【摘要】：目的:初步探討癌-睪丸抗原OY-TES-1致敏樹(shù)突狀細(xì)胞誘導(dǎo)的細(xì)胞毒性T淋巴細(xì)胞對(duì)肝癌細(xì)胞的殺傷作用,為肝癌的免疫治療提供實(shí)驗(yàn)依據(jù)。 方法:(1)靶細(xì)胞篩選:通過(guò)流式細(xì)胞儀篩選HLA-A2+和HLA-A2-的肝癌細(xì)胞株;運(yùn)用RT-PCR法和免疫組織化學(xué)篩選OY-TES-1表達(dá)陽(yáng)性的肝癌細(xì)胞株。(2)運(yùn)用HLA-A2陽(yáng)性健康人外周血,密度梯度法分離單個(gè)核細(xì)胞,經(jīng)rhGM-CSF、rhIL-4和rhTNF-α聯(lián)合誘導(dǎo)培養(yǎng)為樹(shù)突狀細(xì)胞(dendritic cell, DC),通過(guò)光學(xué)顯微鏡和電子顯微鏡及流式細(xì)胞儀進(jìn)行表型鑒定;分別用OY-TES-1融合蛋白(OY-MBP)、麥芽糖結(jié)合蛋白(MBP)和增強(qiáng)型綠色熒光蛋白(EGFP)致敏DC并激活同體T淋巴細(xì)胞,使之增殖、分化為細(xì)胞毒性T淋巴細(xì)胞(CTL);采用CCK-8法檢測(cè)蛋白致敏DC后刺激同種混合淋巴細(xì)胞增殖(MLR)能力;ELISA檢測(cè)致敏DC與T淋巴細(xì)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(cè)CTL對(duì)肝癌細(xì)胞的細(xì)胞毒作用。 結(jié)果: (1)靶細(xì)胞篩選:流式細(xì)胞儀檢測(cè)結(jié)果顯示肝癌細(xì)胞株HepG2為HLA-A2+, Bel-7404為HLA-A2-;RT-PCR和免疫細(xì)胞化學(xué)法結(jié)果顯示Bel-7404和HepG2均表達(dá)OY-TES-1。 (2)DC的鑒定和CTL殺傷效應(yīng):PBMC經(jīng)細(xì)胞因子聯(lián)合誘導(dǎo)培養(yǎng)1周后,出現(xiàn)典型的DC形態(tài),并高表達(dá)HLA-DR、CD86、CD83和CD80;經(jīng)不同蛋白致敏的DC均能促進(jìn)T淋巴細(xì)胞的增殖活化,其中OY-MBP致敏的DC促進(jìn)T淋巴細(xì)胞增殖的能力明顯強(qiáng)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(dǎo)的CTL對(duì)靶細(xì)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(xì)胞后,CTL對(duì)其殺傷作用下降。 結(jié)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(qiáng)的抗肝癌細(xì)胞毒活性效應(yīng),提示OY-TES-1可作為肝癌免疫治療的靶點(diǎn)。
[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(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392

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