fas基因与bcl-2反义RNA转导胃癌耐药细胞的药敏上调效应

Transduction of fas gene or bcl-2 antisense RNA sensitizes cultured drug resistant gastric cancer cells to chemotherapeutic drugs

目的比较ｆａｓ基因和ｂｃｌ２基因在胃癌耐药细胞与非耐药细胞表达的差异，将ｆａｓ基因和ｂｃｌ２反义核酸导入胃癌耐药细胞，并分析转导前后的细胞在ｍＲＮＡ与蛋白的表达水平，研究转导株与非转导株对化疗药物敏感性的区别．方法采用分子克隆技术将ｆａｓ基因和反义ｂｃｌ２片段分别插入真核表达载体ｐＢＫＣＭＶ和ｐＤＯＲＳＶ４０的多克隆克隆位点之间，以脂质体介导法将两个重组表达载体分别转染受体细胞ＳＧＣ７９０１／ＶＣＲ，Ｇ４１８筛选克隆细胞，Ｎｏｒｔｈｅｒｎｂｌｏｔ，Ｗｅｓｔｅｒｎｂｌｏｔ检测耐药细胞与非耐药细胞以及转导细胞中ｆａｓ基因和ｂｃｌ２基因ｍＲＮＡ及其蛋白的表达，ＭＴＴ法药敏实验检测转导细胞与非转导细胞对ＶＣＲ、顺铂、５ＦＵ的敏感性．结果真核表达载体ｐＢＫｆａｓｃＤＮＡ和ｐＤＯＲｂｃｌ２ｃＤＮＡ转导胃癌耐药细胞后，分别从２×１０５细胞中筛选出大约８０和１２０个抗性克隆，转导率各为０４‰和０６‰，随机各挑选２个克隆继续筛选与扩增培养，均获得了稳定的抗性细胞，我们将此命名为ＳＧＣ７９０１ｆａｓ／ＶＣＲｃｅｌ和ＳＧＣ７９０１ａｎｔｉｂｃｌ２／ＶＣＲｃｅｌ．杂交结果表明，胃癌耐药细胞ＳＧＣ７９０１／?

AbstractAIM To compare the expression level of fas gene and bcl-2 gene in gastric cancer cells SGC7901 and in gastric cancer MDR cells SGC7901/VCR, to transduce fas cDNA and bcl-2 antisense nucleic acid into SGC7901/VCR cells respectively, and to observe the expression of two genes in transfectants and nontransfectants as well as their drug sensitivity.METHODS The eukaryotic expression vector pBKfas and pDORanti bcl-2 was constructed, and then transfected into SGC7901/VCR cells by lipofectamine, respectively. The expression of mRNA and protein in SGC7901/VCR cells and SGC7901 cells and transfectants was detected using Northern blot and Western blot and the drug sensitivity of transfectants for VCR, CDDP and 5FU was analysed with MTT assay.RESULTS After gene transfection, 80 for fas or 120 for antisense bcl-2 drugresistant clones were selected from 2×10~5 cells, the transfection efficiency was 0.04% and 006%, respectively. Two clones, SGC7901 fas/VCR cells and SGC7901 anti-bcl-2/VCR cells, were randomly selected for further incubation. Hybridization results showed that the expression level of fas mRNA and protein in SGC7901/VCR cells was much lower, but the expression level of bcl2 mRNA and protein was higher than that in SGC7901 cells, the expression level of fas mRNA and protein in SGC7901 fas/VCR cells was higher, and the expression level of bcl2 mRNA and protein was lower in SGC7901 anti bcl-2/VCR cells than that in nontransfectants. MTT assay showed that transfectants were more sensitive to VCR, CDDP and 5FU than nontransfectants.CONCLUSION bcl-2 gene displayed high expression and fas gene displayed low expression in drug resistant gastric cancer cells. Expression of bcl-2 protein was effectively blocked in SGC7901 anti bcl-2/VCR cells by gene transfection, in contrast, the expression of fas mRNA and protein in SGC7901 fas/VCR cells increased. Fas gene and bcl-2 antisense nucleic acid transfection sensitized drug resistant gastric cancer cells to chemotherapeutic drugs.