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1、DC-CIK細(xì)胞對(duì)人肝癌HEPG2細(xì)胞周期、凋亡的影響項(xiàng)穎秦波田小利(重慶醫(yī)科大學(xué)附屬第一醫(yī)院感染科重慶400030)摘要目的探討細(xì)胞因子從肝癌患者外周血貼壁單個(gè)核細(xì)胞(peripheralbloodmononuclearcells,PBMCs)誘導(dǎo)的樹突狀細(xì)胞(dendriticcells,DCs)與殺傷(cytokineinducedkiller,CIK)細(xì)胞對(duì)人肝癌HEPG2細(xì)胞系周期、凋亡的影響。方法用肝癌患者肝癌組織裂解物(腫瘤抗原)致敏經(jīng)GM-CSF、TNF-a及IL-4等誘導(dǎo)該患者PBMC產(chǎn)生的DCs。用IFN-γ、
2、IL-2和人CD3單克隆抗體(humanCD3monoclonalantibody,hCD3mAb)等誘導(dǎo)該P(yáng)BMC的懸浮細(xì)胞產(chǎn)生CIK細(xì)胞。用Tanswell培養(yǎng)小室將DC-CIK細(xì)胞與HEPG2細(xì)胞在同一培養(yǎng)體系內(nèi)經(jīng)該小室膜分隔培養(yǎng)24、48h。流式細(xì)胞儀檢測(cè)該HEPG2細(xì)胞的周期;AnnexinV-PI雙染法檢測(cè)其凋亡。RT-PCR、Westernblot分別檢測(cè)凋亡相關(guān)基因Bax及其編碼蛋白的表達(dá)。結(jié)果DC-CIK細(xì)胞可明顯影響人HEPG2細(xì)胞生長(zhǎng)周期,兩者共培養(yǎng),可將HEPG2細(xì)胞系阻滯于G1期。DC-CIK細(xì)胞能顯著誘
3、導(dǎo)HEPG2細(xì)胞的凋亡(與對(duì)照相比,其凋亡誘導(dǎo)率差異顯著,P<0.01)?;蚺c蛋白水平檢測(cè)表明,DC-CIK細(xì)胞可致HEPG2細(xì)胞系的凋亡相關(guān)基因Bax表達(dá)明顯上調(diào)。結(jié)論肝癌患者DC-CIK細(xì)胞可明顯抑制肝癌HEPG2細(xì)胞系的生長(zhǎng),顯著誘導(dǎo)該細(xì)胞凋亡,且該凋亡誘導(dǎo)具“時(shí)間-依賴”與“細(xì)胞數(shù)量-依賴”特性。關(guān)鍵詞DC-CIK細(xì)胞肝癌HEPG2細(xì)胞細(xì)胞周期細(xì)胞凋亡TheeffectofCIKcellscombinedwithdendriticcellsofpatientwithlivercanceronthecycleandapop
4、tosisofhumanHEPG2celllineXIANGYingQINBoTIANXiaoli(DepartmentofInfectiousDiseases,TheFirstAffiliatedHospitalofChongqingMedicalUniversity,Chongqing400030,China)Abstract:ObjectiveToexploratetheeffectofcytokineinducedkiller(CIK)cellsanddendriticcells(DCs)fromperipheralblo
5、odmononuclearcell(PBMC)ofpatientwithlivercanceronthecycleandapoptosisofhumanHEPG2celllineoflivercancer.MethodsTheadherentPBMCswereinducedbyGM-CSF、TNF-aandIL-4inordertoproduceDCs,which9基金項(xiàng)目:重慶市衛(wèi)生局醫(yī)學(xué)科學(xué)技術(shù)研究項(xiàng)目(2010-2-301)作者簡(jiǎn)介:項(xiàng)穎,男,重慶市人,主任醫(yī)師。主要從事腫瘤發(fā)病機(jī)制及其治療方面的研究。E-mail:xian
6、g519@hotmail.com通信作者:秦波,電話:15923261188,E-mail:cqqinbo@126.comweresensitizedwithantigenofautologoustissueoflivercancer.Meanwhile,CIKcellswereobtainedfromthesuspendedPBMCsinducedbyIFN-γ、IL-2andhumanCD3monoclonalantibody(hCD3mAb).TheDCs,CIKcellsandHEPG2celllinewerecultur
7、edinasamecircumstancewithaculturingcabinnamedTranswell,buttheDCs-CIKcellswereculturedatonesideofthemembraneoftheTranswell,HEPG2celllineatanothersideofthesamemembranefor24hand48hrespectively.SubsequentlythecycleofhumanHEPG2celllineculturedwithDCs-CIKcellsof24hand48hwer
8、etestedthroughflowcytometry(fcm)respectively.TheAnnexinV-PIstainingwasemployedtoanalysistheapoptosisofthesamecellline,andthe