NYD-SP14基因功能的研究

【作者】 赵沐子；

【导师】 李建民；

【作者基本信息】 南京医科大学 ， 细胞生物学， 2009， 硕士

【摘要】 目的:研究NYD-SP14及其相关蛋白相互作用的机制,探讨NYD-SP14在细胞生长,周期调控,精子发生和肿瘤发生和发展过程中的作用机制。方法:运用生物信息学方法,分析NYD-SP14在不同物种中的同源性差异,通过RT-PCR分析NYD-SP14在小鼠多组织和在正常人及不育症患者精子样本中的表达差异,分析NYD-SP14及其相关蛋白在不同细胞中的表达情况;构建一系列NYD-SP14、PP2A的Aα亚单位、HRB2、HSP70的逆转录病毒载体和慢病毒载体,在永生化HEKTER细胞中筛选稳定表达的细胞株,观察其表达定位,验证NYD-SP14与PP2A的Aα亚单位(PP2R1A)、HRB2、HSP70之间的相互作用,通过流式细胞分析、划痕愈合实验和绘制生长曲线研究NYD-SP14及其相关蛋白表达对细胞生长、增殖和细胞周期的影响,推断NYD-SP14可能的生理学功能。利用生物信息学,构建在三联TPR结构的第十个外显子两端插入LOXP位点的NYD-SP14条件型基因敲除小鼠模型,进一步研究其在小鼠胚胎发育和个体发育中的作用。结果:NYD-SP14在不同物种间广泛表达、高度保守;RT-PCR表明NYD-SP14在脑和睾丸中高表达,在正常人精子样本中表达量是不育症患者样本中表达量的18.5倍;进一步分析显示NYD-SP14在HELA、MCF-7等多种细胞中广泛表达,其相关蛋白HSP90Aα,HSP90Aβ,HSP90B,HSP70和HRB2在HEK293T和HEKTER中都有表达,因为NYD-SP14在睾丸和肿瘤细胞中广泛高表达,说明NYD-SP14可能在其相关蛋白的作用下,参与细胞生长调控。通过慢病毒载体和逆转录病毒载体构建pLenti-SP14-HA-IRES-GFP-Blast、pLenti-SP14-HA、pMIG-PP2R1A、MSCV-GFP-HSP70和pLenti-GFP-HRB2稳定表达细胞株,绿色荧光蛋白的亚细胞定位提示NYD-SP14,PP2R1A,HSP70在HEKTER的胞浆中存在共定位,而HRB2在细胞分裂不同时期定位于不同的部位,分裂期前定位在胞浆,分裂期后定位于胞核。通过WESTERNBLOT的方法对稳定表达细胞株进行验证,通过免疫沉淀证明PP2R1A和NYD-SP14存在相互作用。流式细胞周期检测表明过表达的PP2R1A会抑制细胞增殖,弱表达的PP2R1A对细胞增殖没有明显影响,NYD-SP14可以使细胞阻滞在S期,并诱导细胞凋亡,在PP2R1A高表达时,这种阻滞作用会加强。划线实验和细胞生长曲线也证明PP2R1A和NYD-SP14的表达会抑制细胞增殖,过表达PP2R1A的细胞中再感染NYD-SP14时,这种抑制作用最明显。成功构建NYD-SP14CKO质粒,经PCR鉴定,已获得NYD-SP14flox/+,NYD-SP14flox/flox及NYD-SP14+/–小鼠。结论:小鼠NYD-SP14具有C端的三个连续TPR结构域,在小鼠多组织中广泛表达,脑和睾丸及肿瘤细胞中高表达。细胞试验验证它与PP2R1A和HSP70具有共定位,可以与PP2R1A发生相互作用,并诱导PP2R1A引起的细胞凋亡,说明NYD-SP14可能与PP2R1A具有协同作用,在HSP70/90和HRB2的参与下,共同调控细胞的周期生长。这些研究有助于揭示NYD-SP14在精子发生、细胞周期和肿瘤发生等方面的影响,而NYD-SP14CKO小鼠模型的建立也为进一步研究NYD-SP14在胚胎发育和个体发育中的作用奠定基础。更多还原

