腺病毒增强转铁蛋白受体介导的针对突变型p53的大酶转染可促进肝癌细胞凋亡

Maxizyme against mtp53 transfected by adenovirus enhanced transferrin receptor-mediated gene delivery systeminduced apoptosis of hepatoma cells

目的:通过腺病毒增强的转铁蛋白受体介导法(AVET)将针对突变型p53(mtp53)的大酶(Maxizyme)基因转入肝癌细胞后对肝癌细胞凋亡的影响,探索AVET用于肝癌基因治疗的可行性,为肝癌的基因治疗探索一条新途径. 方法:以带有mtp53基因的人肝癌细胞株MHCC97细胞为模型,用腺病毒增强转铁蛋白受体介导法将针对mtp53的pEGFP-Maxizyme基因和空载体pEGFP分别导入MHCC97 细胞,荧光显微镜观察细胞转染情况,逆转录聚合酶链反应(RT-PCR)检测mtp53 mRNA表达水平的变化,DNA琼脂糖凝胶电泳和流式细胞仪俭测转染后对细胞凋亡的影响. 结果:转染后48 h荧光显微镜下可见呈细胞形态的绿色荧光.收集细胞检测,实验组mtp53 mRNA表达水平与空载体组和空白对照组相比,基因扩增条带亮度明显减弱, mRNA表达水平下降(P<0.05);DNA琼脂糖凝胶电泳出现典型的凋亡梯带(DNA Ladder);流式细胞仪分析显示细胞凋亡水平增高,凋亡指数22.95%,显著高于对照组的2.37% (P<0.05). 结论:腺病毒增强转铁蛋白受体介导的基因转移系统可将pEGFP—Maxizyme有效的转染到肝癌细胞株MHCC97 中,Maxizyme在细胞内成功的切割了mtp53 mRNA,促进了肝癌细胞的凋亡,这为腺病毒增强转铁蛋白受体介导法在肝癌基因治疗中的应用提供了实验依据,也为肝癌的基因治疗提供了一条新途径.

AIM: To evaluate the influence of introduction of maxizyme against mtp53 by adenovirus enhanced transferrin receptor-mediated gene delivery system on MHCC97 cells. METHODS: Hepatoma cell line MHCC97 containing mutated p53 gene was served as a model. The maxizyme against mtp53 was transfected to the cells by adenovirus enhanced transferrin receptor-mediated gene delivery system. The level of mtp53-mRNA was detected by means of semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The apoptosis of the hepatoma cells was measured by DNA ladder assay and FCM. RESULTS: After 48 hours of transfection by AVET system, RT-PCR results indicated the expression of mtp53 mRNA in pEGFP-Maxizyme group was significantly lower than that in control group. And DNA 'ladder' with agarose gel elec-trophoresis was observed in pEGFP-maxizyme group. The result of flow cytometry exhibited apoptotic index in pEGFP-maxizyme group was 22.95%, which was higher than those in blank control group and pEGFP group. CONCLUSION: The recombinant ribozyme cDNA eukaryotic expression vector pEGFP-maxizyme can be efficiently transfected into MHCC97 cell by adenovirus enhanced transferrin receptor-mediated gene delivery system and the expression of maxizyme may inhibit the mtp53 gene expression and promote apoptosis of MHCC97 cells. The AVET system may be a useful tool in gene delivery for gene therapy of human HCC.