【作者】 吴迪；

【导师】 姜又红；

【作者基本信息】 中国医科大学 ， 肿瘤学， 2009， 硕士

【摘要】 肿瘤全细胞抗原负载的DC与CIK共培养对肾癌细胞杀伤活性的研究目的采用CCK-8检测自体/异体抗原负载的DC与CIK共培养以及无抗原负载的DC与CIK共培养、单独培养CIK对自体原代培养肾癌细胞的杀伤活性,探讨肾癌免疫治疗体外不同免疫细胞杀瘤活性差异,为临床上肿瘤抗原不明确的肾癌免疫治疗提供一种新的研究思路。方法一、肾癌细胞培养及肿瘤胞抗原制备采用组织块贴壁法、机械分散法及差速贴壁分离法、混合酶消化法及差速贴壁分离法分离肾癌细胞,比较这几种方法培养肾癌细胞的优劣并优化,体外培养出实验所需短期原代肾癌细胞。超声破碎法获取原代培养肾癌细胞自体肿瘤细胞全抗原,反复冻融法获取异体肾癌细胞系769-P肿瘤细胞全抗原。二、DC、CIK的体外诱导扩增、抗原负载及表型检测分离自体及健康供外周血单个核细胞体外诱导制备DC、CIK细胞。于第8d天收集DC、CIK细胞,进行共培养,分为无抗原DC组,自体肿瘤全细胞抗原递呈DC组,异体肿瘤细胞裂解物抗原递呈DC组,以10:1的比例与培养至第8d的CIK混合培养,三天后进行靶细胞杀伤实验。流式细胞仪检测DC表面成熟标志CD83、CD80、CD86、HLA-DR的表达,CIK细胞检测CD3、CD4、CD8、CD56的表达。三、DC-CIK诱导的特异性杀伤作用检测应用CCK-8法检测CIK,CIK-DC,CIK-DC-自体A,CIK-DC-异体A分别对肾癌细胞系769-P及原代培养肾癌细胞的杀瘤活性。结果一、肾癌细胞培养三种培养方法比较,组织块贴壁法分离的细胞损伤最小,且细胞状态好,但细胞种类多,纯化困难,生长时间长;机械分离法细胞损伤最重,细胞种类多,但生长时间比组织块贴壁法短;混合酶消化法所得的细胞损伤程度次之,分步贴壁后可获得较纯的肿瘤细胞,培养生长时间最短,后续时间均采用此法提取培养肾癌细胞。所有实验均采用生长状态最佳的第三代细胞;100mg肾癌组织经超声破碎后可获取30～50mg全细胞抗原;1瓶长满的肾癌细胞(1～3×10~7)提取出的全抗原约为30～60mg。二、DC、CIK的体外培养及抗原检测外周血单个核细胞,经GM-CSF、IL-4诱导可获得5%～10%的成熟DC。加入肿瘤裂解物抗原及诱导成熟因子后,DC表面成熟标志CD83、CD80、CD86、HLA-DR的表达明显增加。与DC共培养CIK表面抗原CD3~+、CD3~+CD4~+、CD3~+CD56~+、CD3~+CD8~+均比单独培养CIK高。三、DC-CIK诱导的特异性杀伤作用结果显示,自体全抗原刺激的CIK-DC-自体A组与与异体全抗原刺激的CIK-DC-异体A组杀瘤活性最大,两者之间无差异,未经抗原刺激的CIK-DC组次之,单纯CIK组杀瘤活性最低;四组效应细胞组间杀伤率相关性分析显示,除CIK-DC-自体A组与CIK-DC-异体A组之间差异无统计学意义外,各组间差异均显著,各组之间杀伤率比较具有正相关关系;健康组与患者组效应细胞杀伤活性比较,肾癌患者外周血杀瘤活性比健康供者杀瘤活性低,有统计学意义;原代培养肾癌细胞患者自体效应细胞杀伤实验组与原代培养肾癌细胞患者异体健康效应细胞杀伤实验组比较,肾癌细胞患者自体效应细胞与异体健康效应细胞差别无统计学意义;此外,各实验组间两两比较,差异显著,有统计学意义(P<0.05)。结论1、自体全抗原或异体全抗原刺激的DC以及无处理因素刺激的DC与CIK共培养后,CIK细胞在免疫表型,增殖及杀瘤能力都有明显提高;2、DC细胞经自体全抗原或异体全抗原刺激后,免疫表型及增值能力均提高,与CIK细胞共培养后杀瘤活性比无处理因素刺激的DC-CIK共培养杀瘤活性高;3、CIK细胞与DC细胞共培养后较单纯CIK细胞杀瘤活性高,可为临床生物治疗选择提供有力依据。更多还原

