细胞生物学的研究思路

细胞生物学研究思路的探讨李家大医学遗传学国家重点实验室Email：[e-mail]研究背景•Waardenburg综合征（Waardenburgsyndrome，WS）是最常见的综合征型遗传性耳聋，主要遗传方式是单基因致病的常染色体显性遗传伴不全外显。•主要临床特征：耳聋与着色异常（虹膜、皮肤、毛发）分型致病基因临床表型特征WS1PAX3耳聋、着色异常、眼距增宽WS2MITF、SOX10、SNAI2耳聋、着色异常WS3PAX3耳聋、着色异常、上肢发育异常(Klein-Waardenburg综合征)WS4SOX10、END3、ENDBR耳聋、着色异常、巨结肠(Shah-Waardenburg综合征)•神经嵴发育不全：由于神经嵴细胞（neuralcrestcells，NCC）发育缺陷或障碍而导致神经嵴出现异常的增殖、生存、迁徙和分化。由于NCC发育不良导致的这些组织和细胞的病变统称为神经嵴病。研究背景研究背景分型致病基因氨基酸改变耳聋虹膜异色皮肤色素改变内眦异位巨结肠WS2MITFR217I先天性亮蓝色虹膜面部大量雀斑——WS2MITFT192fs先天性完全性虹膜异色———WS1PAX3H80D先天性完全性虹膜异色—+—WS1PAX3H186fs先天性亮蓝色虹膜—+—WS2SOX10E248fs先天性亮蓝色虹膜———WS2SOX10G37fs先天性亮蓝色虹膜———WS2SOX10G38fs先天性亮蓝色虹膜———WS2SOX10R43X先天性完全性虹膜异色面部少量雀斑——WS4SOX10W85X先天性完全性虹膜异色——+研究背景本研究Waardenburg综合征病例基因型和表型特征研究背景基因组水平：WS致病基因突变检测后基因组水平：WS致病基因功能检测分子水平：WS发病机制临床水平：WS基因型如何导致表型研究目的前期研究本文研究表型基因型基因型表型？研究内容蛋白表达水平PAX3与SOX10相互作用活性检测蛋白亚细胞定位蛋白与DNA的结合MITFPAX3SOX1012345invitro课程提纲蛋白质加标签(Tag);定点突变；转染亚细胞定位；Reporterassay；DNA-蛋白质相互作用；蛋白质相互作用；蛋白质半衰期。蛋白质加上标签(Tag)Why：便于检测；市场上有很好的针对标签蛋白的抗体。What：Flag:DYKDDDDK；HA：YPYDVPDYA；c-Myc：EQKLISEEDL；V5：GKPIPNPLLGLDSTHow：前提是不能影响蛋白质的功能课程提纲蛋白质加标签(Tag);定点突变；转染亚细胞定位；Reporterassay；DNA-蛋白质相互作用；蛋白质相互作用；蛋白质半衰期。定点突变site-directedmutagenesisQuikChange®Site-DirectedMutagenesisKit(Stratagene公司)突变引物设计=Tool&SubPageType=ToolQCPD&PageID=15GoogleSearch“Quikchangeprimerdesign”原始质粒原始质粒改造突变质粒构建DNA测序鉴定课程提纲蛋白质加标签(Tag);定点突变；转染;亚细胞定位；Reporterassay；DNA-蛋白质相互作用；蛋白质相互作用；蛋白质半衰期。转染-transfectionTransfectionisamethodoftransportingDNA,RNAand/orvariousmacromoleculesintoaneukaryoticcellbyusingchemical,lipidorphysicalbasedmethods.TransfectionmethodsDEAEdextranCationicpolymerCalciumphosphate磷酸钙Electroporation电穿孔Microinjection显微注射CationicliposomeDEAE-dextranDiethylaminoethyl(DEAE)-dextranisoneoftheoldestmethodsforintroducingnucleicacidsintoculturedmammaliancells.ThepositivelychargedDEAE-dextranmoleculeinteractswiththenegativelychargedphosphatebackboneofthenucleicacid.TheDNA–DEAEdextrancomplexesappeartoadsorbontothecellsurfaceandbetakenupbyendocytosis.优缺点：1.仅适合瞬转（transienttransfection），不适合稳转；2.细胞毒较高；3.重复性较好。CationicpolymerBranched(A)andlinear(B)polyethyleneimine(PEI)商品名：ExGen500，TurbofectCalciumPhosphate磷酸钙TheprincipleinvolvesmixingDNAinaphosphatebufferwithcalciumchloride.Theresultingcalcium-phosphate–DNAcomplexesadheretothecellmembraneandenterthecytoplasmbyendocytosis.优缺点：1.适合稳转；2.重复性较差，主要影响因素包括溶液的pH值；3.一些细胞不适合磷酸钙转染。