实例分析分子生物学技术的应用课件

生物化学与分子生物学实验技术2021/8/51课程安排：32学时讲课：三次(1117,1124,1201)实验：四个/四组？/加样器？/时间？2021/8/52

实例分析分子生物学技术的应用以interleukin24为例2021/8/53二、基因功能研究1.mRNA水平和蛋白质水平表达谱2.体外生物学活性：细胞模型，调节表达水平

细胞活力、细胞增殖、细胞周期、抑瘤活性、等等3.体内生物学活性：抑瘤活性、成血管活性、等等内容一、基因的基本信息：发现、序列、染色体定位、表达调控、同源性分子、修饰、降解、细胞内定位、空间结构等等

三、作用机制：信号转导途径和相互作用分子四、应用2021/8/54235documentsIF5.977.57.22.765.03.069.384.35.521.34.15.256.482021/8/55Oncogene1995,11:2477-2486CancerBiolTher2003;2:S23–S37β-干扰素＋密执霉素(mezerein)人黑色素瘤细胞HO-1

消减杂交mda(melanomadifferentiationassociatedgene)mda-5，mda-6(p21)，mda-7/IL-24，mda-9/synteninSubtractionhybridization一、mda-7/IL-24PaulB.FisherColumbiaUni1.发现2021/8/56杂交产物的克隆、筛选和测序HO-1HO-1IFN-β+MEZ生长阻滞终末分化失去致瘤潜能消减杂交探针Biotin-21-UTP除去杂交体、过量探针、盐和小片段CancerBiolTher2003;2:S23–S372021/8/572.提交序列NCBI/Genbank2021/8/582021/8/592021/8/5103.染色体定位DatabaseanalysisCytoGrowthFactorRev.2003,14:35–51IL-10Family2021/8/511MolMed.2001;7(4):271-2822021/8/512

基因

物种其他名称氨基酸序列同源性MDA-7Mob-5FISPhIL-24HumanMDA-7100%68%69%rIL-24RatMOB-5,C49A68%100%93%mIL-24MouseFISP69%93%100%4.与IL-24同源的分子一致的(*)保守的(:)相似的(.)CytokineGrowthFactorRev.2003;14:35–51Immunology.2005;114(2):166-70NCBIBlast,KEGG2021/8/5132021/8/5145.基因的表达调控JCellPhysiol.2000;185(1):36-46基因组文库IL-24cDNA探针PCR测序启动子：GCG启动子搜索转录起始位点：引物延伸和作图转录水平2021/8/515启动子序列JCellPhysiol.2000;185(1):36-46AP-1:PKC-activatedTFC/EBP:growtharrestandterminaldifferentiationassociatedTF2021/8/516TBPTAF

TFIIH转录起始前复合物：真核生物RNA聚合酶不与DNA分子直接结合，而需依靠众多的转录因子。转录前起始复合物拼板理论：一个真核生物基因的转录需要3至5个转录因子。转录因子之间互相结合，生成有活性和专一性的复合物，再与RNA聚合酶搭配而有针对性地结合、转录相应的基因。2021/8/517RNA聚合酶保护法分析启动子结构2021/8/518LuciferaseassaysystemJCellPhysiol.2000;185(1):36-46NdeI-NheI是启动子转录活性的重要区域，C/EBP-b和cJUN/AP-1提高转录活性。重要区域2021/8/519nuclearlysatesarrow1：C/EBP-barrow2：AP-1JCellPhysiol.2000;185(1):36-46EMSAC/EBP-b和cJUN/AP-1结合在mda-7/IL-24启动子上。celllysates2021/8/520IFN-β和MEZ单独或联合处理不能显著改变启动子活性

Luciferaseassaysystem,NBOncogene.2000;19(10):1362-82021/8/5213’-UTR的ARE调节该基因在终末分化过程中的mRNA水平ARE：AU-richelements

LuciferaseassaysystemOncogene.2000;19(10):1362-82021/8/522dayNCI-H12997.分泌蛋白MolTher.2004;9(3):355-67

