反义热休克蛋白hsc70对突变型p53蛋白稳定性的影响

Preliminary study of effect of antisense hsc70 effect on the stability of mutant p53 protein

目的 探讨反义hsc70RNA对突变型p5 3稳定性的影响。方法 利用DNA重组技术构建反义hsc70真核表达质粒pXAhsc70。脂质体介导将重组质粒转染至MCF 7/Adr人乳腺癌细胞 (突变型p5 3) ,经PCR确定转染细胞中外源DNA存在后 ,以Westernblot检测hsc70蛋白的抑制程度。p5 3稳定性实验分析反义hsc70RNA对突变型p5 3半衰期的改变。结果 建立稳定表达反义hsc70RNA的细胞株MAc70 ,hsc70蛋白表达下降 42 % ,其胞内突变型p5 3含量下降 ,半衰期缩短 ,稳定性下降。结论 反义hsc70RNA能够明显地降低突变型p5 3稳定性。

Objective To investigate the effect of antisense hsc70 RNA on the stability of mutant p53 protein.Methods Using DNA recombination technique, retroviral recombinant expressing antisense hsc70 RNA was constructed and transfected into MCF7/Adr cells. After the existence of foreign DNA had been identified by the polymerase chain reaction (PCR) method, the protein level of hsc70 was detected by Western blot. The half life of mutant p53 was detected by p53 stability assay.Results With the stable expressive transfectant cell strains obtained through G418 selection, the foreign DNA in transfectant cells were confirmed by PCR method and the repression rate of hsc70 protein was 42%. The half life of mutant p53 in MAc70 cell was 10 hours which was significantly lower than the control cells.Conclusion Antisense hsc70 RNA is able to lower the hsc70 protein level and significantly increase the instability of mutant p53 protein in MCF7/Adr human breast cancer cells. [