肝再生磷酸酶-1稳定转染肝癌细胞株的建立及其生物学功能  

Establishment of phosphatase of regenerating liver-1 stably-transfected HCC cell lines and the biological functions in Huh7 cells

作　　者：金少文[1] 王开美 徐康[1] 徐鋆耀[1] 褚忠华[3] 王捷[1] 

机构地区：[1]中山大学孙逸仙纪念医院肝胆外科,广州510120 [2]中山大学孙逸仙纪念医院儿科,广州510120 [3]中山大学孙逸仙纪念医院胃肠外科,广州510120

出　　处：《中华实验外科杂志》2015年第2期302-305,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81372565）;广东省科技攻关计划资助项目（2008A030201005）;巾山大学临床研究5010项目（2010009）

摘　　要：目的 构建肝再生磷酸酶-1（PRL-1）真核表达载体,建立稳定过表达PRL-1的肝细胞癌Huh7细胞株,研究PRL-1在Huh7细胞中的生物学功能.方法 应用反转录-聚合酶链反应（RT-PCR）技术从人肝癌组织总RNA中扩增PRL-1基因编码区域（CDS）全长序列,与双酶切的pMSCV-PIG质粒连接,经PCR及测序鉴定pMSCV-血细胞凝集素（HA）-PRL-1载体构建成功.用293T细胞包装病毒,并感染Huh7细胞,1 mg/L嘌呤霉素持续加压筛选2周后,荧光显微镜、流式细胞仪、实时荧光定量聚合酶链反应（FQ-PCR）及Western blot检测PRL-1在Huh7中的表达.PRL-1稳定转染细胞株构建成功后,用细胞计数试剂盒（CCK-8）法进行细胞增殖实验、平板克隆形成实验和Transwell侵袭实验研究PRL-1在Huh7细胞中的生物学功能.结果 测序结果显示克隆的PRL-1基因CDS序列完全正确,且正确插入到pMSCV-PIG载体中,荧光显微镜及流式细胞仪检测结果显示细胞稳定转染效率在90％以上,FQ-PCR结果显示PRL-1稳定转染组其mRNA表达是对照组的（4.4±1.5）倍（P＜0.05）,Western blot结果显示PRL-1在Huh7细胞中过表达.CCK-8细胞增殖实验结果显示PRL-1促进Huh7细胞增殖（P＜0.01）.PRL-1稳定转染细胞2周克隆形成率为（26.7±1.9）％,明显高于对照组[（7.3±0.8）％,P＜0.01].PRL-1稳定转染组较对照组侵袭细胞数量增多（P＜0.01）.结论 PRL-1肝细胞癌Huh7稳定转染细胞株构建成功,PRL-1促进Huh7细胞增殖、克隆形成及侵袭.Objective To construct phosphatase of regenerating liver-1 （PRL-1） eukaryotic expression vector,establish stably-transfected Huh7 cell lines and investigate biological functions of PRL-1 in Huh7 cells.Methods The coding sequences （CDS） fragment of PRL-1 was amplified by reverse transcriptase-polymerase chain reaction （RT-PCR）.Bgi Ⅱ/Xho Ⅰ restriction sites were introduced into the flank of the target fragment.Then,pMSCV-Hemagglutinin （HA）-PRL-1 vector was constructed by cloning the target fragment into pMSCV-PIG plasmid.PCR and DNA sequencing verified recombinant plasmid was successfully constructed.Huh7 cells were infected by retrovirus supernatant which was packaged using 293T cells.Following infection,cells were selected with 1 mg/L puromycin for 2 weeks.Then the expression of PRL-1 in Huh7 cells was detected by fluorescence microscope,flow cytomety,real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） and Western blotting.Biological functions of PRL-1 in Huh7 cells were investigated by cell counting kit-8 （CCK-8） assay,plate colony formation assay and Transwell invasion assay.Results DNA sequencing demonstrated target fragment inserted into pMSCV-PIG vector was exactly correct.Fluorescence microscope and flow cytometry showed stably-transfected efficiency was over 90%.FQ-PCR assay showed the mRNA level of PRL-1 in overexpression group was （4.4 ±-1.5） times as compared with that in control group （P 〈 0.05）.Western blotting assay indicated PRL-1 was exogenously overexpressed in Huh7 cells.Compared with control cells,the proliferation rate increased in the PRL-1 stable transfected cells （P 〈 0.01）.Colony formation efficiency was higher in PRL-1 stably tranfected cells [（26.7 ± 1.9） % vs.（7.3 ± 0.8） %,P 〈 0.01],and overexpression group had much stronger invasive capability （P 〈 0.01）.Conclusion PRL-1 stably-transfected Huh7 cell line is successfully established and PRL-1 enhances proliferation,colony formation and invasion in Huh7 cells

关 键 词：肝细胞癌 肝再生磷酸酶-1 反转录病毒载体 生物学功能 

分 类 号：R735.7[医药卫生—肿瘤]