17526.穩(wěn)定表達(dá)人gmcsf、il4的ea.hy926細(xì)胞株建立

1、始性T細(xì)胞(rcrcell)的專職抗原提呈細(xì)胞(Antigenpresentingcell，Arc)，能攝取各類抗原，體內(nèi)、外均能激發(fā)T細(xì)胞增殖，誘導(dǎo)特異性細(xì)胞殺傷性T淋巴細(xì)胞(eytotoxicTlymphocytes，CTU生成，產(chǎn)生抗腫瘤效應(yīng)，在免疫應(yīng)答中處于中心地位。DC獨(dú)特的免疫調(diào)節(jié)作用使之成為免疫學(xué)研究的熱點(diǎn)，并在細(xì)胞治療領(lǐng)域具有廣闊的應(yīng)用前景。臨床和研究中使用的DC通常為人外周血單核細(xì)胞(p耐pheralbloodmono

2、uclearcdl，PBMC)在多種細(xì)胞因子作用下體外誘導(dǎo)一周得到。目前已有多個(gè)實(shí)驗(yàn)室正致力于發(fā)展一種體外培養(yǎng)系統(tǒng)，在體外模擬體內(nèi)微環(huán)境來誘導(dǎo)單核細(xì)胞向DC分化，以重建體內(nèi)單核細(xì)胞向DC分化時(shí)細(xì)胞間相互作用和信號(hào)通路，并且取得了很好的結(jié)果。本論文成功構(gòu)建了兩種包含GMCSF和IL4的慢病毒表達(dá)質(zhì)粒；包裝獲得的重組慢病毒可以高效的感染內(nèi)皮細(xì)胞系EAhy926細(xì)胞，感染后的重組細(xì)胞經(jīng)RTPCR、Westernblot及ELISA等多種方法進(jìn)

3、行蛋白翻譯表達(dá)的鑒定，經(jīng)鑒定感染后細(xì)胞分別可以高效表達(dá)細(xì)胞因子GMCSF和IL4，感染后細(xì)胞的生長狀態(tài)均良好，能夠在體外進(jìn)行長期傳代培養(yǎng)，可以用于DC誘導(dǎo)實(shí)驗(yàn)；絲裂霉素C(MMC)可以抑制細(xì)胞增殖，但不改變細(xì)胞分泌蛋白的性質(zhì)，是體外常用的制備飼養(yǎng)層的方法之一，我們通過試驗(yàn)最終確定了MMC的作用時(shí)間和濃度，以便以后將重組的EAhy926細(xì)胞制備成飼養(yǎng)層細(xì)胞來促使人外周血單核細(xì)胞分化成為DCs。本論文共分兩部分，具體如下：一、建立兩種分別表

4、達(dá)GMCSF和IL_4基因的重組EAhy926內(nèi)皮細(xì)胞系利用PCR方法擴(kuò)增出目的GM—CSF基因和IL4基因。將目的基因分別構(gòu)建入慢病毒表達(dá)載體pBPLV，得到兩種重組的慢病毒表達(dá)載體。該兩種重組慢病毒分別經(jīng)雙酶切和測序鑒定顯示重組質(zhì)粒構(gòu)建成功。重組慢病毒載體和慢病毒包裝質(zhì)粒、包膜質(zhì)粒共轉(zhuǎn)染293T細(xì)胞，轉(zhuǎn)染24h后經(jīng)熒光顯微鏡觀察轉(zhuǎn)染效率達(dá)到90％以上；收集細(xì)胞培養(yǎng)上清然后高速離心濃縮病毒原液，濃縮后的病毒可以有效的感染內(nèi)皮細(xì)胞系EA

5、hy926細(xì)胞，感染后的細(xì)胞經(jīng)過擴(kuò)大培養(yǎng)后感染效率依然可達(dá)到90％以上，說明目的基因可以在感染細(xì)胞內(nèi)穩(wěn)定的傳代，重組EAhy926內(nèi)皮細(xì)胞系構(gòu)建成功。二、重組EAhy926內(nèi)皮細(xì)胞系細(xì)胞性質(zhì)的確定對感染后細(xì)胞進(jìn)行生長曲線的測定，結(jié)果顯示感染后細(xì)胞的生長趨勢沒有受r，——————一一AbstractDendriticcells(DCs)arethemostpotentantigenpresentingcellswithauniqueabi

6、litytostimulatenaiveTcellsandinitiateprimaryimmuneresponsewhichphagocytesmanykindsofantigen，andexpresseslotsofMHC，eostimulatorymoleculesadhesionmoleculeandstimulatescytotoxiciticTcellstokilltumorcells，itisinacentralposit

7、ionintheimmuneresponsesystemDC’SuniqueimmunoregulationmakesitbecomingthehotstudyfieldinimmunologyandithasawildapplicationprospectinthefieldofthetreatmentofcellsDCsforresearchandclinicalapplicationsareusuallyobtainedbycul

8、turingPBMCderivedmonocytesinacocktailofcytokinesforabout1weekHoweverseverallaboratorieshaveworkedtodevelopinvitrosystemswhichmorecloselYrecapitulatethecellinteractionsandsignalingpathwaysthattriggermonocyteStoDCdifferent

9、iationinvivoInthispaperwehavesucessfullyconstructedtwecombinantlentiviralexpressionplasmidswhichseperatelycarryingtheGMCSFgeneandtheIL4geneandthenestablishedtwoEAhy926celllineswhichCallrespectivelyexpreSSGM—CSFandIL4stab

10、lybylentiviralsystemTheabilitytoexpressGM—CSFandIL4inthetwoinfectedcelllineswasindentifiedbyRTPCR、WesternblotandELISA，andtheresultstoldUSthatthetwoinfectedEAhy926eel1linesCallrespectivelyexpressGM—CSFandIL4inahighlevelan

11、dCanbeculturedinalongtermThetwocelllineswecontructedwillbeofgreatsignificancetothecultureofDCWealsodeterminedtheactiontimeandconcentrationofMMCwhichiSusedtotheestablishmentoffeedercellsInthelast，theDCswereobtainedbyPBMCe

12、oculturingwitllfeedercellsThisthesisiscomposedoftwoparts嬲followed：1TheestablishmentoftwecombinantEAhy926cell1inesThegeneGMCSFandthegeneIL4WasobtainedbyPCRandtheninseftintolentiviralexpressionplasmidpBPLVTherecombinantlen

13、tiviralplasmidWasidentifiedbyDoubleenzymedigestionandthentransfectedinto293TeellswithlentiviralpackagingplasmidsTheefficiencyoftransfectionWasabove90％Thencollectedthecellculturesupematant，andthenhighspeedcentrifugationof