肝癌細(xì)胞的原代分離及肝癌干細(xì)胞的初步篩選的開題報(bào)告

肝癌細(xì)胞的原代分離及肝癌干細(xì)胞的初步篩選的開題報(bào)告摘要：肝癌是一種具有高度侵襲性和復(fù)發(fā)性的惡性腫瘤，其發(fā)病率和死亡率較高。肝癌干細(xì)胞是一種表現(xiàn)出強(qiáng)大的自我更新、增殖和分化的細(xì)胞群體，是肝癌復(fù)發(fā)和耐藥的主要原因。本研究旨在通過原代肝癌細(xì)胞的分離和肝癌干細(xì)胞的初步篩選，為深入研究肝癌干細(xì)胞的生物學(xué)機(jī)制和尋找肝癌新的治療靶點(diǎn)提供實(shí)驗(yàn)基礎(chǔ)。研究內(nèi)容：1.原代肝癌細(xì)胞的分離根據(jù)臨床肝癌組織樣本的要求，利用外科手術(shù)切除癌癥肝組織，將組織樣本放入磷酸鹽緩沖液中，利用組織塊分離酶和較強(qiáng)的機(jī)械分離強(qiáng)制的處理方法將肝癌組織分離為小碎片。再利用胰酶和膽汁酸酶等多重酶處理，將得到的細(xì)胞用15%FBS/PBS洗滌，免疫熒光染色對肝癌細(xì)胞和其他非肝癌細(xì)胞進(jìn)行鑒定，篩選出符合要求的原代肝癌細(xì)胞。2.肝癌干細(xì)胞的初步篩選利用CD133抗體分選技術(shù)，將原代肝癌細(xì)胞中的CD133+細(xì)胞和CD133-細(xì)胞分別分離出來。并對CD133+細(xì)胞和CD133-細(xì)胞進(jìn)行基因表達(dá)譜分析。并通過體外的腫瘤球形體形成性和裸鼠移植實(shí)驗(yàn)證明CD133+細(xì)胞具有肝癌干細(xì)胞的特性。預(yù)期結(jié)果：1.成功分離出符合要求的肝癌細(xì)胞。2.具有肝癌干細(xì)胞特性的CD133+細(xì)胞被篩選出來。3.對于CD133+細(xì)胞的分子機(jī)制和腫瘤生物學(xué)特性的研究提供了實(shí)驗(yàn)基礎(chǔ)。關(guān)鍵詞：肝癌；原代細(xì)胞；肝癌干細(xì)胞；CD133+細(xì)胞Abstract:Livercancerisamalignanttumorwithhighinvasivenessandrecurrencerate,andtheincidenceandmortalityratearehigh.Livercancerstemcellsareagroupofcellsthatexhibitstrongself-renewal,proliferation,anddifferentiation,andarethemaincauseoflivercancerrecurrenceanddrugresistance.Thisstudyaimstoprovideexperimentalbasisforin-depthstudyofthebiologicalmechanismsoflivercancerstemcellsandfindingnewtherapeutictargetsforlivercancerthroughprimaryisolationoflivercancercellsandpreliminaryscreeningoflivercancerstemcells.Researchcontent:1.IsolationofprimarylivercancercellsAccordingtotherequirementsofclinicallivercancertissuesamples,cancerouslivertissuewasremovedbysurgicalresection,andthetissuesamplewasplacedinaphosphate-bufferedsolution.Thelivercancertissuewasseparatedintosmallfragmentsusingatissueblockseparationenzymeandstrongmechanicalseparation.Theobtainedcellsweretreatedwithmultipleenzymessuchastrypsinandbileacidenzymes,washedwith15%FBS/PBS,andimmunofluorescencestainingtoidentifylivercancercellsandothernon-livercancercells,andscreenoutqualifiedprimarylivercancercells.2.PreliminaryscreeningoflivercancerstemcellsCD133antibodysortingtechnologywasusedtoseparateCD133+cellsandCD133-cellsfromprimarylivercancercells.GeneexpressionprofileanalysiswasperformedonCD133+cellsandCD133-cells.InvitrotumorspheroidformationandnudemousetransplantationexperimentswereperformedtodemonstratethatCD133+cellshavethecharacteristicsoflivercancerstemcells.Expectedresults:1.Successfullyisolatedqualifiedlivercancercells.2.CD133+cellswithlivercancerstemcellcharacteristicswerescreenedout.3.Provideexperimentalbasisforthestudyofthemolecularmechanis