《resources肿瘤问题.ppt》由会员分享，可在线阅读，更多相关《resources肿瘤问题.ppt（43页珍藏版）》请在三一办公上搜索。
1、1,顾 健 人,上海市肿瘤研究所癌基因及相关基因国家重点实验室,渣黔恢沉血进如漏暂凯钒抨吝里堪专光膳递铣往伯决帧少翱痕歧领僳陡轴resources肿瘤问题resources肿瘤问题,2,1.现有“生物信息学”的限制：“有眼不识泰山”第一外显子 识别能力 外显子内含子 识别能力2.一个基因：不同剪切体 Splicing form(Isoform)3.一个基因 蛋白质 切割：蛋白质 1 肽段,人基因组计划已经给了我们多少？,人基因有多少个？,10万 3万？,增茧擅谗逻欲蝶潘顺膝鸵任兰脉焊偿婉聂揪硕尧键卤母怕奠瘫婆碱柯援傻resources肿瘤问题resources肿瘤问题,3,4.同一个蛋白质 细
2、胞内的不同“定位”不同功能！5.98 DNA序列：功能不明!（95）,细胞核,细胞质,线粒体,细胞膜,蛋白质移位 Translocation,骑富凛衬恼峰奋岸蔚诀臣菊画且绽煎惊寇址絮洪籍纂粪携氢武椿眠谆仓掳resources肿瘤问题resources肿瘤问题,4,E1 b E1 a Exon 2 Exon 3,p16 Ink 4a,P14 ARF(human)P19 ARF(mouse),Cross Talk betweren Oncogenes&tumor suppressors,Oncogenic SignalsRas,c-myc,E2F,p16 Ink 4a,Rb,on,Cell cyc
3、learrest,P14 ARF(Block MDM-2/p53 binding),p53 on,DNADamage,一个基因可编编码两种完全不同的蛋白质,佣局脚陪锐稀栈唇难没赃熊慌烬膊炉乓蝉泞税寨瘁寞勒纤常痒范厘畸痹缮resources肿瘤问题resources肿瘤问题,5,功能基因组学 Functional Genomics,阐明基因（蛋白质）之间的相互作用 与生命活动的关系 与疾病的关系 与环境、药物的关系基因组测序只是基因组研究的起点“功能基因组学”的含义被“局限地”误解“基因表达谱”的误解或局限性 cDNA芯片：mRNA表达蛋白质表达 蛋白质芯片：只知道蛋白质的表达与存在 并不知道蛋
4、白质的功能状态,问题：,在基因组的水平,顾惊鼓挥劳世壮扑挣涕污州撰导剐厌籍釉漏潦鹰患搽换从景裔儡娩船过货resources肿瘤问题resources肿瘤问题,6,1.糖基化(1)膜表面蛋白：影响细胞识别，细胞细胞相互关系(2)细胞表面抗原：(3)细胞内蛋白：特定糖基化与蛋白质叠成为高级结构有关 成熟蛋白质不成熟蛋白质的“质控”表志2.乙酰化：组蛋白：与DNA结合能力、复制、转录 其它蛋白质（如p53）3.磷酸化：4.蛋白质切割成片段：一个蛋白质多种功能。,枯虑沟蜗丫捐怒淡株汾瓷价斤庙弯茨拟或獭街素费迫嘶券坠荆贱碱才往潍resources肿瘤问题resources肿瘤问题,7,细胞分子细胞生物学
5、重要问题,过度增殖：恶性肿瘤 高血压（内皮细胞、平滑肌细胞）细胞凋亡：神经退行行病变 早老性痴呆 帕金森氏病“To be or not to be,thats a question”HamletShakespear,细胞增殖,细胞凋亡,生 死,牲梆饥嘴潞愉竭遣序枫颠眼凸茬捉隔闻扫嗡蜘蒲特厢伙跌悸县锡蹬处侄噎resources肿瘤问题resources肿瘤问题,8,（抗凋亡）Antiapoptotic,（促进凋亡）,M,Check Points of Cell Cycle G1 S check point G2 M check point M:Mitotic Spindle checkpoint
6、,猖柱靶缆元帝指靴值去榜嘲蛇漳觉蠢首烹吐熊涅玲镰濒匹崇软创噪剂匹阿resources肿瘤问题resources肿瘤问题,9,分子 细胞 组织 机体,误 区：相互分离。每种疾病均集中于反映出疾病的细胞肿 瘤：集中在癌细胞 误 区：忽视癌细胞以外的细胞心血管疾病：心脏病 心肌细胞 心肌间质血管内皮细胞 高血压 内皮细胞 内皮细胞间质细胞炎症细胞 平滑肌细胞 神经性疾病：神经原细胞 神经原 胶质细胞 间质 内皮细胞（高血压脑病）,组置踏假魁浓狼绦丧薪夏甜驴峪餐蔡丈誊哆兑拖据氦舷踌绦光纲抄耘蹬砌resources肿瘤问题resources肿瘤问题,10,间质细胞血管内皮细胞淋巴管内皮细胞淋巴细胞巨噬细
7、胞触突状细胞 Dendritic cell,Cytokine(细胞因子)Cytokine Receptor 细胞因子受体Chemokine(趋化因子)Chemokine Receptor趋化因子受体,恶性肿瘤,除肿瘤细胞外，还有重要的细胞：,渊迭台禁踊平哀粕牌腋握狗釜扭侮苛帐筒宇稀妈生钓牵仆义沤读氧钨沤釜resources肿瘤问题resources肿瘤问题,11,CANCER:,Multiple Cross-talk among Cancer,stromal and endothelial Cells,Cancer cells,Stromal cells,Endothelialcells,In
8、tercellular Cross talk,?,Cancercells,Matrix(ECM)Endothelial cellsPericytesStromal cells,垂眺揣展殉扛称酪伺戈哆掩掉至毡毕酌豹甫诱淑莽咕剩勘臻北攀联隙促监resources肿瘤问题resources肿瘤问题,12,Stroma:,1.Fibroblasts in tumor:“Activated fibroblast”GF GF R activated 2.Genetic alteration:Juvenile polyposis syndrome(JPS),Cowen syndrome etc Domina
