癌—睾丸抗原OY-TES-1致敏树突状细胞的体外抗肝癌研究

发布时间：2019-03-08 10:19

【摘要】：目的:初步探讨癌-睾丸抗原OY-TES-1致敏树突状细胞诱导的细胞毒性T淋巴细胞对肝癌细胞的杀伤作用,为肝癌的免疫治疗提供实验依据。方法:(1)靶细胞筛选:通过流式细胞仪筛选HLA-A2+和HLA-A2-的肝癌细胞株;运用RT-PCR法和免疫组织化学筛选OY-TES-1表达阳性的肝癌细胞株。(2)运用HLA-A2阳性健康人外周血,密度梯度法分离单个核细胞,经rhGM-CSF、rhIL-4和rhTNF-α联合诱导培养为树突状细胞(dendritic cell, DC),通过光学显微镜和电子显微镜及流式细胞仪进行表型鉴定;分别用OY-TES-1融合蛋白(OY-MBP)、麦芽糖结合蛋白(MBP)和增强型绿色荧光蛋白(EGFP)致敏DC并激活同体T淋巴细胞,使之增殖、分化为细胞毒性T淋巴细胞(CTL);采用CCK-8法检测蛋白致敏DC后刺激同种混合淋巴细胞增殖(MLR)能力;ELISA检测致敏DC与T淋巴细胞共同培养后上清液IFN-γ的含量,并用乳酸脱氢酶(LDH)释放法检测CTL对肝癌细胞的细胞毒作用。结果: (1)靶细胞筛选:流式细胞仪检测结果显示肝癌细胞株HepG2为HLA-A2+, Bel-7404为HLA-A2-;RT-PCR和免疫细胞化学法结果显示Bel-7404和HepG2均表达OY-TES-1。 (2)DC的鉴定和CTL杀伤效应:PBMC经细胞因子联合诱导培养1周后,出现典型的DC形态,并高表达HLA-DR、CD86、CD83和CD80;经不同蛋白致敏的DC均能促进T淋巴细胞的增殖活化,其中OY-MBP致敏的DC促进T淋巴细胞增殖的能力明显强于其他各组(P0.05),IFN-γ分泌量也明显高于其他各组(P0.05);经OY-MBP致敏DC诱导的CTL对靶细胞HepG2有杀伤作用,其杀伤率明显高与其他各组(P0.05);用HLA-ABC抗体封闭靶细胞后,CTL对其杀伤作用下降。结论:在体外用OY-TES-1融合蛋白致敏的DC可有效激发CTL,产生较强的抗肝癌细胞毒活性效应,提示OY-TES-1可作为肝癌免疫治疗的靶点。
[Abstract]:Aim: to investigate the cytotoxicity of cytotoxic T lymphocytes induced by dendritic cells sensitized with cancer-testis antigen (OY-TES-1) on hepatocellular carcinoma (HCC) cells in order to provide experimental basis for immunotherapy of HCC. Methods: (1) screening of target cells: the hepatoma cell lines of HLA-A2 and HLA-A2- were screened by flow cytometry. OY-TES-1 positive hepatoma cell lines were screened by RT-PCR method and immunohistochemistry. (2) HLA-A2 positive healthy human peripheral blood mononuclear cells were isolated by density gradient method, then rhGM-CSF, was used to isolate the mononuclear cells. Dendritic cells (dendritic cell, DC),) were induced by rhIL-4 and rhTNF- 伪 and identified by light microscope, electron microscope and flow cytometry. OY-TES-1 fusion protein (OY-MBP), maltose binding protein (MBP) and enhanced green fluorescent protein (EGFP) were used to sensitize DC and activate homosome T lymphocytes to proliferate and differentiate into cytotoxic T lymphocytes (CTL);). CCK-8 assay was used to detect the ability of (MLR) to stimulate the proliferation of allogeneic mixed lymphocytes after protein sensitized DC. ELISA was used to detect the content of IFN- 纬 in the supernatant of sensitized DC and T lymphocytes, and the cytotoxic effect of CTL on hepatoma cells was detected by lactate dehydrogenase (LDH) release assay. Results: (1) screening of target cells: the results of flow cytometry showed that HepG2 was HLA-A2, Bel-7404 was HLA-A2-;RT-PCR, and immunocytochemistry showed that both Bel-7404 and HepG2 expressed OY-TES-1.. (2) Identification of DC and killing effect of CTL: after 1 week of co-culture with cytokines, PBMC showed typical DC morphology and high expression of HLA-DR,CD86,CD83 and CD80;. DC sensitized by different proteins could promote the proliferation and activation of T lymphocytes, and the ability of DC sensitized by OY-MBP to promote the proliferation of T lymphocytes was significantly stronger than that of other groups (P0.05). The secretion of IFN- 纬 was also significantly higher than that of other groups (P0.05). CTL induced by OY-MBP sensitized DC had a killing effect on target cell HepG2, and its killing rate was significantly higher than that of other groups (P0.05). After blocking target cells with HLA-ABC antibody, the killing effect of CTL on target cells was decreased. Conclusion: DC sensitized with OY-TES-1 fusion protein in vitro can effectively stimulate CTL, to produce strong cytotoxicity against HCC, suggesting that OY-TES-1 can be used as a target for immunotherapy of HCC.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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