携带hIL-2cDNA的逆转录病毒载体的构建及其在人肝癌细胞中的特异表达研究

Expression of human interleukin-2 gene in the human hepatoma cells

将人白细胞介素２（ｈＩＬ-２）ｃＤＮＡ克隆到逆转录病毒载体ＭＮＳＭ的ＰＬ位点 ，分别构建了转录受ＳＶ４０早期启动子和人甲胎蛋白增强子调控的重组逆转录病毒载体ＭＮＳＩ和ＭＮＳＩＡ。用脂质体转染法将ＭＮＳＩ和ＭＮＳＩＡ分别转导ＰＡ３１７包装细胞，测质粒转染率为（５～２０）×１０^(－３)克隆／μｇＤＮＡ·１０~６细胞，病毒感染率为（５．４～４５０）×１０~４ＣＦＵ／ｍｌ。重组病毒在４μｇ／ｍｌ ｐｏｌｙｂｒｅｎｅ存在条件下感染人肝癌细胞、肾癌细胞和黑色素瘤细胞，Ｎｅｏ~Ｒ克隆经Ｓｏｕｔｈｅｒｎｂｌｏｔ分析证明ｈＩＬ－２ｃＤＮＡ转入人肿瘤细胞并整合， Ｒ ＮＡ斑点杂交及ＩＬ－２活性表达分析证明，人甲胎蛋白增强子可促进异源启动子启动ｈＩＬ－２ｃＤ－ＮＡ在合成甲胎蛋白的人肝癌细胞中高效特异转录和表达。该研究对肝癌特异性免疫增强基因治疗有重要意义。

Human interleukin-2 complementary DN A (hlL-2 cDNA ) was inserted into polylinker site of MNSM retroviral vector to construct retroviral vectors MNSI , in which the transcription of hlL-2 cDNA was drived by SV 40 early region promoter ,and MNSIA ,in which the transcription of hlL -2 cDNA drived by SV 40 early region promoter was regulated byα-fetoprotein enhancer. The recombinant retroviruses were used to infect huamn hepatoma , renal carcinoma and melanoma cell lines in the presence of 4μg/m1 poly- brene. Southern hybridization of DNA from NeoR colonies showed that hlL-2 cDNA was transfered and in- tergrated into the genome of the modified tumor cells. Dot hybridization of total RNA from the Neo^R. colonies and expression assay indicated that humanα-fetoprotein enhancer could induce efficient and specific transcription and expression of hlL-2 gene drived by the promoter of different origins in the human hep- atoma cells which can produce a-fetopratein.