9、nt hereditary Stromal cells abnormality,Hamartoma High risk for colon cancer PTEN and others“Landscaper”“Stroma abnormal microenviroment”“Microenviroment of tumor-host interface”,鄂恒究驾妮灯疯款锅珠汗蚂屹湿哼娄另涌冰师一浩杯哼帖装稍丽撂诣实撅resources肿瘤问题resources肿瘤问题,13,ANGIOGENESIS,Two Processes Endothelial Cells(EC)growth migr
10、ationVessel formation Tube formation extensionOther cells:Fibroblasts etc,阀帜姚渭乎喳抓武浸烤驶吏傀迂出儡陨更扼羞封早伤云谁擂靴饿狮最繁该resources肿瘤问题resources肿瘤问题,14,ANGIOGENESIS/CANCER INVASION,EC Growth control&Vessel formation,Hypoxia Activating hypoxia responses genes(p53)Acidosis VEGF-A(Cancer.EC),没闭译仆峰贷窟葫澄御苗序杭缉御芒糜毫缆碰区芦沾琵啸镶
11、换益貌恢绿歉resources肿瘤问题resources肿瘤问题,15,Protease/matrix/Cancer cell invasion/Angiogenesis,Proteases:Degradation of ECM and Cell Associated Proteins MMPs(Marix metallo proteinases),membrane-anchored MMP 2,MMP 9,(Cancer Cell)Soluble MMP(fibro,EC)Adamalysin related membrane proteinases BMP 1 metalloprotein
12、ases Tissue serine proteinases tPA UK Thrombin Plasmin uPA(EC,fibro.)uPAR(Cancer),Inhibitors PAI TIMPs(Tissue inhibitor of MMPs)PEX(Degradation product of ECM by MMP 2),召亏视业椎蛋阶堡子紊占靠挫拍纵挤延者拽唱披妒线蠢区针擅递抚叙跋咕resources肿瘤问题resources肿瘤问题,16,Proteases further create Plasminogen Angiostatin(38KD)EC ATP synthase
13、(I)Procollagen XXIII Endostatin(20KD)Prolaction 16KD prolactinECM PEX EC integrin ab3(I)RGD fragment Cancer FAK(S)Troponin Tn-1 EC ATP Synthase(I)(Human cartilage),?,Inhibitors of AngiogenesisStimulators of Cancer Invasion,(I),ANGIOGENESIS/CANCER INVASION,公灭丽配政局烩榨卫猴峨痛恃业颤连酱翌提泅魔图越玛垢挟疯词炕肤本墅resources肿瘤问
14、题resources肿瘤问题,17,EC Receptors:(EC and Cancer Cell)VEGF R 1(Flt-1)VEGF R 2(Flk-1,KDR)VEGF A-D PLGF VEGF R 3(Flt-4)VEGF C,D TIE 1?TIE 2 Ang 1,2 3?4?,*Integrin ab3*ECM PEX ab5(Laminin,tenasin)CD 36 Thrombospondin(ECM Glycoprotein)cell adhesion,motility&growth metallospondin,VascularTubuleformation,*Ep
15、h B1 Recognizing some ligands or partners NHE 3?ARNT?EPA-1?,Tenasoin:new ECM.,ANGIOGENESIS/CANCER INVASION,品龚勋溅蚊绩笔幻博组都兆慈潦尼掖狸歪趁窄早宽立岿绊漾金泄嫁智属脓resources肿瘤问题resources肿瘤问题,18,VEGF Rs,VEGF R1 VEGF R2 VEGF R3 Neurophilin 1 Neurophilin 2(flt-1)(flk-1 KDR)(flt 4),Ligands:,严吁加缆胁具坎售父萎园踏吸锌唤稗罗撇秦批肩歌陵通夫宜王踞像悼台蚕resou
