【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(dǎo)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(xué)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(dǎo)培養(yǎng)為樹突狀細胞(dendritic cell, DC),通過光學(xué)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(jié)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(jié)果: (1)靶細胞篩選:流式細胞儀檢測結(jié)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(xué)法結(jié)果顯示Bel-7404和HepG2均表達OY-TES-1。 (2)DC的鑒定和CTL殺傷效應(yīng):PBMC經(jīng)細胞因子聯(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(dǎo)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(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)生較強的抗肝癌細胞毒活性效應(yīng),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(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前10條

1 賈軍;任軍;張紅梅;張利旺;郭曉彤;;造血干細胞來源的樹突狀細胞負載p53基因誘導(dǎo)對HMCC97肝癌細胞特異性殺傷[J];北京大學(xué)學(xué)報(醫(yī)學(xué)版);2008年05期

2 馬昌云;吳芳;孔繁義;周文艷;;樹突狀細胞的體外培養(yǎng)及其抗癌作用[J];中國肺癌雜志;2010年05期

3 ;Treatment of established colon carcinoma-bearing mice by dendritic cells pulsed with lysates of heat-treated tumor cells[J];Science in China(Series C:Life Sciences);2009年09期

4 吳鳴宇;蔡兵;趙翼;謝平;張濱;;MAGE-1多肽負載樹突狀細胞對人肝癌細胞的體外殺傷作用[J];中國臨床醫(yī)學(xué);2008年02期

5 陳紅松;張華剛;梅銘惠;費然;叢旭;覃理靈;魏來;陳慰峰;;NY-ESO-1和MAGE-A3抗原肽誘導(dǎo)肝癌患者的免疫應(yīng)答研究[J];免疫學(xué)雜志;2006年06期

6 范蓉;肖紹文;黃巍;羅彬;李頎;徐麗湘;謝小薰;;癌-睪丸抗原OY-TES-1多克隆抗體的制備及鑒定[J];中國生物制品學(xué)雜志;2009年08期

7 范蓉;黃巍;肖紹文;羅彬;何少健;羅國容;謝小薰;;OY-TES-1在肝細胞癌中的表達及抗體血清學(xué)分析[J];世界華人消化雜志;2009年32期

8 陳紅松,蔡勝利,王瑜,趙海濤,彭吉潤,龐學(xué)文,朱繼業(yè),叢旭,芮靜安,冷希圣,杜如昱,王宇,陳慰峰;Expression of the MAGE-1 gene in human hepatocellular carcinomas[J];Chinese Medical Journal;2000年12期

9 張文敏;陳裕慶;張萌;凌航;文劍明;;NY-ESO-1致敏樹突狀細胞誘導(dǎo)的CTL對肝癌細胞株的特異性殺傷作用[J];中國腫瘤生物治療雜志;2009年01期

10 ;Biological functions of melanoma-associated antigens[J];World Journal of Gastroenterology;2004年13期

相關(guān)博士學(xué)位論文 前1條

1 張堯;基于樹突狀細胞的膀胱腫瘤疫苗的實驗研究[D];第三軍醫(yī)大學(xué);2008年

，

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