论文部分内容阅读
目的探讨U6嵌和型Maxizyme对肝癌突变抑癌基因p53的抑制作用。方法应用计算机设计针对肝癌突变抑癌基因p53(mtp53)249位密码子(AGG→AGT)Maxizyme(Mz)的基因片段,构建其真核表达载体,使抗mtp53的Mz嵌于U6表达系统中,细胞外由T7 RNA聚合酶转录,细胞内由RNA聚合酶Ⅲ高效转录。检测Mz在细胞外对mtp53的切割效率。在LipofectamineTM2000介导下转染肝癌细胞MHCC97,应用逆转录聚合酶链反应(RT-PCR)检测Mz对肝癌细胞mtp53的抑制作用,western blot分析mtp53蛋白表达水平,并用四甲基偶氮唑盐(MTT)法观察肿瘤细胞的生长情况。结果在细胞外,Mz可特异性切割靶基因mtp53,切割效率为42%,而野生型p53(wtp53)没有被切割。RT-PCR和western blot检测结果提示转染Mz的MHCC97内mtp53 mRNA和蛋白(5.3 ×104)表达均明显下降,转染Mz的MHCC97增殖速度明显降低。结论U6表达系统能高效启动核酶等小分子RNA的转录,U6嵌和型Maxizyme在细胞内外均可特异性抑制mtp53表达,抑制肝癌细胞增殖,有望开发为肝癌基因治疗的新方法。
Objective To investigate the inhibitory effect of U6-embedded Maxizyme on the p53 tumor suppressor gene in liver cancer. Methods The gene fragment of mutated tumor suppressor p53 (mtp53) 249 (AGG→AGT) Maxizyme (Mz) was designed by computer to construct its eukaryotic expression vector and the anti-mtp53 Mz was embedded in the U6 expression system. It is transcribed extracellularly by T7 RNA polymerase and efficiently transcribed within the cell by RNA polymerase III. The efficiency of mtp53 cleavage by Mz outside the cell was examined. The MHCC97 cells were transfected into liver cancer cells by LipofectamineTM2000. The inhibitory effect of Mz on hepatoma cells mtp53 was detected by reverse transcription polymerase chain reaction (RT-PCR). The expression of mtp53 protein was detected by western blot and tetramethyl azolinium salt was used. MTT assay was used to observe the growth of tumor cells. As a result, Mz specifically cleaves the target gene mtp53 extracellularly, with a cleavage efficiency of 42%, whereas wild-type p53 (wtp53) is not cleaved. The results of RT-PCR and western blot indicated that the expression of mtp53 mRNA and protein (5.3 × 104) was significantly decreased in Mz-transfected MHCC97 cells, and the proliferation of MHCC97 transfected with Mz was significantly decreased. Conclusion The U6 expression system can effectively start the transcription of small RNA such as ribozymes. U6 inlay Maxizyme specifically inhibits the expression of mtp53 both inside and outside the cell, inhibits the proliferation of hepatoma cells, and is expected to be a new method for gene therapy of liver cancer.