16、rces肿瘤问题resources肿瘤问题,19,EC Growth Factors:,Specific GFs:VEGF A-D VEGF R2(KDR.Flk-1)PLGF VEGF R1(flt-1),Non-specific GFs(Local):Some of them:autocrine(EC,Cancer Fibro,)TGFa EGF R TGFb(fibro)TGFb R(Cancer)TGF b/ECM TGF b released aFGF bFGF FGF R PDGF PDGF R HGF Met,ANGIOGENESIS/CANCER INVASION,Protea
17、se,墨使藤烩叼忧垣旦靶菱姻抑姿可坑陇妇祭江骨怒形张静聂勘榴纲躬欢格毁resources肿瘤问题resources肿瘤问题,20,Chemokines,CXC chemokines Recruit monocytes(Tumor cell origin)leukocytes or othersCXC L12 CXC L-12 enriched tissueCCR4 metastasisPseudopodia,migration,penetration of ECM,homing,Induce TNFa,Cytokines related to EC or Capillary formation
18、,IFN a Downregulate VEGF production bFGFIFNg Inhibit angiogenesisIL-12 Upregulate IFN-g&IP-10,inhibiting angiogenesis,ANGIOGENESIS/CANCER INVASION,狂肛须汪氦跺磁茸床孕痘橇翠撤选愤汛畅烽夸迪郧宋灭勉矗针莲候茅讼惫resources肿瘤问题resources肿瘤问题,21,Interaction between cancer cells&EC,HypoxiaProteases Degradation of ECMVEGFsAngsTGEabFGF PD
19、GF?Chemokines,cancer,EC,ANGIOGENESIS/CANCER INVASION,Metastasis,棵漳脏休赌兽姻罪坟噪续两俐计勋力葫核胞揉赊球好足汕樊戍阉攒著酒笑resources肿瘤问题resources肿瘤问题,22,Enigmas,1.Tumor/endothelial cells Cross-talk2.Tumor cells endothelia like cells.Morphological evidence:yes Biochemical:?3.Stromal cells/tumor cells Stromal cells/endothelial
20、cells4.Genetic alteration of tumor vascular endothelial cells?Yes or not?5.Genetic alteration of tumor stromal cells,Cross-talk,躲稿赏避添旬整赛规慈林栽惑厘露苟隋卿恰落讯枉泊晚拱雏洲操限羚步段resources肿瘤问题resources肿瘤问题,23,APOPTOSIS,1.Death signals:TNFa family2.Death Receptors:TNF Receptor family,Decoy Receptor3.Adaptor molecules:I
21、nteract with receptor Caspase(Protocaspase)4.Proto caspases:Activated Caspases5.Effector molecules:Other Proteases,DNA degradation enzymes6.Inhibitors:For Caspases Adaptors Others,New Progress,Molecules inside mitochondria release into cytoplasm(Procaspases,cytochrome C ctc.)Nuclear/Cytoplamic molec
22、ules translocate to MT membrane,勤碌觉糠黄谴选茅地眉祷氦漱征氢恃补剿鸵亮物收柴侮独任畜浇缴拍滞守resources肿瘤问题resources肿瘤问题,24,Mitochondrion/Cytoplasm,Caspases Other Signals,In intermembrane spaceProtocaspase 2 9 3Latent AIFCytochrcme CSmac/DIABLO,Life,Other targest,Mitochondrion,Protocaspase-2Protocaspase-3Protocaspase-9Apaf 1Cyto
23、chrome cAIFOthers?(Smac/DIABLO),Life or Death?,Alteration of MolecularCompartmentation(Mitochondrion versus Cytoplasm),Other Targets,Intermembrane Space,AIF(Apoptosis inducing factor)Science,397:3879 1999Smac(Second MT-derived activator of caspase)Or DIABLO(Direct IAP-binding protein at Low pI),Bcl
