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目的为了解肿瘤抑制基因p53和Rb基因与肝癌对药物敏感性之间的关系,本实验用逆转录病毒介导将野生型p53或Rb基因导入人肝癌细胞株SMMC7721并观察其药物敏感性的改变。方法将野生型p53cDNA或RbcDNA克隆在逆转录病毒载体pLXSN,转染人肝癌细胞株SMMC7721,筛选阳性克隆,同时以空载体pLXSN和反义p53基因作为对照,免疫组化检测p53或Rb基因的表达。加入化疗药物48h后掺入3H-TdR,18h后检测CPM值。结果成功地构建了分别含野生型p53、Rb或反义p53的逆转录病毒载体,转染SMMC7721细胞系后获得了p53或Rb的表达;与亲代7721细胞相比,p53或Rb的表达阳性的7721细胞的生长速度均明显减慢,p53表达阳性的7721细胞对阿霉素的敏感性明显增强,同样条件下,Rb在7721细胞系内的表达则对阿霉素敏感性无影响。结论野生型p53基因转染能提高人肝癌7721细胞对阿霉素的敏感性
Objective To understand the relationship between the tumor suppressor gene p53 and Rb gene and the sensitivity of hepatocarcinoma to drugs, we used retrovirus to transduce wild type p53 or Rb gene into human hepatocellular carcinoma cell line SMMC7721 and observed the change of drug sensitivity . Methods The wild-type p53 cDNA or Rb cDNA was cloned into the retroviral vector pLXSN and transfected into human hepatocellular carcinoma cell line SMMC7721. The positive clones were screened. The empty vector pLXSN and antisense p53 gene were used as controls, and the expression of p53 or Rb gene was detected by immunohistochemistry . Addition of chemotherapeutic agents 48h after 3H-TdR incorporation, CPM value after 18h. Results The retroviral vector containing wild-type p53, Rb or antisense p53 was successfully constructed and the expression of p53 or Rb was obtained after transfected into SMMC7721 cell line. Compared with the parental 7721 cells, the expression of p53 or Rb was positive 7721 cells significantly slowed down the growth of 7721 cells with p53 expression was significantly increased sensitivity to doxorubicin, under the same conditions, the expression of Rb in 7721 cell line had no effect on doxorubicin sensitivity. Conclusion Wild-type p53 gene transfection can enhance the sensitivity of human hepatocellular carcinoma cell line 7721 to doxorubicin