逆转录病毒介导的IL－2和TNF－α融合基因在卵巢癌细胞中的表达及其对肿瘤细胞生长的影响

RETROVIRAL-MEDIATED IL-2AND TNF-αFUSION GENE EXPRESSION IN HUMAN OVARIAN CANCER CELLS AND ITS EFFECTS ON THE GROWTH OF THE CELLS

ＩＬ－４和ＴＮＦ－α是引人注目的抗肿瘤活性因子，两者在抗肿瘤及免疫调节方面具有协同作用。我们利用逆转录病毒载体Ｘｄ１构建了插入ＩＬ－２和ＴＮＦ－α融合基因的重组逆转录病毒载体ＸｄＦ，融合基因包括编码ＩＬ－２前导肽和ＩＬ－２和ＴＮＦ－α成熟肽的序列。重组载体用Ｌｉｐｏｆｅｃｔｉｎ方法引入病毒包装细胞ＰＡ３１７，挑选Ｇ４１８抗性克隆，用ＮＩＨ３Ｔ３细胞测定病毒滴度，获得一滴度为１×１０￣４ＣＦＵ／ｍｌ的高滴度克隆。超速离心浓缩这一重组病毒上清，转导人卵巢癌细胞系ＳＫＯＶ３，经Ｇ４１８筛选获得抗性克隆。ＰＣＲ可从融合基因转导的细胞ＤＮＡ中扩增出１．１ｋｂ的融合基因片断，证明目的基因完整的整合在细胞的基因组ＤＮＡ中，测其上清中的ＩＬ－２和ＴＮＦ－α活性结果显示：ＩＬ－２的活性为８－１５Ｕ／１０￣６ｃｅｌｌｓ／２４ｈ，ＴＮＦ－α的活性为１５０－４００Ｕ／１０￣６ｃｅｌｌｓ／２４ｈ。这一结果表明逆转录病毒介导的融合基因可以在卵巢癌细胞中获得有效表达，表达的融合基因产物在体内和体外部对肿瘤细胞的生长具有抑制作用。

oth IL-2 and TNF-α are cytokines with antitumor activity.In order to observe the feasiblity of cytokine fu-sion gene expression in tumor cells,we used retroviral vector Xdl to construct recombinant retroviral vector XdFwith an inserted IL-2 and TNF-αfusion gene.The fusion gene included the sequences encoding the signal peptide ofIL-2 and mature peptide of IL-2 and TNF-α.The plasmid vector was introduced into packaging cell line PA 317 bylipofectin method.The G418-resistant colonies were selected and the supernatants of the colonies were used to de-termine the virus titers with NIH3T3 cells,A higher-titer colony of 1×1O4CFU/ml was obtained.The supernatantwas used to transduced the human ovavian cancer cell line SKOV 3 after being concantrated 100 times by ultracen-trifugation.The G418-resistant colonies of ovavian cancer cells were obtained by G418 screening and PCR was usedto identify the integration of the fusion gene in genomic DNA of G418-resistant human ovavian cancer cell line.Thebiologic activity assay of IL-2 and TNF-αin the superntant demonstrated that the IL-2 and TNF-α activity was inthe range of 8-15U/1O6cells/24h and 150-400U/106cells/24h,respectively.This rcsults showed that the IL-2 andTNF-α fusion gene could be efficiently expressive in the transduced human ovavian cancar cells and the secreting o-varian tumor cells had a much decreased tumorigenicity in mude mice.