pEGFP--hTERT轉(zhuǎn)染雞胚胎干細(xì)胞建系的研究.pdf

1、黃曉梅pEGFPhTERT轉(zhuǎn)染雞胚胎干細(xì)胞建系的研究pEGFPhTERT轉(zhuǎn)染雞胚胎干細(xì)胞建系的研究研究生：黃曉梅導(dǎo)師：李碧春專(zhuān)業(yè)：特種經(jīng)濟(jì)動(dòng)物飼養(yǎng)(揚(yáng)州大學(xué)動(dòng)物科學(xué)與技術(shù)學(xué)院，揚(yáng)州225009)摘要胚胎干細(xì)胞(Embryonicstemcells，ESC)是一種包含個(gè)體全部遺傳信息且能夠自我更新的發(fā)育全能性細(xì)胞，可以從內(nèi)細(xì)胞團(tuán)或原始生殖細(xì)胞分離獲得。近幾年關(guān)于雞胚胎干細(xì)胞建系的研究越來(lái)越多，其培養(yǎng)的難點(diǎn)就是在使雞胚胎干細(xì)胞無(wú)限增殖的同時(shí)

2、維持其未分化狀態(tài)。端粒是真核細(xì)胞染色體的末端物質(zhì)，細(xì)胞每分裂一次，端粒便隨之縮短50100bp，當(dāng)其縮短到某一特定值時(shí)，細(xì)胞將失去增殖力，進(jìn)入老化、凋亡狀態(tài)。hTERT基因?qū)Χ肆Ｃ讣せ罹哂写龠M(jìn)作用，將外源的hTERT基因?qū)爰?xì)胞，能夠使細(xì)胞增殖時(shí)端粒縮短的速度減慢或停止。本研究成功構(gòu)建真核表達(dá)載體pEGFPhTERT，并轉(zhuǎn)染雞胚胎干細(xì)胞(Chickenembryonicstemcells，cESCs)，使得細(xì)胞內(nèi)部hTERT基因高表達(dá)，

3、再通過(guò)改善外部培養(yǎng)條件，從而使雞胚胎干細(xì)胞在體外能夠進(jìn)行長(zhǎng)期穩(wěn)定的傳代培養(yǎng)，為后續(xù)雞胚胎干細(xì)胞的建系提供了理論依據(jù)和實(shí)踐基礎(chǔ)。主要研究?jī)?nèi)容如下：1pEGFPhTERT真核表達(dá)載體的構(gòu)建及其亞細(xì)胞定位研究。提取質(zhì)粒PCIneohTERT，用EcoRI和SalI雙酶切后回收到的35kb片段即為目的基因hTERT，然后利用T4連接酶與熒光真核表達(dá)載體pEGFPN1連接構(gòu)建重組質(zhì)粒pEGFPhTERT。采用LipofectaminTM轉(zhuǎn)染法進(jìn)行

4、轉(zhuǎn)染DF1細(xì)胞，以5105個(gè)／mL接種到24孔板內(nèi)，轉(zhuǎn)染24h后于熒光倒置顯微鏡下觀察。試驗(yàn)組：轉(zhuǎn)染重組質(zhì)粒pEGFPhTERT；陰性對(duì)照組：轉(zhuǎn)染空載體質(zhì)粒pEGFPN1；空白對(duì)照組：未轉(zhuǎn)染質(zhì)粒。結(jié)果顯示試驗(yàn)組與陰性對(duì)照組均有綠色熒光表達(dá)，表明pEGFPhTERT重組質(zhì)粒成功轉(zhuǎn)入細(xì)胞，空白組未見(jiàn)綠色熒光。2VC(VitaminC，vc)對(duì)雞胚胎干細(xì)胞增殖的促進(jìn)作用。將雞胚胎干細(xì)胞按lxl03個(gè)／孔的密度接種到96孔板，然后分別將VC以0

5、Ⅲnol／L、10Ianol／L、100I，tmol／L以及1000lamol／L的濃度加入到細(xì)胞培養(yǎng)孔中，最后加入CCK08后用酶標(biāo)儀測(cè)定每孔細(xì)胞的OD值來(lái)檢測(cè)細(xì)胞的增殖，OD值與細(xì)胞的數(shù)目成正比，結(jié)果表明添加10I_tmol／LVC的細(xì)胞OD值最大，即細(xì)胞數(shù)目最多，為促進(jìn)雞胚胎干細(xì)胞分裂增殖的最適濃度。黃曉梅pEGFPhTERT轉(zhuǎn)染雞胚胎干細(xì)胞建系的研究IIlResearchonestablishingchickenembryoni

6、cstemcelllinebypEGFPhTERTtransfectionMSCandidate：XiaomeiHuangAdvisor：ProfBichunLiMajor：SpecialEconomicAnimalBreeding(CollegeofAnimalScienceandTechnologyYang出ouUniversityYangzhou225009)AbstractEmbryonicstemcellisakindofun

7、differentiatedpluripotentstemcell，whichwasderivedfromICMorprimordialgermcellandcontainedallthegeneticinformationThereweremoreandmorestudiesonchickenembryonicstemcelllinesbuildinginrecentyears，anddifficultyinmakingitscult

8、ivationishowtomaintaintheirundifferentiatedstatewhilechickenembryonicstemcellsproliferateindefinitelyTelomereisasectionofDNAatthebottomofeverychromosomeineukaryoticcellOnceeverycelldivision，telomereisshorten50100bpWhenit

9、isreducedtoacertainvalue，theproliferationofthecellswillstopproliferating，thenenterintoagingandapoptosisTelomeraseactivationcouldbepromotedbyhTERTgene，SOwhentheexogenoushTERTgeneWastransfecetedintocells，telomereshortening

10、timewillslowdownorstopwhilecellsproliferateTheeukaryoticexpressionvectorpEGFPhTERTwasconstructedsuccessfullyinthisstudyandtransfectedintochickenembryonicstemcells，whichgiverisetohTERTgenehighexpressionwithincellsMeanwhil

11、ethroughtheimprovementoftheexternalcultureconditions，thechickenembryonicstemcellsarecapableofproliferatingforlongtermstabilitywhichcouldprovideatheoreticalbasisandpracticalbasisforthefollowupstudyofchickenembryosstemcell

12、linesbuildingThemaincontentsaleasfollows：1ConstructionofeukaryoticexpressionvectorpEGFPhTERTandstudyonsubcellularlocalizationofitsexpressionproductinvitroPCIneohTERTWasderivedbyplasmidDNAextractionandafterthatthehTERTgen

13、eWasobtainedbyrestrictionenzymedigestionwithEcoRIandSalIAtlastpEGFP—hTERTwasbuiltsuccessfullythroughconnectedtothefluorescenteukaryoticexpressionvectorpEGFPN1byT4ligaseThenitWastransfectedintoDF1cellswiththedensityofthe5