【摘要】 目的 :构建肝癌靶向性 HSV- tk/ GCV基因转移系统 ,并研究其在体外对肝癌细胞的杀伤效应。方法 :体外培养通过基因重组技术构建和表达抗转铁蛋白受体 ( Tf R)人 -鼠嵌合抗体的转染瘤细胞 D2 C5 .6,诱导裸鼠( Balb/ c,nu/ nu)产生腹水并纯化 ;以蛋白质连接因子 -水溶性碳二亚胺 ( EDC)介导 Tf R的抗体与多聚左旋赖氨酸的连接 ( Ab- PL L ) ,与 p EBAF/ tk按比例混匀 ,得到 Ab- PL L - p ETAF/ tk基因转移系统 ;分别转染不同的细胞株进行体外效应研究 :人肝癌细胞株 Hep G2、SMMC772 1,人肺癌细胞株 5 49,观察给予不同浓度的更昔洛韦 ( GCV)随时间延长的细胞改变情况 ,用 MTT法检测 GCV对不同细胞的杀伤作用。结果 :分析型酸性尿素凝胶电泳证实抗 Tf R的抗体与 PL L成功连接 ;体外杀伤实验显示 :经 Ab- PL L - p ETAF/ tk基因转移系统处理的 3种细胞对 GCV表现出不同的敏感性。 Hep G2细胞 ( CD71 ,AFP )对 GCV很敏感 ,低浓度的 GCV( 1mg/ L )处理 3 d,即可使细胞生长抑制率达到 74.8% ,SMMC772 1细胞 ( CD71 ,AFP+ )对 GCV低度敏感 ,A5 49( CD71- ,AFP- )对 GCV不敏感。结论 :Tf R介导的肝癌靶向性 HSV- tk/ GCV基因转移系统构建成功 ;有可能用以肝癌细胞为靶细胞的基因治疗的研究更多还原

【Abstract】 Objective:To construct the gene targeting delivery and expression system, and to investigate the special killing effect of HSV-tk/GCV system on human liver cancer cells in vitro mediated by it.Methods:We injected a number of murine hybridomas D2C5.6, secreting chimeric antibody, into abdominal cavities of nude mouses and got enough ascitic fluid and purified it.Then it was to be coupled to poly-L-lysine mediated by EDC; The vector, carrying HSV-tk gene, was extracted and complexed to the conjugate after determine the combination ratio of plasmid and Ab-PLL to form a complex including three components. To test this complex in vitro ,a model system was used consisting of hepatoma cell lines: HepG2(transferrin receptor , AFP), SMMC7721(,+), and A549(-,-).Results:The HepG2/tk cells that over-secrete AFP were highly sensitive to the GCV, whereas the SMMC7721/tk cell that under-secrete AFP were less sensitive to GCV, and the A549/tk cells that could not secrete AFP were not.Conclusions:The tissue-specific HSV-tk/GCV anti-tumor system mediated by transferrin rceptor has been constructed successfully,and it showed a good capacity of targeting tumor cells.更多还原