野生型Parkin基因对肝癌细胞生长的影响

Effect of wild type parkin gene on growth of hepatocellular carcinoma cells

目的 探讨野生型及突变型Parkin基因表达对人肝癌细胞株Huh-7在体内外生长情况的影响.方法 利用脂质体介导的基因转染法将野生型及突变型Parkin基因真核表达载体转染肝癌细胞株Huh-7,筛选稳定表达细胞株,通过逆转录-聚合酶链反应（RT-PCR）进行鉴定并送测序分析.细胞增殖实验和裸鼠致瘤性实验检测各稳定表达株的体内外生长情况.结果 成功建立了稳定表达野生型和突变型Parkin基因的Huh-7细胞株.以转染空载体的Huh-7细胞作为对照,野生型Parkin基因的表达可明显抑制肝癌细胞在体外的生长（t=3.875,P=0.031）,可显著减缓裸鼠皮下瘤的生长速度并减小其体积（t=8.228,P=0.003）.突变型Parkin基因的表达对肝癌细胞的生长影响不大（P＞0.05）.结论 野生型Parkin的重表达有助于肝癌细胞恶性表型的逆转.野生型Parkin基因是一个肝癌相关的抑癌基因.

Objective To explore the effect of wild type or mutant parkin gene expression on the growth of human hepatocellular carcinoma cell line Huh-7. Methods The parkin （wild type or mutant） expression vector and empty vector were transferred into Huh-7 cell lines with LipofectAMINE 2000 reagents. The positive clones that expressed parkin gene stably were chosen by G418 and checked by reverse transcription-polymerase chain reaction （RT-PCR） to check the DNA sequences. The cytobiological behaviors of those positive clones were analyzed by cell proliferation assay and tumorigenesis in nude mice. Results Huh-7 cell lines that expressed wild type or mutant parkin gene stably were successfully established. The growth of wild type parkin-expressed cells was obviously inhibited compared with the control cells transfected with empty vectors（t= 3. 875, P= 0. 031）.The volume of tumor formed by wild type parkin-expressing cells in nude mice was also significantly reduced （t=8. 228,P=-0. 003）. Mutant parkin gene expression had a slight effect on the growth of Huh-7 cells in vitro and in vivo （P〉0.05）. Conclusion The re-expression of wild type parkin gene can favor the malignant phenotype revision of Huh-7 cells. Therefore, it might be a good candidate for tumor suppressor gene associated with HCC.