【Abstract】 ObjectiveAdopt CCK-8 kit to detect the kill activity of CIK co-culture with DC loaded by autologous or variant tumor antigen,and single culture CIK for autologous renal carcinoma cell,discuss the differences of immunocells for RCC immunotherapy,and provide a new research method for the patients who can’t have an operation or get autologous tumor antigen.Methods1.Renal cell carcinoma cell primary culture and tumor antigen preparationUtilize the culture methods of explant tissue culture adherence,mechanical dispersion,enzymes digestion to establish short-term renal cell line,compare these methods and optimize.With ultrasonic cell disruptor to obtain autologous renal tumor antigen and renal cell carcinoma cell line 769-P whole-cell antigen.2.DC and CIK culture in vitroGather peripheral blood of patients or volunteers,use Ficoll-Paque Plus density gradient centrifugation to get peripheral blood mononuclear cells(PBMC),then add cytokines to induce DC and CIK.By day 8,harvest DC and CIK to co-culture,there are 3 groups in our experiment,DC without tumor antigen group,DC with autologous tumor antigen group,DC with variant tumor antigen group.We detect CD83,CD80, CD86,HLA-DR of DC,CD3,CD4,CD8,CD56 of CIK by flow cytometry.3.DC and CIK culture specific kill effect detection With CCK-8 kit detect the kill activity of CIK,CIK-DC,CIK-DC-autologous tumor antigen,CIK-DC-variant tumor antigen to the autologous renal cell carcinoma primary culture cell and cell line 769-P.Results1.Renal cell carcinoma cell primary culture and tumor antigenpreparationCompare these three methods,we find that enzymes digestion is the best,all the erperiment take on the third generation of primary culture short-term renal cell line. 100mg renal tumor tissue could extract 30～50mg whole-cell tumor antigen,and 1～3×10~7 RCC cells could extract 60～80mg tumor antigen.2.DC and CIK culture in vitroWith adding GM-CSF,by day 7,We could obtain the number of DC of 5～10%PBMC.After adding tumor cell lysate and cytokine,the mature markers of DC increase,such as CD83,CD80,CD86,HLA-DR.The markers of CIK that co-cultured with DC are higher than that single cultured CIK,such as CD3~+,CD3~+ CD4~+, CD3~+CD56~+,CD3~+CD8~+.3.DC and CIK culture specific kill effect detectionThe reaults show that the activity of CIK-DC-autologous tumor antigen group and CIK-DC-variant tumor antigen group are higher than that of other two groups,but there is no difference between them,CIK-DC group is the second,and the CIK group is the lowest.There is a positive correlation.The kill activity of patients’ peripheral blood is lower than that of health volunteers’,and there is significance.The activity of autologous peripheral blood to autologous tumor is equal to that of variant health volunteer,besides,there is significance between every two experimental groups (P<0.05).Conclusion1.DC induced by autologous or variant tumor antigen co-culture with CIK,can improve CIK in immunophenotype,generation and kill activity. 2.After induced by autologous or variant tumor antigen,DC has improvement in immunophenotype and generation.CIK co-culture with DC induced by tumor antigen has higher kill activity than that of other two groups.3.CIK co-cultured with DC has higher kill activity than that of single cultured CIK,this may provide an evidence for choosing clinical biotherapy.更多还原