构建fas基因真核表达载体逆转胃癌耐药细胞MDR表型

Construction of eukaryotic expression vector pBK fas and MDR reversal test of drug resistant gastric cancer cells

目的将ｆａｓ基因转导入胃癌耐药细胞，建立高表达ｆａｓ的耐药株，比较转导前后ｆａｓｍＲＮＡ与蛋白的表达水平，观察转导株对化疗药物的敏感性．方法采用分子克隆技术将人ｆａｓ基因的全长ｃＤＮＡ片段插入真核表达载体ｐＢＫＣＭＶ的多克隆位点之间，并借助脂质体将重组表达载体转导入人胃癌多药耐药细胞ＳＧＣ７９０１／ＶＣＲ，Ｇ４１８筛选克隆细胞，Ｎｏｒｔｈｅｒｎｂｌｏｔ和Ｗｅｓｔｅｒｎｂｌｏｔ观察ｆａｓ基因的表达，ＭＴＴ法检测转导株对ＶＣＲ，ＣＤＤＰ，５ＦＵ的敏感性．结果成功地构建了真核表达载体ｐＢＫｆａｓ；转导细胞后，从２×１０５细胞中筛选出大约１２０个抗性克隆，转导率大于０５‰，随机挑选２个克隆继续筛选与扩增培养，最终获得了１株稳定的抗性细胞，命名为ｆａｓＳＧＣ７９０１／ＶＣＲ；杂交结果表明，转导株与非转导株均有ｆａｓ基因的表达，但转导株ｆａｓｍＲＮＡ及其蛋白水平显著高于非转导株；ＭＴＴ实验结果显示，转导株对ＶＣＲ，ＣＤＤＰ，５ＦＵ的敏感性显著升高．结论在胃癌耐药细胞中ｆａｓ基因处于低表达状态．将ｆａｓ基因全长ｃＤＮＡ导入耐药细胞，能有效增强ｆａｓｍＲＮＡ及其蛋白的表达水平，并使耐药细胞对化疗药物的敏感性显著?

AIM To get fas overexpressing drug resistant gastric cancer cells by gene transduction, to compare the expressing level of fas in gene transfected and non transfected cells, and to observe drug sensitizing effect of fas gene in transfected cells. METHODS With molecular cloning technique, the full length cDNA of fas was inserted into the multiple cloning site of the expressing vector pBK CMV. Drug resistant gastric cancer cell strain SGC7901/ VCR was transpected with recombinant plasmids, and positive clones were selected by G418. Then the expressing level of fas gene was determined by means of Northern-blot and Western-blot, and MTT assay was used to detect drug sensitivity of gene transfected cells. RESULTS The expressing plasmids pBK fas cDNA was successfully constructed. More than 120 positive clones were selected from 2× 10 5 gene transfected cells, the transduction efficiency being more than 0 5‰. Two positive clones were expanded and passaged, and one drug resistant cell ( fas SGC7901/ VCR) was obtained. fas gene was expressed in both gene transfected and non transfected cells. Expression of fas mRNA and protein was enhanced positively in gene transfected cells. MTT assay showed increasing sensitivity of gene transfected cells to CDDP, VCR and 5 FU. CONCLUSION Expressing level of fas gene is very low in gastric cancer drug resistant cells, but it is enhanced efficiently after transfected with fas cDNA and markedly increases the sensitivity of fas gene transfected cells to chemotherapeutic agents, which indicates that inhibition of apoptosis plays an important role in tumor MDR and induced apoptosis may reverse the tumor MDR to some extent.