【Abstract】 Objective:ToinvestigateNYD-SP14proteinandthoseproteinswhichcaninteractwithNYD-SP14,furtherexploretheeffectsofNYD-SP14oncelldivision,growthandthemechanismoftheseeffectsonthespermatogenesisandtumorigenesis.Methods:ThehomologydifferencesofNYD-SP14indifferentspecieswereviewedthroughbioinformaticmethods.TheexpressionprofilesofNYD-SP14wereviewedthroughRT-PCRindifferenttissuesofmouseanddifferentspermsamples,theexpressionprofilesofNYD-SP14andrelatedproteins----thescaffoldsubunitAαofPP2A(PP2R1A),heatshockprotein70(HSP70)andHIV-1revbindingprotein2(HRB2),wereviewedindifferentcelllines.DifferentplasmidsofNYD-SP14andtheinteractingproteinsthatwerebasedonretroviralandlentiviralvectorswereconstructed,thesteadyexpressionHEKTERcelllineswereobtained,thesubcellularlocationsoffusionproteinswereobserved,andtheinteractionofNYD-SP14andPP2R1Awasidentifiedthroughimmunoprecipitation.ThefunctionofNYD-SP14andtheinteractingproteinsoncellgrowth,proliferationandcellcyclewereinvestigatedandthepotentialbiologicalfunctionsofNYD-SP14weresuspected.Forfurtherresearch,NYD-SP14conditionalknockoutmousemodelwasgeneratedtostudytheconcretefunctiononembryogenesisandontogenesis.Resultsls:NYD-SP14iswidelyexpressedandhighlyconservedinvariousspeciesfromprokaryotetoeukaryote.TheexpressionprofileofNYD-SP14showedthatNYD-SP14iswidelyexpressedinmurinetissuesandhumanspermsamplesbuthighlyexpressedinmurinetesticle,brainandallfertilespermspecimens;furtheranalysisshowedthewideexpressionofNYD-SP14andrelatedproteins,suchasHSP90Aα,HSP90Aβ,HSP90B,HSP70andHRB2indifferentcelllines,includingHEK293T,immortalHEKTER,breastcancercelllineMCF-7andHELA,indicatingapotentialroleforNYD-SP14intestisandcancerdevelopmentand spermatogenesis.ThesubcellularlocalizationsoftheGFP-fusionproteinsrevealedNYD-SP14,PP2R1A,andHSP70werelocatedincytoplasmmostly,whilethemitosiscycle-dependentprotein,HRB2wasexpressedinthecytoplasmbeforecelldivision,butinthenucleusaftercelldivision.WesternBlottingidentifiedthestableinfectedcelllinesthatexpressedNYD-SP14anditsrelatedproteinssteadilyandImmunoprecipitationprovedtheinteractionbetweenNYD-SP14andPP2R1A.ThedetectiononcellcycleandcellgrowthcurveindicatedNYD-SP14couldpromoteG1toSphase,blockmotosisinSphaseandinduceapoptosis,whiletheoverexpressionofPP2R1AcouldsuppressthegrowthofHEKTERandenhancetheblockingeffectofNYD-SP14.TheNYD-SP14flox/+andflox/floxmiceaswellastheNYD-SP14+/–mouseweregeneratedandconfirmedwithspecificprimersbyPCR.Conclusion:MurineNYD-SP14,whichwaswidelyexpressedinmulti-tissuesandhighlyexpressedinmurinetestis,brain,fertilehumanspermatozoaspecimenandcancercells,hasaC-terminalthreecontinuousTPRdomains.SubcellularlocationanalysisrevealsthatNYD-SP14isco-locatedwithPP2R1AandHSP70.TheresultsthatNYD-SP14couldblockmitosisinSphase,inducetheapoptosisinimmortalHEKTERcellandPP2R1Acouldenhancetheeffectsuggestingtheinteractionasacellcycleregulator.TheseresultsconducetorevealthefunctionofNYD-SP14onspermatogenesis,cellcycleandtumorigenesis,andlayafoundationforthefunctionalstudyofNYD-SP14.TheCKOmousealsoprovidesnewwaysforthefurtherstudiesofNYD-SP14onembryogenesisandontogenesis.更多还原