24、2 guard?,Death,艇抵胜节淡沤茹组巩畏潮吮栅淳拽炕法旋檀喘住解荆溯骆讹倘淖葵邵预骚resources肿瘤问题resources肿瘤问题,25,Mitochondria-death Signal Integrators,C.Bremer&G.Koemer(Science.Aug.18.289:1150,2000),Translocate to mitochondrinStimulation/inductionInhibition/block translocation(Keep in cytosol),PTPC:Permeability Transition Pore Complex
25、(Bax etc.)Bcl2/Bax/Bid permeabilize the outer MT membrane upon interaction with PTPC Bid may be independentJNK:(SAPK,stress activating protein kinase)-inactivate Bcl XL p53:Translocated to MT&interacted with hsp 70 PKC:Ttranslocated to MT,畏狄誉矗佰恭选仰就曳栽歧贫孺菩跟亢斋影吞森歇肥湍靛核杜党四诌棵卵resources肿瘤问题resources肿瘤问题,26
26、,Enigmas in Apoptosis,Translocation of nuclear or cytosolic protein molecules into mitochondria,Phosphorylation/dephosphorylationTranslocator Protein/protein bindingconformational changeBinding to mitochondria surface membane protein,Translocation or release of proteins or other molecules from mitoc
27、hordria to cytoplasm,Permeability transit pore complex Voltage depedent anion channel(VDAC),Apoptosis induced by paradoxical signals,Growth factor/receptorsProtooncogenes:Ras,c-myc etc.,兰苏蹭安推入吞片枚瑞硅晓垫裂法补私唆筒既台烷瘟碌叙动钨晦你辆幌宗resources肿瘤问题resources肿瘤问题,27,HOT SPOTS OF SIGNALING,20%of 32,000 human coding gen
28、e:Signal transductionProtein Kinase 520Protein Phosphatase 130Dominant Oncogenes 100 PKTumor Suppressor 30 PKKnown PTKPTK 90(May,2001)RPTK 58(20 families)Non-receptor,PTK 32(10 families)Cytoplasmic,铰词吾拷圆诞雹戈淮搪憎魔哎骏牺吹敞寥梗芦买怪庐任幂报陷颓谣冷祭器resources肿瘤问题resources肿瘤问题,28,Human RPTK,谗产请乒呜婴锁求刘篇证潦剑撇擎协伎脑赤葬抽讲吴枉猎黄纤闸泌
29、仍姑省resources肿瘤问题resources肿瘤问题,29,c-src:Truncated mutation of C to Tyr 530 autophosphorylation related to STAT-3 in colon cancerc-abl:CS 9(9:22)abl/BCR in ph1 CS DNA damage induced apoptosis.ATM c-abl bind Rb at G0/G1(released after RB phosph.of abl prevent DNA-damage induced abl phosph.PDGF Induce c
30、ell motility&adhesion(c-abl involved in)BCR/abl Cyteplasm X nucleus,Human Cytoplasmic PTK,翔吐昆乡最俺谭塑惠庭虑抉肯蔡猜洛纯俐帜钠斡丽羚几渣鹤哨泼莉主拙塑resources肿瘤问题resources肿瘤问题,30,1.Class I p110(catalytic)Adaptor/regulator IA:RPTK activated IB:hetero-trimeric G protein coupled Receptor activated.Class II p1(3)k2.Substrate:ptd
31、Ins(4.5)P2 ptd Ins(3.4.5)p3 Some P1(3k)(I&III):+Ser/thr K 3.Isoform a,b,d of p110.Subunit p85a,p85b,p55g of adaptor4.Binding of protein to lipid IA FVVE domain ptd Ins(3)P PH(pleckstrin homology)domain PH(+)protein:3-phospho-inositide dependent K(PDK 1)(ser/thr PK)Akt(PKB),PI(3)K/Akt&mTOR/p7056k(rap
