腺相关病毒介导的肿瘤坏死因子相关细胞凋亡诱导配体的表达及其体外抗肿瘤活性的研究  

Expression of TRAIL_(114-281) mediated by adeno-associated virus and its tumoricidal activity

作　　者：马宏[1] 刘彦信[1] 刘士廉[1] 许瑞安[2] 郑德先[1] 

机构地区：[1]中国医学科学院中国协和医科大学基础医学研究所医学分子生物学国家重点实验室,北京100005 [2]香港大学分子生物学研究所

出　　处：《中华医学杂志》2004年第19期1635-1641,共7页National Medical Journal of China

基　　金：国家"八六三"高技术研究发展计划基金资助项目 (2 0 0 1AA2 17111);国家自然科学基金资助项目(3 0 170 3 62 );北京市科委重大生物医药基金资助项目(H0 10 2 10 2 0 0 12 2 )

摘　　要：目的　研究腺相关病毒 (AAV)介导的重组可溶性肿瘤坏死因子相关细胞凋亡诱导配体(TRAIL114 2 81)在体内外的表达规律及其杀伤肿瘤细胞的生物学活性 ,探讨重组腺相关病毒用于肿瘤基因治疗的应用前景。方法　构建编码TRAIL胞外区 114 2 81位氨基酸的多肽的重组腺相关病毒载体rAAV TRAIL114 2 81,转染HEK2 93人胚肾细胞 ,获得高滴度的重组病毒颗粒后 ,转导人T淋巴细胞白血病细胞株Jurkat、人肝癌细胞株HepG2和SMMC 772 1以及人宫颈癌细胞株HeLa等 ,并采用经C5 7BL/ 6小鼠门静脉输入、皮下注射、肌内注射、腹腔注射或口服等不同给药途径 ,研究重组腺相关病毒介导的TRAIL114 2 81蛋白在体内外的表达规律。采用实时PCR方法测定重组病毒滴度。采用酶联免疫吸附实验 (ELISA)、Western印迹法和免疫组化法测定蛋白表达。采用MTT法测定重组rAAV TRAIL114 2 81杀伤肿瘤细胞的生物学活性。结果　成功地获得了rAAV TRAIL114 2 81重组病毒 ,其滴度为每毫升 7 5× 10 12基因组颗粒数 (Gps/ml)。重组病毒转导使TRAIL114 2 81在宿主细胞中高效表达 ,并可显著诱导Jurkat、HeLa和SMMC 772 1等肿瘤细胞凋亡 ,但不能诱导HepG2细胞凋亡。体内实验中 ,经小鼠门静脉输入重组病毒 ,可使TRAIL114 2Objective To investigate the expression of the soluble tumor necrosis factor related apoptosis inducing ligand (TRAIL) mediated by adeno associated virus (AAV) and its tumoricidal activity in vitro and vivo Methods The recombinant AAV expression vector encoding the extracellular domain (114 281aa peptide, TRAIL 114 281 ) of TRAIL was constructed and transfected into human embriotic kidney cells HEK293 for virus package The human tumor cell lines of T lymphocyte leukemia Jurkat, liver cancer HepG2 and SMMC 7721, and cervical cancer HeLa were trasduted by using the recombinant virus particles respectively The recombinant virus particles were also injected into C57BL/6 mice via the hepatic portal vein or hypodermic, intramuscular, celiac and oral pathways to study the expression of TRAIL 114 281 The recombinant virus titer was determined by real time PCR The expression of TRAIL 114 281 was evaluated by ELISA, Western blotting and immunohistochemistry assay respectively The tumoricidal activity and apoptosis were evaluated by MTT assay Results The recombinant AAV encoding for the soluble TRAIL (114 281aa) were constructed successfully The titer of recombinant virus was 7 5×10 12 genome particles(Gps)/ml Transduction of rAAV TRAIL 114 281 led to high level expression of TRAIL 114 281 and the induction of apoptosis of Jurkat, Hela and SMMC 7721 cancer cells, but not HepG2 cells, in vitro The recombinant peptide TRAIL 114 281 in trimeric active form was highly and constantly expressed in the hepatocytes and secreted into the serum up to 6 months in the of C57BL/6 mice injected with the recombinant virus particles via the hepatic portal vein The peptide TRAIL 114 281 in the livers, but not other tissues, were also detected in the mice administrated with rAAV TRAIL 114 281 particles via subcutaneous, intramuscular, intraceliac or oral pathway Conclusion The long term, stable and liver tropism expression of peptide TRAIL 114 28

关 键 词：TRAIL 介导 重组腺相关病毒 表达规律 重组病毒 滴度 体外抗肿瘤活性 外源 诱导 杀伤 

分 类 号：R73-36[医药卫生—肿瘤]