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1、摘要論文題目：體外外周血培養(yǎng)樹(shù)突狀細(xì)胞的實(shí)驗(yàn)研究專業(yè)：腫瘤學(xué)研究生：郭輝指導(dǎo)教師：趙勇剛教授摘要目的提取神經(jīng)膠質(zhì)瘤患者外周血分離出單核細(xì)胞，進(jìn)行體外培養(yǎng)、誘導(dǎo)產(chǎn)生樹(shù)突狀細(xì)胞，并探討細(xì)胞因子對(duì)樹(shù)突狀細(xì)胞誘導(dǎo)、分化成熟的影響，為最終臨床應(yīng)用樹(shù)突狀細(xì)胞免疫治療神經(jīng)膠質(zhì)瘤打下基礎(chǔ)。方法臨床收集確診為神經(jīng)膠質(zhì)瘤的患者靜脈血共20份，每份20ml，經(jīng)淋巴細(xì)胞分離液梯度分離出單個(gè)核細(xì)胞（PBMC），貼壁法獲得單核細(xì)胞后，每個(gè)樣本均分為兩份，共兩組，分別加入GM-CSF+IL-4和GM-CSF+IL-4+Flt3-L進(jìn)行誘導(dǎo)，在相同條件下進(jìn)行體外培
2、養(yǎng)，隔日半量換液，補(bǔ)充細(xì)胞因子，共7天，用倒置顯微鏡觀察其形態(tài)，并進(jìn)行流式細(xì)胞儀檢測(cè)其表面標(biāo)志，對(duì)培養(yǎng)結(jié)果在獲得率、純度和細(xì)胞表型三方面進(jìn)行對(duì)比。結(jié)果應(yīng)用GM-CSF+IL-4和GM-CSF+IL-4+Flt3-L兩種培養(yǎng)方法均可誘導(dǎo)產(chǎn)生樹(shù)突狀細(xì)胞,而且在樹(shù)突狀細(xì)胞的獲得率和純度方面無(wú)統(tǒng)計(jì)學(xué)差異，經(jīng)流式細(xì)胞儀檢測(cè)，應(yīng)用GM-CSF+IL-4組所獲得的樹(shù)突狀細(xì)胞符合不成熟樹(shù)突狀細(xì)胞的表型，而GM-CSF+IL-4+Flt3-L組所誘導(dǎo)的樹(shù)突狀細(xì)胞表達(dá)更高比例的成熟標(biāo)志CD83，趨向于成熟的樹(shù)突狀細(xì)胞表型。結(jié)論從膠質(zhì)瘤患者外周血中分離出
3、的單核細(xì)胞，經(jīng)GM-CSF和IL-4刺激后，可以誘導(dǎo)、擴(kuò)增出大量的樹(shù)突狀細(xì)胞，該細(xì)胞具有典型的樹(shù)突狀細(xì)胞的形態(tài)；由GM-CSF和IL-4誘導(dǎo)產(chǎn)生不成熟的樹(shù)突狀細(xì)胞，存在Flt3-L時(shí)可誘導(dǎo)生成成熟的樹(shù)突狀細(xì)胞。關(guān)鍵詞：樹(shù)突狀細(xì)胞；Fms樣酪氨酸激酶3配體（Flt3-L）；體外培養(yǎng)；外周血；膠質(zhì)瘤論文類型：應(yīng)用基礎(chǔ)研究I摘要Subject:TheStudyofInductionOfDendriticCellsFromHumanPeripheralBloodMonocytesInVitroSpecialty:OncologyName:G
4、UOhuiSupervisor:ProfessorZHAOyong-gangABSTRACTObjectiveWeseparatethemonocytes(MNC)fromtheperipheralbloodofpatientswithmalignantglioma.Thenthemonocytes(MNC)areaddeddifferentcombinedcytokinestoinducethegenerationofdendriticCells(DCs)invitro.Theaimofthestudyistoinvestigat
5、etheeffectofcytokinesonthegenerationofDCs.ItwillprovidetheexperimentalfoundationforclinicalgliomaimmunotherapyofDCs.MethodsWegatheredtheperipheralblood20mlfrom20patientswithmalignantglioma,theperipheralbloodmononuclearcells(PBMC)wereseparatedfromthebloodbycentrifugalin
6、densitygradient.Theperipheralbloodmononuclearcells(PBMC)wereculturedinbottles,thentheadhesioncellscollectedfromtheperipheralbloodmononuclearcells(PBMC)fromeachpatientwhichweredividedintotwoparts,thendividedintotwogroups.OnegroupwasaddedcombinedcytokinesGM-CSF+IL-4,theo
7、therwasaddedcombinedcytokinesGM-CSF+IL-4+Flt3-L.Afterculturedforsevendaysinthesamecondition,collectadherentcells,observetheirmorphologybyinvertedmicroscopeanddetectimmunefactorswithflowcytometry.Wecomparedthetwogroupsintermsofpick-uprate、purityandphenotypeofcells.Resul
8、tsThedendriticCells(DCs)canbeinducedfromthemonocytes(MNC)culturedwithcombinedcytokineseitherGM-CSF+IL-4orGM-CSF+IL-4+