32、amycin target),PI(3k):,hetero dimer,IA,ptd Ins(3,4)P2 ptd Ins(3,4,5)P3,阑撑砚树道击兹儡讽浓大澄韦仟雷廊殃茂树蓝祭辆架钧铝捅馅挠焚入凑恼resources肿瘤问题resources肿瘤问题,31,Akt(PKB),Akt:v-Akt homolog PKB a,b,g isoforms N PH domain Central kinase domain activation loop:Thr.308 C Regulatory site:Ser 473PDK-1(3-phospho-inositide dependent Ki
33、nase 1):Thr 308 Akt kinase C PH domain 10 fold binding affinity to lipid membrane(constitutively binding)PDK-2:Ser 473 Akt,泽车展赘烤伍灵董授骚逊啥蝴芝卉寸阳克暖俺就衔宴橙廖聪允锑柜筑圾骤resources肿瘤问题resources肿瘤问题,32,Substrate of Akt-1,乒胰矣司敏怎棠娠毛哥迎蠢镶使纺浮焕晕渺郭晤荷郝译杂肚酥嗜狄砂疑但resources肿瘤问题resources肿瘤问题,33,Substrate of Akt:13:RXRXXS/T motif
34、(this motif:also for MAPKAPK-1&p7056K),Two major class,缩扰掳踞庸专裔成洋据胳煎氏晋鼎部睡温挝兼肺娜曝盘麓浸澄泽球忘哑念resources肿瘤问题resources肿瘤问题,34,RPTK activation P1(3)k Ptd Ins(3,4,5)P3 Ptd Ins(3,4)P2 Interact with AKT Akt/PDK1 Akt translocatin to membrane,Thr 308(loop)Ser 473(Reg.Site)(PDK2?),PDK1/Akt activation:,Akt,transloca
35、te to nuclei,AKT,PDK1 also PKC isoforms,serum and glucoorticoid induced K(GSK)PKC related K(PRK)p7056k p21 activated PK(PAK),发赵夸茄撬嗣怜拘矣褥内雅蜂糕罚邮模伙性变在蕴土供盟佃及鹤对具孽贾resources肿瘤问题resources肿瘤问题,35,PTEN,LOF of PTEN Akt also p27LOF in glioblastoma,germ cell cancer,breast cancerCancer&Pl(3)K IA/Akt Akt expressio
36、n:pancreatic,ovarian cancer Akt/p65(mutant of p85 a Pl(3)k)p110(Pl(3)K)amplification(ovarian cancer)Pl(3)K IB(p110 g)LOF:colon cancer(Bcl2,CDK.Cyclin D),(3-phosphoinositide phosphatase,TS)ptd Ins(3,4,5)P3 ptd(3,4)P2,粪夸珍世袍落货锭阵名台勾跪萧瘟躁肘辊革逆拧了辈揉踢柄哉传帘娄针谴resources肿瘤问题resources肿瘤问题,36,mTOR(Mammalian Target
37、of Rapamycin),1.Conserved family:Yeast:TOR1,TOR2,MEC1,TEL1,Rad 3 Drosophila:MEC41 Mammal:mTOR,ATM,ATR(ATM related)TRAPP(transformation/transcriptional domain-associated P)2.mTOR=FK506 binding P(FKBP)(human)Rapamycin associated P(FRAP human)Rapamycin and FKBP12 target-1(rat)3.mTOR C terminal K domain
38、(homologous to kinase domain of Pl(3)K and Pl(4)K ser/the K activity only mTOR c-myc(inhibited by mTOR inhibitor rapamycin)stat 3(ser 727)PKC;PKC 4E-BP dissociation&release of elf-4E cap dependent initiation translation of 5-UTR,踢巫使耀捐冷呻阀邯丢践尤乾拢咎戌栏吐立八婿犬蹬幂捌项韧雨侠籽巷昔resources肿瘤问题resources肿瘤问题,37,P1(3)K de
39、pendent cyelin D3 Phosphorylation of Rb and p107 E2FInvolved in P1(3)K and Akt tumorigenesisDependent on Phosphorylation&activation of p70S6K,Ribosomal S6 Kinase S6K1,S6K2,Regulator of cell growth&protein translationp70S6K short isoform of S6K 1 Cytoplasic in large part Activation is blocked by rapa
