腺病毒介导的HBsAg基因修饰树突状细胞瘤苗对HepG2．2．15肝癌细胞的免疫效应  

Immune response of HBsAg gene-modified dendritic cell-based vaccine in HepG2.2.15 hepatocellular carcinoma cells

作　　者：杨静悦[1] 刘文超[1] 曹大勇[2] 斯小明[1] 腾增辉[3] 

机构地区：[1]第四军医大学西京医院肿瘤科,西安710032 [2]第四军医大学西京医院肝胆外科,西安710032 [3]第四军医大学药理教研室

出　　处：《中华肿瘤杂志》2007年第10期728-732,共5页Chinese Journal of Oncology

基　　金：国家自然科学基金资助项目(30572100);全军医药卫生科研基金资助项目(06MA205)

摘　　要：目的研究腺病毒介导的HBsAg基因修饰树突状细胞(DC)体外诱导对HepG2.2.15肝癌细胞特异性的细胞毒效应。方法将HBsAg基因亚克隆到pIND和pShuttle载体中,构建重组载体pShuttle-S。将用PI-SceⅠ和Ⅰ-CeuⅠ双酶切后所获得的HBsAg基因片段与线性化的腺病毒载体pAdeno-X连接,构成腺病毒表达载体AdVHBsAg。经HEK293细胞包装腺病毒后,收获病毒并做滴度测定,再将AdVHBsAg转染人单核细胞来源的DC构建AdVHBsAg-DC肝癌瘤苗。采用Western blot法鉴定转染基因表达;流式细胞仪检测表面分子;3H-胸腺嘧啶核苷(3H-TdR)法检测T细胞增殖反应的能力;乳酸脱氢酶(LDH)法检测CTL活性。结果穿梭质粒插入片段为HBsAg基因;包装的腺病毒载体具有良好的感染性,可在HEK293细胞中形成病毒颗粒;HBsAg基因表达于AdVHBsAg- DC中,表明腺病毒介导的HBsAg基因转染的有效性;AdVHBsAg-DC可高表达CD1a、CD11c、CD80、CD86和HLA-DR;AdVHBsAg-DC刺激自体T细胞增殖功能明显高于AdVLacZ-DC组和未转染的DC组(P<0.05);AdVHBsAg-DC体外诱导CTL能够对表达HBsAg肝癌细胞起到特异性杀伤作用。结论AdVHBsAg可在DC中表达HBsAg肝癌相关抗原;AdVHBsAg-DC瘤苗可诱导T细胞产生高效的针对HepG2.2.15肝癌细胞的细胞毒效应。Objective To study the cytotoxic T lymphocyte （CTL） response induced by dendritic cells （DC） transduced with recombinant adenovirus vector bearing hepatitis B virus surface antigen （HBsAg） gene in hepatocellular carcinoma HepG2.2. 15 cells in vitro. Methods Full length HBsAg cDNAs were subcloned into plND vector, followed by being cloned into pShuttle vector. The HBsAg gene fragments resulted from the pShuttle-S digested with PI-Sce and I -Ceu were linked to the linear adeno-X virus DNA. After packaged with HEK293 cells, the adenovirus expression vector was obtained. Then the recombinant adenovirus expression plasmid AdVHBsAg was transfected into human monocyte-derived dendritic cells, to construct AdVHBsAg hepatocareinoma tumor vaccine. The effectiveness of transfection was detected by Western blot. Surface molecules of AdVHBsAg-DC were detected by FACS. Autologous T cell proliferation stimulated by AdVHBsAg-DC was detected by 3 H-TdR assay. Cytotoxic CTL activity induced by AdVHBsAg-DC in vitro was detected by LDH assay. Results HBsAg gene in the inserted DNA of AdVHBsAg was confirmed by PCR, and predictive fragments proved by restriction enzyme digestion analysis were exhibited. Cell pathological changes appear after 10 days HEK293 cells transfected AdVHBsAg. Western blot analysis showed that HBV surface antigen gene was expressed in transfected DC, indicating that the transfection was effective. AdVHBsAg-DC was able to upregnlate CDla, CDllc, CD80, CD86 and HLA-DR. Autologns T cell proliferation induced by AdVHBsAg-DCs was significantly higher than that in DC control group and LacZ-DC group （ P 〈 0.05 ）. AdVHBsAg-DC activated CTL presented the specific killer ability to the hepatocellular carcinoma cells expressing HBsAg. Conclusion DC transduced with recombinant adenovirus HBsAg can express HBV-related hepatocellular carcinoma antigen （HBsAg） , andAdVHBsAg-DC can induce potent immune response against HBsAg-positive hepatocellular carcinoma cells in vitro.

关 键 词：HBsAg基因 树突状细胞 HepG2.2.15肝癌细胞 

分 类 号：R735.7[医药卫生—肿瘤]