人天冬氨酸-β-羟化酶树突状细胞疫苗对肝癌细胞系的体外作用研究
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摘要
目的:通过对携带人天冬氨酸-β-羟化酶(aspartate-β-hydroxylase,AAH)基因的树突状细胞杀伤肝癌细胞系的研究,探索可用于防治肝癌的树突状细胞疫苗。方法:重组腺病毒(Ad-AAH-IRES2-EGFP)感染C57BL/6小鼠骨髓未成熟树突状细胞(dendritic cells,DCs)制备携带AAH基因的DCs(AAH-DCs),与同体小鼠脾脏T淋巴细胞共培养后作为效应细胞,以肝癌细胞株HepG2和Huh7分别作为靶细胞,效应细胞分别和靶细胞共培养24 h,CCK-8法检测效应细胞对靶细胞增殖的影响;流式细胞术检测效应细胞对靶细胞凋亡的影响。将AAH-DCs连续3次皮下注射免疫C57BL/6小鼠,乳酸脱氢酶释放法检测免疫后小鼠脾脏T淋巴细胞对靶细胞的裂解效应。结果:AAH-DCs与T淋巴细胞共培养组靶细胞的增殖能力明显低于对照组(P=0.000),凋亡率明显高于对照组(P=0.000);实验组靶细胞的裂解率明显高于对照组(P=0.000)。结论:荷载AAH基因的树突状细胞疫苗能够诱导产生细胞毒性T淋巴细胞从而杀伤表达AAH的肝癌细胞。
Objective:To explore the antitumor effect of dendritic cells(DCs)vaccine targeting aspartate-β-hydroxylase(AAH)gene against hepatocellular carcinoma(HCC)cells HepG2 and Huh7 for searching DCs vaccine for hepatocarcinoma. Methods:AAH-DCs vaccine was prepared in vitro via infecting DCs with recombinant adenovirus vector encoding AAH. T cells co-cultured with AAH-DCs vaccine were served as the effector cells and human HCC cell lines HepG2 and Huh7 were regarded as the target cells. The proliferation and apoptosis of target cells,after co-culturing with the effector cells for 24 h,were detected by CCK-8 and FCM analysis respectively. C57 BL/6 mice were immunized with AAH-DCs vaccines by three continuous subcutaneous injections,and the cytotoxic effect of T cells against target cells was observed by lactate dehydrogenase(LDH)release assay. Results:Compared with the control groups,the declined proliferation activity of target cells in AAH-DCs vaccine group was found by CCK-8,while increased apoptosis rate of target cells in AAH-DCs vaccine group was tested by FCM(P=0.000). Additionally,LDH release assay showed that the cytolysis effect of target cells in AAH-DCs vaccine group was higher than that in other control groups(P =0.000). Conclusion:DCs vaccines targeting AAH can induce the production of cytotoxicity T lymphocytes for fulfilling the killing of HCC cells expressing AAH gene.
Objective:To explore the antitumor effect of dendritic cells(DCs)vaccine targeting aspartate-β-hydroxylase(AAH)gene against hepatocellular carcinoma(HCC)cells HepG2 and Huh7 for searching DCs vaccine for hepatocarcinoma. Methods:AAH-DCs vaccine was prepared in vitro via infecting DCs with recombinant adenovirus vector encoding AAH. T cells co-cultured with AAH-DCs vaccine were served as the effector cells and human HCC cell lines HepG2 and Huh7 were regarded as the target cells. The proliferation and apoptosis of target cells,after co-culturing with the effector cells for 24 h,were detected by CCK-8 and FCM analysis respectively. C57 BL/6 mice were immunized with AAH-DCs vaccines by three continuous subcutaneous injections,and the cytotoxic effect of T cells against target cells was observed by lactate dehydrogenase(LDH)release assay. Results:Compared with the control groups,the declined proliferation activity of target cells in AAH-DCs vaccine group was found by CCK-8,while increased apoptosis rate of target cells in AAH-DCs vaccine group was tested by FCM(P=0.000). Additionally,LDH release assay showed that the cytolysis effect of target cells in AAH-DCs vaccine group was higher than that in other control groups(P =0.000). Conclusion:DCs vaccines targeting AAH can induce the production of cytotoxicity T lymphocytes for fulfilling the killing of HCC cells expressing AAH gene.
引文
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[16]符少月,焦庆昉,黄启彬,等.带有HPV-16 E6/E7基因的树突状细胞疫苗对裸鼠人宫颈CaSki细胞移植瘤的抗肿瘤作用[J].重庆医科大学学报,2013,38(5):512-516.
[17]李成敏,刘飞飞,伍春霞,等.人ASPH基因修饰树突状细胞介导CTLs对肝癌细胞SMMC-7721的体外杀伤作用[J].重庆医科大学学报,2016,41(6):320-326.
[18] Noda T,Shimoda M,Ortiz V,et al. Immunization with aspartate-β-hydroxylase-loaded dendritic cells produces antitumor effects in a rat model of intrahepatic cholangiocarcinoma[J]. Hepatology,2012,55:86-97.
