV-sport6(阴性对照)、7个截短型TRAIL变异体质粒(pCMV载体)、RefSeq(pCMV载体，阳性对照)按照说明书，使用VigoFect(Vigorous公司)阳离子转染试剂将质粒转入HEK293T细胞。继续培养24小时后收集细胞培养上清于离心管，胰酶消化细胞，一并转入离心管，1,500rpm离心5分钟，弃上清，PBS洗涤细胞2次，加入2％FBS的FACS封闭液(含有2％FBS的PBS)，将待测细胞制成密度大约为5×105/ml的单细胞悬液。随后将anti-Myc抗体(购自MBL公司)按1∶200稀释加入单细胞悬液，充分混匀，于室温孵育约1小时，然后用PBS洗涤2次，用100μl FACS封闭液重悬细胞，加入FITC标记的羊抗鼠二抗((购自MBL公司))，继续室温孵育30min，然后PBS洗涤2次，最后加入2％多聚甲醛或冷PBS 400μl重悬细胞进行流式检测。
结果参见图3，可以看出7个截短型TRAIL变异体在细胞膜表面均有表达，其中粗线条表示变异体的表达强度，细线条表示空载体对照。
实施例4双萤光素酶报告系统的检测
HEK293T细胞(购自美国ATCC)常规传代培养于含10％胎牛血清(HyClone，SH30084.03)的DMEM培养基(HyClone，SH30022.01B)中，37℃，5％CO2孵箱中培养。按照1.1×106细胞/ml的密度，接种于96孔板，每孔100μl，置于孵箱培养18小时～24小时，使细胞密度达到40％～60％，以备转染。按照说明书，使用VigoFect阳离子转染试剂(威格拉斯生物技术(北京)有限公司)分别将50ng pNF-κB-Luc、pIL8(-162)-Luc、pCCL4-Luc、pCCL20-Luc、5ngpRL-TK-Luc和50ng待筛基因或者对照质粒共转入HEK293T细胞。转染pCMV-sport6空载体的细胞作为空白对照。转染pCMV-RefSeq基因的细胞作为阳性对照。每种待筛质粒设3个复孔。培养24小时后检测萤光素酶活性。
按照双萤光素酶报告系统(Promega，E1960)的说明书裂解细胞后，利用多功能微孔板检测系统GENios ProTM(Tecan)检测萤光素酶强度。转染空载体对照pCMV-sport6的细胞的相对萤光值设为100％。结果取3个复孔的平均值。
结果参见图4和7，由图4可以看出除了BX439之外，都有不同程度的活化NF-κB的能力，尤其是DA，BX424和E4，它们活化NF-κB的能力甚至强于RefSeq；由图7可以看出7个截短型变异体均不同程度的活化巨噬细胞炎性蛋白-1β(MIP-1β/CCL4)、巨噬细胞炎性蛋白-3α(MIP-3α/CCL20)以及白介素8(IL8/CXCL8)启动子报告基因，其中以DA，BX424和E4活化效果显著，RefSeq和其它TRAIL变异体的活化能力相对较弱。
实施例5荧光显微镜观察7个截短型变异体对NF-κB的影响
借助于荧光显微镜，利用NF-κB-GFP可以很方便地观察到7个截短型变异体在细胞对NF-κB的影响。实验中使用pCMV-sport6空载体作为阴性对照，以转染了RefSeq后为阳性对照。
293T细胞(购自美国ATCC)常规传代培养于含10％胎牛血清(HyClone，SH30084.03)的DMEM培养基(HyClone，SH30022.01B)中，37℃，5％CO2孵箱中培养。按照1.1×106细胞/ml的密度，接种于96孔板，每孔100μl，置于孵箱培养18小时～24小时，使细胞密度达到40％-60％，以备转染。按照说明书，使用VigoFect阳离子转染试剂(威格拉斯生物技术(北京)有限公司)分别将200ng pCMV-sport6(阴性对照)和NF-κB-GFP、7个截短型变异体质粒(pCMV载体)转入293T细胞。继续培养24h后利用荧光显微镜观测。
结果如图5，可以看出7个截短型TRAIL变异体能够不同程度的活化NF-κB-GFP，使其荧光增强，其中BX424、DA、E4和RefSeq活化能力最强。
实施例6用共培养的方法检测7个截短型变异体NF-κB的激活情况