Overexpression,WBAd.IL-24celllysateAd.IL-24supernatant2021/8/523分泌作用的抑制剂MolTher.2004;9(3):355-67

WB2021/8/524CytokineGrowthFactorRev2003;14:35–51二硫键糖基化位点C2021/8/525PNGaseF:

endoglycanaseFendo-O:内-O糖苷酶6.翻译后修饰Overexpression,WBJBiolChem.2002;277(9):7341-7

MDA-7/IL-24蛋白可被糖基化修饰MolTher.2004;9(3):355-67

glycopeptidaseFLSN2021/8/5267.细胞内定位MolTher.2004;9(3):355-67IFC2021/8/527MolTher.2004;9(3):355-67SNL2021/8/528QuantitativereceptorbindinganalysisJBiolChem.2002;277(9):7341-7

AP:HumanPlacentalAlkalinePhosphatase293TIL-20R2,IL-20R1/IL-20R2,IL-22R1/IL-20R2结合AP的活性高。8.受体2021/8/529JBiolChem.2002;277(9):7341-7

Cellsurfacestainingassay2021/8/530RT-PCRExpDermat.2006;15:991–10042021/8/531CancerBiolTher.2003;2:S23–S37RT-PCRPCNC2021/8/532Cytokine.2008;41:16–23FACS2021/8/533IL-22R1/IL-20R2IL-20R1/IL-20R2Immunology.2005;114(2):166-70

2021/8/534CytoGrowthFactorRev.2003,14:35–512021/8/5353.前列腺4.睾丸5.卵巢6.小肠7.结肠1.mRNA表达谱：分布在人的免疫系统NB二、基因功能的研究Oncogene2001;20:7051–7063.1.脾脏2.胸腺8.外周血白细胞2021/8/536CytoGroFacRev.2003;14:35–51−：untreated+：IFN-β+MEZ,24hNBOncogene.2001;20:7051–70632021/8/537Real-timeRT-PCR在黑色素瘤恶性发展过程中，mda-7/IL-24的mRNA表达水平逐渐降低，直至完全丧失。PharmacolTher.2006;111(3):596-628

2021/8/538Kaplan-MeiersurvivalanalysisCancerCellInter.2010;10:292021/8/5392.蛋白水平：IHCCytokine.2008;41:16–23类风湿关节滑膜NCsubliningmononuclearcellsendothelialcellsofbloodvessels2021/8/540Cytokine.2008;41:16–23ELISA2021/8/541免疫系统：脾、胸腺、外周血白细胞特定细胞：黑色素细胞、痣、早期黑色素瘤细胞、平滑肌细胞、皮肤成纤维细胞、皮肤伤口边缘和基底类成纤维细胞样细胞

肿瘤细胞：50多种，无内源性MDA-7/IL-24蛋白表达。分布：局限性2021/8/542基因在靶细胞中的过表达Adenovirus2021/8/543JCellPhysiol.2007;210(2):549-59

CAR检测(Coxsackie-adenovirusreceptors)FACS,WB2021/8/544人正常卵巢上皮细胞卵巢癌细胞靶细胞中重组蛋白的检测Western-blot卵巢癌细胞MolCancer.2007;6(1):112021/8/545CancerImmunolImmunother.2007;56:205–2152021/8/5467d3.细胞活力CrystalvioletstainingCancerGeneTher.2006;13(11):1011-22TRAIL：肿瘤坏死因子相关的凋亡诱导配体2021/8/547MolMed.2001;7(4):271-282Trypanblueexclusionassay2021/8/548CancerImmunolImmunother.2007;56:205–215TrypanblueexclusionassayAd.mda7能够降低细胞活力。2021/8/549特异性抑制多种肿瘤细胞生长4.体外抑瘤活性：细胞增殖CancerGeneTher.2006;13(11):1011-22