[19] Shimoda M,Yoshito T,Kevin PC,et al. Tumor progression-related transmembrane protein aspartate-b-hydroxylase is a target for immunotherapy of hepatocellular carcinoma[J]. Journal of Hepatology,2012,56:1129-1135.
[20] Yoshito T,Sasmita M,Howard S,et al. Aspartate-β-hydroxylase induces epitope-specific T cell responses in hepatocellular carcinoma[J]. Vaccine,2015,33:1256-1266.
[21] Radford KJ,Tullett KM,Lahoud MH. Dendritic cells and cancer immunotherapy[J]. Curr Opin Immunol,2014,27:26-32.
[2] Armengol C,Sarrias MR,Sala M. Hepatocellular carcinoma:present and future[J]. Med Clin(Barc),2017[Epub ahead of print].
[3] Lavaissiere L,Jia S,Nishiyama M,et al. Overexpression of human aspartyl(asparaginyl)beta-hydroxylase in hepatocellular carcinoma and cholangiocarcinoma[J]. J Clin Invest,1996,98(6):1313-1323.
[4] Xue T,Su J,Li HM,et al. Evaluation of HAAH/humbug quantitative detection in the diagnosis of hepatocellular carcinoma[J]. Oncol Rep,2015,33(1):329-337.
[5] Xian ZH,Zhang SH,Cong WM,et al. Expression of aspartyl betahydroxylase and its clinicopathological significance in hepatocellular carcinoma[J]. Mod Pathol,2006,19(2):280-286.
[6] Wang K,Liu J,Yan ZL,et al. Overexpression of aspartyl-(asparaginyl)-β-hydroxylase in hepatocellular carcinoma is associated with worse surgical outcome[J]. Hepatology,2010,52(1):164-173.
[7] Aihara A,Huang CK,Olsen MJ,et al. A cell-surfaceβ-hydroxylase is a biomarker and therapeutic target for hepatocellular carcinoma[J].Hepatology,2014,60(4):1302-1313.
[8] Rosenberg SA,Yang JC,Restifo NP. Cancer immunotherapy:moving beyond current vaccines[J]. Nat Med,2004,10(9):909-915.
[9] Nencioni A,Brossart P. Cellular immunotherapy with dendritic cells in cancer:current status[J]. Stem Cells,2004,22(4):501-513.
[10] Ballestrero A,Boy D,Moran E,et al. Immunotherapy with dendritic cells for cancer[J]. Adv Drug Deliv Rev,2008,60(2):173-183.
[11]冯鹏飞,韩双印.树突状细胞疫苗治疗恶性肿瘤研究进展[J].中华实用诊断与治疗杂志,2015,29(5):420-422.
[12] Steffen Z,Irina N,Torsten W,et al. A critical role for Notch Signaling in the formation of cholangiocellular carcinoma[J]. Cancer Cell,2013,23:784-795.
[13] Diana LB,Gao JS,Tong M,et al. Potential role of phosphorylation as a regulator of aspartyl-(asparaginyl)-β-hydroxylase:relevance to infiltrative spread of human hepatocellular carcinoma[J]. Liver Cancer,2015,4:139-153.
[14] Cantarini MC,de la Monte SM,Pang M,et al. Aspartyl-asparagyl beta hydroxylase over-expression in human hepatoma is linked to activation of insulin-like growth factor and notch signaling mechanisms[J].Hepatology,2006,44(2):446-457.
[15] Wu XM,Liu X,Jiao QF,et al. Cytotoxic T lymphocytes elicited by dendritic cell-targeted delivery of human papillomavirus type-16E6/E7 fusion gene exert lethal effects on CaSki cells[J]. Asian Pac J Cancer Prev,2014,15(6):2447-2451.
[16]符少月,焦庆昉,黄启彬,等.带有HPV-16 E6/E7基因的树突状细胞疫苗对裸鼠人宫颈CaSki细胞移植瘤的抗肿瘤作用[J].重庆医科大学学报,2013,38(5):512-516.
[17]李成敏,刘飞飞,伍春霞,等.人ASPH基因修饰树突状细胞介导CTLs对肝癌细胞SMMC-7721的体外杀伤作用[J].重庆医科大学学报,2016,41(6):320-326.
[18] Noda T,Shimoda M,Ortiz V,et al. Immunization with aspartate-β-hydroxylase-loaded dendritic cells produces antitumor effects in a rat model of intrahepatic cholangiocarcinoma[J]. Hepatology,2012,55:86-97.
[19] Shimoda M,Yoshito T,Kevin PC,et al. Tumor progression-related transmembrane protein aspartate-b-hydroxylase is a target for immunotherapy of hepatocellular carcinoma[J]. Journal of Hepatology,2012,56:1129-1135.
[20] Yoshito T,Sasmita M,Howard S,et al. Aspartate-β-hydroxylase induces epitope-specific T cell responses in hepatocellular carcinoma[J]. Vaccine,2015,33:1256-1266.
[21] Radford KJ,Tullett KM,Lahoud MH. Dendritic cells and cancer immunotherapy[J]. Curr Opin Immunol,2014,27:26-32.