将HEK293T细胞(购自美国ATCC)和HCT116细胞(购自美国ATCC)常规传代培养于含10％胎牛血清(HyClone，SH30084.03)的DMEM培养基(HyClone，SH30022.01B)中，37℃，5％CO2孵箱中培养。按照1.1×106细胞/ml的密度，接种于96孔板，每孔100μl，置于孵箱培养。共培养时，293T细胞分为两个转染组(1)单独转染TRAIL各变异体重组质粒；(2)共转pCMV-sport6(阴性对照)，pNF-κB-Luc以及pRL-TK-Luc基因质粒；转染6小时后，将上述(1)组细胞消化处理后加入到上述(2)组细胞中继续培养24小时后进行测定。
结果参见图6，从图中可以看出只有RefSeq在共培养的模式下能够活化NF-κB。
实施例7流式技术检测7个截短型变异体对细胞凋亡影响
细胞凋亡的主要生化指标是细胞膜脂酰丝氨酸(PS)外翻和线粒体膜电位的下降，碘化丙啶(PI)是一种核酸染料，它不能透过完整的细胞膜，但在凋亡中晚期的细胞和死细胞，PI能够透过细胞膜而使细胞核红染。因此将Annexin-V与PI匹配使用就可以将凋亡早晚期的细胞以及死细胞区分开来。将HEK293T细胞(购自美国ATCC)和HCT116细胞(购自美国ATCC)以30万/孔铺于六孔板(NEST Biotecl，703001)中常规培养18小时，分别将3μgpCMV-sport6(阴性对照)、7个截短型变异体质粒(pCMV载体)、RefSeq(pCMV载体，阳性对照)按照说明书，使用VigoFect(Vigorous公司)阳离子转染试剂将质粒转入HEK293T细胞和HCT116细胞。继续培养，分别于24小时后和48小时后收集细胞培养上清于离心管，胰酶消化细胞，一并转入离心管，1,500rpm离心5分钟，弃上清，PBS洗涤细胞2次，重悬于200μl结合缓冲液(10mM HEPES，pH 7.4，140mM NaCl，1mM MgCl2，5mM KCl，2.5mMCaCl2)，加入终浓度为0.5μg/ml的FITC-Annexin-V(购自美国BD公司556547)，室温避光反应20分钟后加入终浓度为1μg/ml的PI(购自美国BD公司556547)，根据细胞浓度补上200-400μl结合缓冲液，立即进行流式细胞术分析。
结果如图8所示，可以看出，7个截短型变异体质粒与对照相比没有明显的诱导细胞凋亡的作用，这说明截短型变异体已经丧失了促进细胞凋亡的能力。
Claims (10)
1.氨基酸序列为SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ IDNO：5、SEQ ID NO：6和SEQ ID NO：7所示的截短型TRAIL变异体在细胞中活化NF-κB和治疗炎症中的应用。
2.如权利要求1所述的应用，其中所述应用为活化NF-κB和炎性蛋白-1β(MIP-1β/CCL4)、巨噬细胞炎性蛋白-3α(MIP-3α/CCL20)以及白介素8(IL8/CXCL8)的启动子。
3.如权利要求1所述的应用，其中所述应用不是通过细胞凋亡引起的。
4.如权利要求1或2所述的应用，其中所述细胞为截短型TRAIL变异体普遍表达的人源细胞，包括人B淋巴细胞瘤细胞、组织相容性淋巴瘤细胞、人急性单核细胞性白血病细胞、神经胶质母细胞瘤细胞、肝癌细胞、人胚肾细胞、结肠癌细胞。
5.如权利要求1或2所述的应用，其中所述细胞为293T和HCT116细胞。
6.根据权利要求1-3中任一项所述的截短型TRAIL变异体在治疗炎症反应药物制备中的应用。
7.根据权利要求6所述的应用，其特征在于：活化NF-κB和炎性蛋白-1β(MIP-1β/CCL4)、巨噬细胞炎性蛋白-3α(MIP-3α/CCL20)以及白介素8(IL8/CXCL8)的启动子。
8.根据权利要求5所述的用途，其特征在于：所述应用不是通过细胞凋亡引起的。
9.根据权利要求7所述的用途，其中所述细胞为截短型TRAIL变异体普遍表达的人源细胞，包括人B淋巴细胞瘤细胞、组织相容性淋巴瘤细胞、人急性单核细胞性白血病细胞、神经胶质母细胞瘤细胞、肝癌细胞、人胚肾细胞、结肠癌细胞。
10.根据权利要求7所述的用途，其中所述细胞为293T和HCT116细胞。
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