MTT2021/8/5503H-thymidineAssayMolMed.2001;7(4):271-2822021/8/551CancerRes.2006;66(16):8182-8191Colonyformation2021/8/552缺失信号肽的突变体(SP-mda-7)具有抗瘤活性CancerRes.2004;64:2988–2993.WB,MTT2021/8/553MatrigelInvasionAssay,ColonyformationC8161C8161CancerRes.2004;64:2988–2993.2021/8/554Xenografttumorsinnudemice5.体内抑瘤活性SW620CancerGeneTher.2006;13(11):1011-222021/8/555CancerBiolTher2003;2:S23–S372021/8/556SW620CancerGeneTher.2006;13(11):1011-22Xenografttumorsinnudemice2021/8/557IHCNoAb1IL-24CancerGeneTher.2006;13:1011–1022SubcutaneousSW620tumors2021/8/558抗瘤活性源自MDA-7/IL-24蛋白的表达MolCancer.2007;6(1):11

MDAH2774tumor-bearinganimal2021/8/559对增殖无影响影响细胞增殖Mammaryepithelialcells(HuMEC)Prostateepithelialcells(HuPEC)Melanocytes(NHuMel)Bronchialepithelialcells(HNBE)Fetalastrocytes(PHFA)Skinfibroblasts(MJ90)Skinfibroblasts(HF)Lungfibroblasts(NHLF)Endothelialcells(HuVEC)RenalepithelialcellsMesothelialcellsBreastcarcinomaProstatecarcinomaMelanomaLungcancerGlioblastomamultiformeOsteosarcomaColoncarcinomaNasopharyngealcarcinomaPancreaticcarcinomaCervicalcarcinomaOvariancarcinomaPharmacolTher.2006;111(3):596-628特异性抑制多种肿瘤细胞生长2021/8/560MDA-MB453CancerImmunolImmunother.2007;56:205–215HUVECOncogene.2002;21(29):4558-66

5.细胞周期分析FACS使肿瘤细胞G2/M期阻滞2021/8/561CancerImmunolImmunother.2007;56:205–215WB2021/8/5626.细胞凋亡CancerGeneTher.2006;13(11):1011-22

FACSanalysisPIstaining2021/8/563AnnexinVstainingMolMed.2001;7(4):271–2822021/8/564FO-1cellsSB203580：p38MARK的抑制剂DNALadder

PNAS.2002;99:10054–10059.2021/8/565TUNELPBSIL-24CancerGeneTher.2006;13:1011–1022SubcutaneousSW620tumors2021/8/566H1299tumor-bearinganimalOncogene.2002;21:4558–4566IHC,TUNEL2021/8/567PCR,RT-PCR2021/8/568IHC,TUNEL2021/8/569TUNELMCF7MolMed.2001;7(4):271-2822021/8/570CancerImmunolImmunother.2007;56:205–215Caspase3

analysisMDA-MB4532021/8/571人正常卵巢上皮细胞卵巢癌细胞caspase-3、caspase-8和PARP被切割MolCancer.2007;6(1):11

2021/8/572腺病毒感染结直肠癌细胞48h后CancerGeneTher.2006;13(11):1011-22

2021/8/573肺癌细胞JCellPhysiol.2007;210(2):549-59具有剂量依赖效应2021/8/574Z-VAD.fmk:Caspase的广谱抑制剂CancerGeneTher.2006;13(11):1011-22

2021/8/575保护细胞免受凋亡与Bcl-2家族基因相关CancerGeneTher.2006;13(11):1011-22刺激特定细胞凋亡参与caspase9活化和自切割腺病毒感染结直肠癌细胞48h2021/8/576HUVEC+Ad.mda7PositivecontrolNegativecontrolHUVEC+Ad.mda7+MatrigelOncogene.2002;21(29):4558-66Tubeformation7.抗血管形成作用：抑制内皮细胞形成管状结构2021/8/577Oncogene.2002;21(29):4558-66血管内皮细胞标志物表达降低H1299tumor-bearinganimals2021/8/5781.p38MAPK途径三、作用机制MolCancer.2007;6(1):11

卵巢癌细胞人正常卵巢上皮细胞卵巢癌细胞WBPKR:double-strandedRNA-activatedproteinkinase2021/8/579PNAS.

