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Wnt信号通路的异常活化与人类多种恶性肿瘤的发生密切相关,为探索作为Wnt蛋白拮抗剂sFRP(Secreted frizzled related protein)家族成员之一的Frzb/sFRP3在肝癌细胞中的抗癌潜力,本研究主要通过RT-PCR以及慢病毒表达系统进行了Frzb的cDNA克隆、重组慢病毒构建以及肝癌稳定表达细胞株的建立.从人正常肝细胞HL-7702中提取总RNA,通过RT-PCR获得Frzb cDNA片段并经测序证实;与此同时,将Frzb cDNA分别构建至以绿色荧光蛋白或嘌呤霉素抗性为报告基因的慢病毒真核表达载体pLVX-IRES-ZsGreen1-Frzb及pLVX-Puro-Frzb,通过酶切和测序获得正确的慢病毒重组表达质粒;其次,通过Lenti-X?Lentiviral Expression Systems将慢病毒重组表达质粒与慢病毒包装质粒psPAX2、pMD2.G共转染HEK293T细胞,48 h后制备重组慢病毒悬液,随后感染HEK293T细胞,分别通过荧光显微镜观察绿色荧光蛋白的表达以及Western blot验证了Frzb(FLAG标签)在细胞中的正确表达;最后,将Frzb重组慢病毒悬液感染肝癌HepG2细胞,经2μg/mL嘌呤霉素筛选成功地获得了稳定表达Frzb的细胞株.以上结果表明慢病毒表达体系对Wnt信号通路的分子靶向药物研究具有重要价值.
Abnormal activation of Wnt signaling pathway is closely related to the occurrence of many human malignancies. To explore the anti-cancer potential of Frzb / sFRP3, a member of the Secreted frizzled related protein family, in this study mainly by RT-PCR as well as lentiviral expression system of the cDNA clones Frzb the recombinant lentivirus and the establishment of cell line HCC stable expression. total RNA was extracted from human normal liver cells HL-7702, obtaining Frzb cDNA by RT-PCR Then the Frzb cDNA was constructed into lentiviral eukaryotic expression vectors pLVX-IRES-ZsGreen1-Frzb and pLVX-Puro-Frzb with green fluorescent protein or puromycin resistance as the reporter gene respectively. The lentiviral recombinant plasmids were obtained by restriction enzyme digestion and sequencing. Secondly, Lenti-X? Lentiviral Expression Systems were co-transfected with lentivirus packaging plasmid psPAX2 and pMD2.G into HEK293T cells for 48 h Recombinant lentivirus suspension was transfected into HEK293T cells and the expression of green fluorescent protein (EGFP) was observed by fluorescence microscopy. Western blot showed that Frzb (FLAG tag) was Cells were infected with Frzb recombinant lentivirus.Finally, Frzb cells were stably transfected with HepG2 cells by 2μg / mL puromycin.The above results indicated that the lentiviral expression system could effectively inhibit Wnt signaling pathway Molecular-targeted drug research is of great value.