Maxizyme对肝癌突变抑癌基因p53的抑制作用

Inhibitory effect of maxizyme on mutant-type p53 in hepatocellular carcinoma

目的:通过Maxizyme胞外、胞内及整体水平对肝癌突变抑癌基因p53的抑制作用,为肝癌的基因治疗探索一条新途径. 方法:应用计算机设计针对人肝癌突变抑癌基因p53 (mtp53)249位密码子的Maxizyme(Mz)和对照大酶asMz,应用分子克隆技术构建核酶的细胞外转录载体和真核表达载体.应用RiboMAXTM large Scale RNA production systems胞外转录出核酶和靶基因,进行细胞外切割反应,检测大酶对mtp53的切割作用,同时检测对野生型p53(wtp53)是否具有切割作用.在脂质体LipofectanfineTM2000的介导下稳定转染含有突变型p53, 且突变位点在249位密码子的人肝癌细胞MHCC97,应用Northern Blot,Western Blot等方法检测大酶在细胞内的表达及对肝癌细胞mtp53的抑制作用.建立人肝癌裸鼠皮下移植瘤模型,将荷瘤裸鼠随机分为空白对照组、空载体组和基因治疗组.观察肿瘤生长情况并记录动物死亡时间,绘制肿瘤生长曲线和生存期图.RT- PCR检测mtp53mRNA表达水平的变化. 结果:成功构建核酶基因并将其正确克隆入细胞外转录载体pBSKU6和真核载体pEGFPC1中.Mz在细胞外具有良好的切割靶基因mtp53活性,切割效率为49%,而对野生型p53无切割作用,asMz对突变型p53及野生型p53 均无明显切割作用.在脂质体LipofectAMINETM2000的介导下成功转染肝癌细胞MHCC97,嵌于U6表达系统中的Mz在细胞内高效表达.Northern Blot检测证实pEGFPMz组mtp53的mRNA水平明显下调,Mz在细胞内的切割效率为65%.Western Blot结果提示基因治疗组mtp53的蛋白表达明显下降,切割效率为67%.动物试验证实Mz可减慢裸鼠肿瘤生长速度,Mz治疗组荷瘤鼠生存期明显延长.PT—PCR检测到Mz治疗组裸鼠肿瘤组织mtp53的mRNA水平明显下调(mtp53 mRNA: 0.95±0.13 vs 1.44±0.14.1.47±0.12;P<0.05). 结论:Mz可有效切割mtp53,抑制其mRNA和蛋白质的表达,有效抑制肝癌细胞的生长.

AIM： To investigate the inhibitory effect of maxizyme on the mutant-type p53 （mtp53） gene at codon 249 in exon 7 （AGG→AGT） in cell-free system, hepatocellular carcinoma （HCC） cell line MHCC97, and nude mice bearing human HCC, and to explore a new method for gene therapy of HCC. METHODS： Anti-mtp53 and control mutant maxizyme were designed and then cloned into the vector pBSKU6 and pEGFPC1, respectively. The ^32p-labeled mtp53 transcript was the target mRNA. Cold maxizyme transcript was incubated with ^32p-labeled target RNA in cell-free system. The products were quantified by measuring the radioautographed count per minute （cpm） in 1 μL solution. The MHCC97 cells were the target cells which contained a mutation at the third-base position of codon 249 of the p53 gene（AGG→AGT）. PEGFPMz （recombinant eukaryotic vector） was transfected into MHCC97 cells by LipofectamineTM2000. The expression of mtp53 was analyzed by Northern Blot and Western Blot. The nude mice bearing human liver cancer were prepared and divided into blank control pEGFP and pEGFPMz group. The growth curve of the tumor in mice and the survival rate of mice were obsewed. The expression of mtp53 mRNA were detected by reverse transcription polymerase chain reaction （PT-PCR）. RESULTS： The established pEGFPMz had the correct structure. Maxizyme had a specific cleavage activity for mtp53 with a cleavage efficiency of 49% extracellularly, while the wild type p53 was not cleaved. The control maxizyme had no significant effect on both mutant and wile type p53. After pEGFPMz were transfected into MHCC97 cells, the expression of mtp53 mRNA and protein in pEGFPMz group were 65% and 67% respectively, which were significantly lower than those in blank control and pEGFP group （P〈0.05）. The tumor size decreased and mtp53 mRNA was down-regulated in mice treated with pEGFPMz as compared with those in mice of blank control and pEGFP group （mtp53 mRNA： 0.95±0.13 vs1.44 ± 0.14, 1.47 ± 0.12; P〈0.05）, and also the survivals of the mice in pEGFPMz were improved. CONCLUSION： Maxizyme can effectively inhibit the expression of mtp53 mRNA and protein as well as the growth of hepatocellular carcinoma cells.