CationicLiposome脂质体Theliposomescurrentlyinusetypicallycontainamixtureofcationicandneutrallipidsorganizedintolipidbilayerstructures.Transfection-complexformationisbasedontheinteractionofthepositivelychargedliposomewiththenegativelychargedphosphategroupsofthenucleicacid.Theuptakeoftheliposome–DNAcomplexesmaybemediatedbyendocytosis.品牌：Lipofectamine(Invitrogen)。优缺点：1.转染效率高；2.重复性好；3.转染时需降低血清浓度，从而提高了细胞毒性；ElectroporationCellsinasuitablecuvettearesubjectedtoashorthigh-voltagepulsethatcausesthemembranepotentialofthecellstobreakdown.Asaresult,poresareformedthroughwhichmacromoleculessuchasDNAcanenter.优缺点：1.适合几乎所有的细胞；2.瞬转、稳转；3.需要DNA量大；4.高达50–70%细胞死亡；Microinjection•DirectMicroinjection:Useofafineneedle.HasbeenusedfortransferofDNAintoembryonicstemcells.研究结果野生/突变PAX3蛋白表达野生/突变SOX10蛋白表达AB课程提纲蛋白质加标签(Tag);定点突变；转染;亚细胞定位；Reporterassay；DNA-蛋白质相互作用；蛋白质相互作用；蛋白质半衰期。ReporterAssay:fortranscriptionalfactors(TF)PromoterGeneofinterestTFDBDADPromoterReportergeneTFDBDADTF:transcriptionalfactorAD:activationdomainDBD:DNA-bindingdomainReporterGene:Agenewithareadilymeasurablephenotypethatcanbeeasilydistinguishedoverabackgroundofendogenousproteins.Reportergenes1.CAT：chloramphenicolacetyltransferase2.-gal：(-galactosidase)3.SEAP：(secretedalkalinephosphatase)4.Luciferase5.GFP：GreenFluorescentProtein……CAT：chloramphenicolacetyltransferase氯霉素乙酰转移酶1.1streportergeneusedtomonitortranscriptionalactivityincells;：2.Bacterialenzymethattransfersacetylgroupsfromacetyl-CoAtochloramphenicol,detoxifyingit;3.Reactionquantifiedusingradiolabeledsubstrates(14C-chloramphenicol)orbyELISA(non-radioactive).CATassay：acetylated&non-acetylatedchloremphenicolarechechedbyTLCCATassay：ELISA-gal(-galactosidase):•E.colienzyme(encodedbylacZ)thathydrolyzessugarssuchaslactose•Manyassayformats:colorimetric,fluorescent,chemiluminescentSEAP(secretedalkalinephosphatase):•Secretedoutsidethecell(canassaysamplerepeatedlyandnon-destructivelybysamplingculturemedium)•Thisproteinisquantifieddirectlybymeasuringtheenzymeactivityinthesupernatantoftheculturemedium.•Fluorescenceandchemiluminescenceassaysareavailablefordetection.Luciferase:•Firefly(Photinuspyralis)luciferase•Seapansy(Renillareniformis)luciferase•FireflyluciferaseproduceslightbyATP-dependentPhotinuspyralisRenillareniformisGFP:GreenFluorescentProtein•DerivedfromjellyfishAequoreavictoria•AutofluorescentuponUVirradiation(doesn’trequirecofactorsorsubstrates)•Retainsactivityinpresenceofheat,denaturants,detergents,mostproteases•Allowsfornon-invasivemonitoringofgeneexpressioninlivingtissues研究方法荧光素酶活性检测（Luciferaseactivityassay）研究结果ABMITFpromoterMITFprom