發(fā)布時間：2019-03-08 10:19

【摘要】：目的:初步探討癌-睪丸抗原OY-TES-1致敏樹突狀細胞誘導的細胞毒性T淋巴細胞對肝癌細胞的殺傷作用,為肝癌的免疫治療提供實驗依據(jù)。 方法:(1)靶細胞篩選:通過流式細胞儀篩選HLA-A2+和HLA-A2-的肝癌細胞株;運用RT-PCR法和免疫組織化學篩選OY-TES-1表達陽性的肝癌細胞株。(2)運用HLA-A2陽性健康人外周血,密度梯度法分離單個核細胞,經(jīng)rhGM-CSF、rhIL-4和rhTNF-α聯(lián)合誘導培養(yǎng)為樹突狀細胞(dendritic cell, DC),通過光學顯微鏡和電子顯微鏡及流式細胞儀進行表型鑒定;分別用OY-TES-1融合蛋白(OY-MBP)、麥芽糖結合蛋白(MBP)和增強型綠色熒光蛋白(EGFP)致敏DC并激活同體T淋巴細胞,使之增殖、分化為細胞毒性T淋巴細胞(CTL);采用CCK-8法檢測蛋白致敏DC后刺激同種混合淋巴細胞增殖(MLR)能力;ELISA檢測致敏DC與T淋巴細胞共同培養(yǎng)后上清液IFN-γ的含量,并用乳酸脫氫酶(LDH)釋放法檢測CTL對肝癌細胞的細胞毒作用。 結果: (1)靶細胞篩選:流式細胞儀檢測結果顯示肝癌細胞株HepG2為HLA-A2+, Bel-7404為HLA-A2-;RT-PCR和免疫細胞化學法結果顯示Bel-7404和HepG2均表達OY-TES-1。 (2)DC的鑒定和CTL殺傷效應:PBMC經(jīng)細胞因子聯(lián)合誘導培養(yǎng)1周后,出現(xiàn)典型的DC形態(tài),并高表達HLA-DR、CD86、CD83和CD80;經(jīng)不同蛋白致敏的DC均能促進T淋巴細胞的增殖活化,其中OY-MBP致敏的DC促進T淋巴細胞增殖的能力明顯強于其他各組(P0.05),IFN-γ分泌量也明顯高于其他各組(P0.05);經(jīng)OY-MBP致敏DC誘導的CTL對靶細胞HepG2有殺傷作用,其殺傷率明顯高與其他各組(P0.05);用HLA-ABC抗體封閉靶細胞后,CTL對其殺傷作用下降。 結論:在體外用OY-TES-1融合蛋白致敏的DC可有效激發(fā)CTL,產(chǎn)生較強的抗肝癌細胞毒活性效應,提示OY-TES-1可作為肝癌免疫治療的靶點。
[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- 偽 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- 緯 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- 緯 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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

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