摘要
目的构建重组逆转录病毒载体pMSCV-ANGPTL4,并检测该重组逆转录病毒载体所介导的人ANGPTL4基因的抗肿瘤作用。方法将ANGPTIA cDNA亚克隆至逆转录病毒载体质粒 pMSCV。用脂质体法将重组病毒pMSCV-ANGPTL4转染HepG2细胞。流式细胞术和荧光显微镜检测 HepG2细胞绿色荧光蛋白的表达。RT-PCR法检测ANGPTL4 mRNA的表达。检测HepG2细胞体内外生长情况。结果重组病毒pMSCV-ANGPTL4和空病毒pMSCV的病毒滴度分别为1．4×106和 1．5×106感染性病毒颗粒／ml。表达GFP的HepG2-ANGPTL4细胞组和HepG2-pMSCV细胞组的阳性细胞率分别为68．45％和77．72％。HepG2-ANGPTL4细胞组和HepG2-pMSCV细胞组的平均荧光强度比对照组HepG2细胞分别高31．68倍和64．87倍。HepG2-ANGPTL4细胞组ANGPTL4 mRNA的表达分别是HepG2-pMSCV细胞组和HepG2细胞组的154％和161％。HepG2-ANGPTL4细胞组的体外增殖速度较之HepG2-pMSCV细胞组和HepG2细胞组明显降低(P<0．01)。HepG2-ANGPTL4细胞荷瘤裸鼠肿瘤的平均体积和重量均较HepG2-pMSCV细胞组和HepG2细胞组明显降低(P<0．01)。结论获得稳定转染ANGPTL4基因的人肝癌细胞株HepG2-ANGPTL4。重组逆转录病毒载体所介导的人 ANGPTL4基因转移很可能是一种有效的肝癌基因治疗方式。
Objective To construct recombinant retroviral vector containing human hepatocellular carcinoma-related gene ANGPTL4 （angiopoietin-like 4 ） cDNA and to evaluate antitumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfer. Methods ANGPTL4 cDNA was cloned in vitro from human liver cell lines HL-7702 and subcloned into plasmid vector pMSCV and sequenced. High-titer recombinant retrovirus pMSCV-ANGPTL4 and blank retrovirus pMSCV packaged under mediation of lipofectamine infected HepG2 cells in vitro, respectively. Flow cytometry and fluorescence microscopy detected expression of GFP （green fluorescence protein） in HepG2 cells. The expression of ANGPTL4 mRNA in HepG2 cells was determined. Results Recombinant retroviral vector pMSCV- ANGPTL4 was constructed successfully, Titer of recombinant retrovirus pMSCV-ANGPTL4 packaged is 1.4 x 10^6 infective viral grains/ml, Titer of blank retrovirus pMSCV packaged was 1.5 x 10^6 infective viral grains/ml. Positive cell rate of HepG2-ANGPTL4 cells group expressing GFP was 68.45% , and average intensity of fluorescence of HepG2-ANGPTL4 cells group was 31. 67 -fold as that of HepG2 cells group. Positive cell rate of HepG2-pMSCV cells group expressing GFP was 77.72% , and average intensity of fluorescence of HepG2-pMSCV cells group was 64. 87 -fold as that of HepG2 cells group. The expression of ANGPTL4 mRNA in HepG2-ANGPTL4 cells group was higher than that in HepG2-pMSCV cells group （154%） and HepG2 cells group（ 161% ）. The proliferation rate of HepG2-ANGPTL4 cells group in vitro was lower than HepG2-pMSCV cells group and HepG2 cells group（P 〈 0. 01 ）. The mean volume and weight of tumors in HepG2-ANGPTL4 cells bearing nude mice were lower than HepG2 cells group and HepG2- pMSCV cells group （P 〈 0. 01 ）. Conclusions A stable ANGPTL4-transfected human liver cancer cell line HepG2-ANGPTL4 was obtained and gene transfer of human ANGPTL4 mediated by retroviral vector is possibly effective against liver cancer.
出处
《中华普通外科杂志》
CSCD
北大核心
2006年第1期51-54,共4页
Chinese Journal of General Surgery
