中文摘要：

Wnt信号的异常持续激活可导致肝癌的发生发展,跨膜受体Frizzled-7在绝大多数肝癌临床肿瘤组织中呈现高度表达特征.为探索可溶性跨膜受体sFZD7在肝癌细胞中的抗癌活性,本研究主要通过分子克隆手段构建sFZD7的重组慢病毒表达载体,实现其在肝癌细胞Mahlavu中的超量表达,并通过抗生素筛选获得稳定表达sFZD7的肝癌细胞株,从而为研究sFZD7在肝癌中的生物学功能奠定基础.具体方法是首先提取人正常肝细胞HL-7702的总RNA,通过RT-PCR获取sFZD7 cDNA片段,分别将其插入慢病毒表达载体p LVX-IRES-Zs Green1与p LVX-Puro中,并通过酶切鉴定及测序分析以验证该慢病毒重组表达质粒的成功构建.其次,包装sFZD7重组慢病毒,并将制备的重组慢病毒悬液感染人胚肾HEK293T细胞,分别通过荧光显微镜与Western blot技术检测sFZD7的表达.最后,通过病毒感染与嘌呤霉素筛选获取稳定表达sFZD7的Mahlavu细胞株.实验结果表明,通过分子生物学手段已经成功地获得sFZD7慢病毒重组表达质粒p LVX-IRES-Zs Green1-sFZD7与p LVX-Puro-sFZD7;与此同时,已获得sFZD7重组慢病毒,感染HEK293T细胞后已通过荧光显微镜技术及Western blot证实了sFZD7的正确表达;此外,通过该sFZD7重组慢病毒感染肝癌Mahlavu细胞并经嘌呤霉素筛选手段成功地获得了稳定表达sFZD7的肝癌细胞株.上述结果可为sFZD7在肝癌细胞中的生物学功能与分子机制后续研究奠定基础.

英文摘要：

WNT/β-catenin signaling contributes to embryonic development and may lead to disease including cancer. Aberrant activation of WNT/β-catenin signaling is an important molecular signaling event in the tumorigenesis of hepatocellular carcinoma（HCC）. Consequently, targeting the Wnt signaling pathway may be an emerging strategy in prevention and treatment of liver cancer. Frizzled-7（FZD7） highly overexpresses in clinical HCC tissues, although it is reported that s FZD7, the extracellular domain of Frizzled-7, may antagonize FZD7-mediated Wnt signaling in some cancer cells. So far the anticancer potential of s FZD7 in liver cancer remains largely unknown. This study investigated the lentiviral expression and stable hepatocellular carcinoma Mahlavu cell line of s FZD7. Firstly, the s FZD7 c DNA frag ments were amplified from total RNA of human liver cells HL-7702 by RT-PCR and subsequently inserted into the lentivirus expression vectors p LVX-IRES-Zs Green1 and p LVX-Puro, respectively. Then the recombinant lentiviral expression plasmids were confirmed by double enzyme digestion and sequencing. Secondly, the recombinant lentiviral particles for s FZD7 were obtained by Lenti-XTM lentiviral expression system and the overall titers were evaluated by f luorescence microscopy and Western blot in the infected HEK293 T cells. Finally, Mahlavu cells were infected with the recombinant lentiviral particles, the stable s FZD7-transducted cell line then successfully established by puromycin selection and identified by Western blot.