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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. Methods The antisense hsc70 eukaryotic expression plasmid pXAhsc70 was constructed by recombinant DNA technology. Lipofectamine-mediated transfection of the recombinant plasmid into MCF 7/Adr human breast cancer cells (mutant p53) and the presence of foreign DNA in the transfected cells was confirmed by PCR. The inhibition of hsc70 protein was detected by Western blot. P5 3 stability assay analyzes the change in half-life of mutant p53 by antisense hsc70 RNA. Results The establishment of a cell line MAc70 stably expressing antisense hsc70 RNA reduced the expression of hsc70 protein by 42%, decreased the intracellular mutant p53 content, shortened the half-life, and decreased the stability. Conclusion Antisense hsc70 RNA can significantly reduce the stability of mutant p53.