【作者】 戴玮；

【导师】 钱海鑫； 刘建夏；

【作者基本信息】 苏州大学 ， 普外科， 2007， 硕士

【摘要】 第一部分:唑来膦酸刺激人外周血γδT细胞增殖目的:探讨唑来膦酸刺激人外周血γδT细胞的增殖作用方法:抽取10例健康捐献者外周血10ml,以密度梯度离心法分离外周血单个核细胞。选用第三代双膦酸盐类药物唑来膦酸作为刺激因子,以不同浓度的唑来膦酸联合IL-2刺激外周血γδT细胞增殖。细胞培养12-14天后,收集细胞,以流式细胞仪检测相关表型, 3H-TdR掺入法测定细胞增殖程度。结果:培养12-14天后,外周血γδT细胞增殖明显,与对照组相比较,实验组γδT表型比例升高显著(对照组9.7%、实验组80.8%、P<0.01)。CD3升高显著(对照组71.2%、实验组96.2%、P<0.01)。3H-TdR掺入法测定显示实验组细胞增殖显著:(空白对照组CMP值:111.1、实验组CMP值:17488.6、P<0.01)。在所有唑来膦酸浓度中,以1μΜ浓度时,γδT细胞增殖最为明显。结论:用唑来膦酸能促进人外周血γδT细胞的增殖,得到大量高纯度的γδT细胞。第二部分:扩增的γδT细胞对人肝癌细胞的杀伤效应目的:探讨以唑来膦酸刺激增殖的γδT细胞对人肝癌细胞的杀伤作用方法:同实验一法获取健康人外周血γδT细胞,以γδT细胞作为效应细胞,分别以人肝癌细胞株SMCC-7721、MHCC-97、HepG2作为靶细胞。按照效靶比20:1、10:1共同培养48小时后终止实验,以SRB法测定效应细胞对靶细胞的杀伤效能。结果:γδT细胞对三种肿瘤细胞均表现出强大的杀伤作用,效靶比20:1时,γδT细胞对肝癌细胞杀伤率最高达98.7%,效靶比10:1时,γδT细胞对肝癌细胞杀伤率最高达92.6%。同一效靶比时,γδT细胞对不同细胞株杀伤作用无显著差异(P>0.05),不同效靶比时,γδT细胞对同一细胞株杀伤作用有显著差异(P<0.05)。结论:以唑来膦酸刺激扩增的γδT细胞在体外对肝癌细胞有显著的杀伤作用更多还原

【Abstract】 PartⅠγδT lymphocyte multiplication stimulated by zoledronic acidObjective To study the effect ofγδT lymphocyte multiplication stimulated by zoledronic acid .Methods: Take 10ml peripheral blood of healthy adults, obtain peripheral blood mononuclear cell by centrifugation on a Ficoll-Hypaque density gradient. Take zoledronic acid the Third-generation Biphosphonate as the stimulating factor.Exert different concentration zoledronic acid combined with IL-2 to stimulate peripheral bloodγδT cells proliferation. Collecting cells after 12-14 days, analyze correlated phaenotypes by flow cytometry. The quantity of cells was determined by method of 3H-TdR incorporation.Results: After cultured 12-14 days ,peripheral bloodγδT cells obviously multiplied. Confer with the control group, proportion ofγδT phaenotype in experimental gtoup obviously heightened(control group 9.7%、experimental gtoup 80.8%、P<0.01). proportion of CD3 phaenotype in experimental gtoup also obviously heightened(control group 71.2%、experimental gtoup 96.2%、P<0.01)。Cells in experimental gtoup obviously multiplied determined by method of 3H-TdR incorporation(CMP of control group 9961.5、CMP of experimental gtoup 17488.6、P<0.01).In all of the zoledronic acid concentration,γδT cells multiplied most obviously in 1μΜzoledronic acid.γδT cells.Conclusions: Peripheral bloodγδT lymphocyte can be stimulated and multiplied by zoledronic acid ,and can obtain quantity of highly purifiedγδTcells. PartⅡThe cytotoxic activity of the multipliedγδT lymphocyte to hepatoma carcinoma cell.Objective To investigate the cytotoxic activity ofγδT lymphocyte stimulated and multiplied by zoledronic acid to hepatoma carcinoma cell.Methods: Obtain peripheral bloodγδT cells of healthy adults by the method of experiment one .TakeγδT cell as effector cell,and hepatoma carcinoma cell(SMCC-7721、MHCC-97、Hep - G2) as target cell. Mixed culture such cells according to the efffector/target cell ratio(E/T 10:1,20:1).Stop experiment after 48 hours. Determine the cytotoxic activity of effector cells to target cells by method of SRB.Results:γδT cell display powerful cytotoxic activity to the three tumour cells. The percentage of cytotoxin reached 98.7% when E/T was 20:1 and reached 92.6% when E/T was 10:1. The cytotoxic activity ofγδT cell to different tumour cells has no significant deviation in same E/T(P>0.05). The cytotoxic activity ofγδT cell to same tumour cells has significant deviation in different E/T(P<0.05)。Conclusions: TheγδT lymphocyte stimulated and multiplied by zoledronic acid has significant cytotoxic activity to hepatoma carcinoma cell in vitro.更多还原