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目的 探讨野生型 /突变型RASSF1A基因的表达对人肝癌细胞株QGY 770 3在体内外生长的影响。方法 构建突变型RASSF1A基因的真核表达载体 ,通过脂质体介导的基因转染法将野生型 /突变型RASSF1A基因的真核表达载体及空载体转染肝癌细胞株QGY 770 3 ,G418筛选稳定表达株 ,并以逆转录 聚合酶链反应 (RT PCR)和Westernblot进行鉴定。通过细胞生长曲线、软琼脂集落形成试验和裸鼠致瘤性试验对各稳定表达株的生物学行为进行检测。结果 成功建立稳定表达野生型和突变型RASSF1A基因的肝癌细胞株。以转染空载体的QGY 770 3细胞为对照 ,野生型RASSF1A基因的表达可明显抑制肝癌细胞在体外的生长 ,显著降低肝癌细胞在软琼脂中形成的克隆数目 (P <0 .0 1)和大小 ,显著减慢裸鼠皮下瘤的生长速度和重量 (P <0 .0 1)。突变型RASSF1A基因的表达对肝癌细胞的生长影响不大。结论 野生型RASSF1A的重表达有助于QGY 770 3恶性表型的逆转 ;野生型RASSF1A基因是一个肝癌相关的抑癌基因。
Objective To investigate the effect of wild type / mutant RASSF1A gene expression on the growth of human hepatoma cell line QGY 770 3 in vitro and in vivo. Methods The eukaryotic expression vector of mutant RASSF1A gene was constructed. The eukaryotic expression vector and empty vector of wild type / mutant RASSF1A gene were transfected into hepatoma cell lines QGY 770 3 and G418 by liposome-mediated gene transfection Strains were stably expressed and identified by reverse transcription polymerase chain reaction (RT PCR) and Western blotting. The biological behavior of each stable expression strain was tested by cell growth curve, soft agar colony formation assay and nude mouse tumorigenicity test. Results The hepatoma cell lines stably expressing wild-type and mutant RASSF1A genes were successfully established. The expression of wild-type RASSF1A gene was significantly inhibited by QGY 7703 cells transfected with empty vector, which significantly inhibited the growth of hepatocarcinoma cells in vitro and significantly reduced the number of colonies formed by hepatoma cells in soft agar (P <0.01) and Size, significantly slowed down the growth rate and weight of the nude mouse’s subcutaneous tumor (P <0 01). The expression of mutant RASSF1A gene had little effect on the growth of HCC cells. Conclusion The re-expression of wild-type RASSF1A contributes to the reversal of the malignant phenotype of QGY7703. The wild-type RASSF1A gene is a hepatocellular carcinoma-related tumor suppressor gene.