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应用PCR等方法从含有p53基因突变的人肺癌细胞系GL总RNA中扩增并克隆了人突变型p53基因(mt)cDNA.序列分析证实该cDNA含有人p53基因全部编码序列,在codon245出现了G→T突变,导致由甘氨酸变为半胱氨酸.构建了其逆转病毒表达载体(pD53M),并将该载体导入没有p53突变的药物敏感癌细胞系GAO.免疫组织化学证实pD53M转染的GAO细胞有突变型p53蛋白表达,说明重组逆转病毒表达载体pD53M在GAO细胞中可以稳定表达.体内和体外实验表明pD53M转染的GAO细胞对几种抗癌药的耐药性有不同程度的增加,最高增加了4倍.用定量PCR和FCM分析发现转染细胞MDR1基因表达没有增高,Pgp功能没有加强,MRP基因表达轻度增高,Mrp功能稍微加强,这提示突变型p53导致的耐药性可能与MDR1/Pgp无关,而与MRP/Mrp可能有一定关系.
The human mutant p53 gene (mt) cDNA was amplified and cloned from human lung cancer cell line GL total RNA containing p53 gene mutation by PCR and other methods. Sequence analysis confirmed that this cDNA contains the entire coding sequence of human p53 gene and appeared in codon 245. A G→T mutation resulted in the conversion of glycine to cysteine. A retroviral expression vector (pD53M) was constructed and introduced into a drug-sensitive cancer cell line GAO without p53 mutation. Immunohistochemistry confirmed the pD53M transfection. GAO cells have mutant p53 protein expression, indicating that the recombinant retroviral expression vector pD53M can be stably expressed in GAO cells. In vivo and in vitro experiments show that pD53M transfected GAO cells have increased resistance to several anticancer drugs to varying degrees , The highest increase of 4 times. Quantitative PCR and FCM analysis found that the transfected cells MDR1 gene expression did not increase, Pgp function is not enhanced, MRP gene expression is slightly increased, Mrp function is slightly strengthened, suggesting that mutant p53 caused by drug resistance It may not be related to MDR1/Pgp, but may have some relationship with MRP/Mrp.