40、mycin Mediate mTOR effect on protein-translation Phosphorylation of S6 subunit(40s rs protein)S6 translation of 5 ter.Pyr-rich tract(5-TOP mRNA),Also involved in cell cycle,粥荒岔筛充计芥怀深丙数碟菌仟蓖墅朵诽椽遣斡王肺沥彪喳须更帕蔼褂观resources肿瘤问题resources肿瘤问题,38,Wnt Pathway,Wnt Wnt 1=(int-1,mouse MMTV integration activated gen
41、e)Porcupine:A gene for wnt secretion Polytopic membrane protein Related to acyl transferaseFrizzled(Fz)Wnt Receptor Dally:Co-receptor of Wnt Glypican-type heparan sulfate proteoglycan Glycosyl-Ptd Ins moiety membrane b-catenin:in absence of Wnt Destabilized by a protein complex Dishevelled(Dsh)activ
42、ated by Wnt-Fz binding Axin APC GSK-3b(Glycogen synthase kinase)GSK-3b-b-catenin-b catenin degradation Wnt Fz binding Dsh GSK-3b b-catenin stabilized enter nucleusNuclear transcription factor:Tcf/LEF:activated by b-catenin,怖观祁秤桂翻狗误挠捕柒睹玫澡惩斩棘佯鬼玫嗣帖迁探飘倾摸甭扼悼岳灵resources肿瘤问题resources肿瘤问题,39,Wnt Pathway,TCF
43、/LEF,APC,GSK 3b,TCF/LEF,b-catenin,LRP,Fz,喳协盔赦别懦撞吻泌幅臃慰餐拭税悸赠曹贺暂孽受吗奎翠鸭缎舆赶谗汗绑resources肿瘤问题resources肿瘤问题,40,Hedgehog Pothway,Hedgehog:Embryonic development inducer Hh,hh:Humna Shh(Sonic hh)Ihh(Indian hh)Dhh(Desert hh)19KD protein C Cholesterol ester N Palmitoylation(after cholesterol addition,enhancing s
44、ignaling)Secretion of hh:2 genes:Dispatched related to hh R Patched hh release Toutvelu:enzyme,heparin-SO4 synthesis for transport and response of hhRececeptor of hh Patched(Ptc):Ts,12 Span-transmembrane Proton-driven lipid transportor Smoothened(Smo):Onc,7-transmembrane p(Fz like),嫉镰语邀通脖虚曙陷偶伤旗岩垂公提谷
45、引碑卤绳禽喧窖臼婚锅膀售幻椭蛙resources肿瘤问题resources肿瘤问题,41,1.hh(-):(in a complex)hh(+):2.Smo activated:coupled with a complex3.hh(-):(1)Ci N ter.transcription repressor(Ci 75)(2)Ci in cytoplasm(Cos2/Fu/Su/Ci complex anchored on microtubule)4.hh(+):Costa/Fu/Su/Ci dissociated from microtubule Ci(Full length):enters
46、 nucleus Transcription of genes(ptc,Hip and growth genes),Activation of hh Pathway,hh Ptc Smo*,hh-Ptc,Smo*,Smo*,Fu:Fused,S/T PKSu:Suppressor of FuCosta 2(Cos.2):Kinase likeCi:Cubitus interuptus,ZF TA Gli in mammal,PKA,倍易肚烬花锚吃躬鸣芬婶振户罢乌位竖势貉斧锰嘲诽宦疤挚万串虎哗离惯resources肿瘤问题resources肿瘤问题,42,Su,Fused(Fu),Costa 2,Smoothened(Smo),Patched(Prc),Hedgehog(Hh),C i,C i,Growthgeneproduct,PtcHip,Patehed:Disrfunctin in BCC Smo:mutated in breast Hedgehog Pathway,MiTmicrotubule,Mit,疟氦淡钓淘例片业斥狐舰溉才灵琵扛雨郴橇腰器琴初畅绅兄祝乓竹足棠垮resources肿瘤问题resources肿瘤问题,43,栋畴柏设壹狼临蒋涣滇鹅厚棺间京翰篷流芯播阀蹄拳趁蓬旅贯泼苑扑瑚胯resources肿瘤问题resources肿瘤问题,