2002;99(15):10054-9

SB：p38MARK抑制剂p38DN：p38负显性突变体黑色素瘤细胞FO-12021/8/580PNAS.

2002;99(15):10054-9

NB，WBGADD：生长阻滞和DNA损伤诱导的基因2021/8/581NB，WB3dPNAS.

2002;99(15):10054-9

2021/8/582Antisense,MTTPNAS.

2002;99(15):10054-9

Ad.vec(whitebars)Ad.mda-7(blackbars)2021/8/583p38MAPK途径和GADD家族成员的关系PNAS.2002;99(15):10054-92021/8/5842.PI3K信号途径MolTher.2003;8(2):207-19H1299/Ad.mda-7+2400cDNAMicroarray2021/8/585conAd-lucAd-mda7H1299conAd-lucAd-mda7HUVECMolTher.2003;8(2):207-19WB2021/8/586H1299TrypanblueexclusionassayMolTher.2003;8(2):207-192021/8/587Biotechniques.2002;Suppl:30–39.2021/8/5883.选择性诱导肿瘤细胞凋亡具有多种机制CancerRes.2004;64:2988–2993.2021/8/589CancerRes.2003;63(23):8138–8144.MDA-7蛋白诱导前列腺癌细胞凋亡的可能机制2021/8/590CancerRes.2005;65(22):10128-101382021/8/591AdMDA-7/IL-24免疫细胞ES/MDA-7/IL-24mAb-MDA-7/IL-24四、临床应用研究CancerRes.2005;65(22):10128-101382021/8/592Cycle1Day30Ⅰ期临床试验证明mda-7/IL-24使用安全、有临床疗效。INGN241(重组腺病毒):Introgen公司,Ⅰ期：28例;Ⅱ期：进行中INGN241(Ad.mda-7)treatmentofamelanomametastasisinarightsupraclavicularlymphnode.MolTher.2005；11,149–1592021/8/5932.Ad.mda-7/IL-24与化疗药物联合使用高表达：25–30%原发乳腺癌和8–11%宫颈癌相关性强：肿瘤的发生、发展和不良预后抗体：4D5,trastuzumab,HerceptinHer-2:humanepidermalgrowthfactorreceptor-2Her-2+转移乳腺癌30%Her-2

+有效

CancerGeneTher.2006;13(10):958-68

2021/8/594

CancerGeneTher.2006;13(10):958-68

FACS2021/8/595

CancerGeneTher.2006;13(10):958-68

Trypanblueexclusionstaining2021/8/596L=Ad-luc

CancerGeneTher.2006;13(10):958-68

Ad.mda-7/IL24+Herceptin2021/8/597

CancerGeneTher.2006;13(10):958-68

Herceptin(1and2mg/ml)Trypanblueexclusionstaining2021/8/598MCF7MDA-MB-453MDA-MB-453

CancerGeneTher.2006;13(10):958-68

2021/8/599与Herceptin联合使用，抗瘤效果更好，并扩大抗瘤谱。

CancerGeneTher.2006;13(10):958-68

MCF-7-Her-18:Her-2-overexpressing2021/8/5100EGFR:epidermalgrowthfactor-receptor2021/8/5101(EGFR-Wildtype)Ad.mda-7＋GefJCellPhysiol.2007;210(2):549-59

MTTGefitinib:anorallyactiveselectivereversibleinhibitoroftheEGFRtyrosinekinase.2021/8/5102JCellPhysiol.2007;210(2):549-59

delE746-A750L858R+T790MEGFR-Wildtype2021/8/5103与gefitinib联合使用，抗瘤效果提高。JCellPhysiol.2007;210(2):549-59

2021/8/5104sMDA-7/IL-24(1)抑制血管内皮细胞成管能力CancerRes.2003;63(16):5105-13

3.基因工程重组MDA-7/IL-24蛋白药物2021/8/5105CancerRes.2003;63(16):5105-13