摘要
背景与目的:肝细胞生长因子(hepatocytegrowthfactor,HGF)通过其受体c-Met的激活,在调节肿瘤侵袭和转移和血管形成中具有重要作用。NK4不仅是HGF的拮抗剂,也是血管形成的抑制剂,阻断HGF/c-Met途径和肿瘤血管的形成可成为抗肿瘤治疗的策略之一。为此,我们构建NK4基因真核细胞表达载体并进行转染研究,探讨NK4基因在胰腺癌细胞中的表达及其对胰腺癌细胞生物学特性的影响。方法:对重组pcDNA3/hNK4质粒进行酶切,将NK4基因克隆到真核表达载体pRC/CMV2,应用脂质体将重组pRC/CMV2-hNK4质粒转入胰腺癌SW1990细胞中,采用逆转录聚合酶链反应(RT-PCR)及免疫印迹(Westernblot)分别检测转染的肿瘤细胞中NK4mRNA和蛋白的表达并筛选出高表达的细胞克隆。采用Transwall小室和Matrigel侵袭小室测定转染NK4基因对胰腺癌细胞运动和侵袭的影响。结果:转导NK4基因的SW1990细胞可表达并分泌NK4,RT-PCR扩增出预期的453bp片段,Westernblot显示有50000的NK4蛋白,转染NK4基因对胰腺癌细胞SW1990的生长无抑制作用(P>0.05),但可显著抑制HGF或成纤维细胞所诱导胰腺癌细胞的运动和侵袭(P<0.01),而转染空载体则无此作用(P>0.05)。结论:转染NK4基因可抑制胰腺癌细胞的运动和侵袭,在胰腺癌抗转移治疗中可能具有重要作用。
BACKGROUND &OBJECTIVE: Hepatocyte growth factor (HGF) plays an imp ortant role in the regulation of migration, invasion,and angiogenesis of cancer via the activation of its receptor, c-Met. NK4 is not only an antagonist of HGF but also an angiogenesis inhibitor. The blockade of HGF/c-Met signal pathway a nd tumor angiogenesis may be a new strategy for cancer treatment. This study was designed to construct eukaryotic expressing vector of NK4 gene, transfer it int o human pancreatic cancer cell line SW1990, and observe the effect of transfecte d NK4 gene on the biological behaviors of SW1990 cells,and its expression in SW1 990 cells. METHODS: The recombinant of pcDNA3/hNK4 plasmid was digested by restr ictive enzyme,NK4 gene was cloned into a high effective eukaryotic expressing ve ctor pRC/CMV2, and the recombinant of pRC/CMV2-hNK4 plasmid was transiently int roduced into SW1990 cells by lipofectamine. Reverse transcriptase-polymerase ch ain reaction (RT-PCR),and Western blot were used to detect the expression of NK 4 at mRNA,and protein levels,respectively. Migration, and invasion capabilities of the transfected cells were evaluated by Transwall chamber, and Matrigel invas ion chamber, respectively. RESULTS: Expressions of NK4 gene after lipofectamine mediated transfection were observed in SW1990 cells, expected fragment of 453 bp has been amplified by RT-PCR,and Western blot analysis showed positive express ion of NK4 protein (50 KDa). NK4 gene had no inhibitory effect on the growth of SW1990 cells (2.2×105 vs 2.5×105, P >0.05), while it had significantly suppres sive effect on the migration and invasion of SW1990 cells driven by HGF or fibro blasts (P< 0.01). CONCLUSION: NK4 gene transfection may inhibit spreading and in vasion of pancreatic cancer cells, which would play an important role in the ant i-metastasis therapy for pancreatic cancer.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2004年第10期1134-1138,共5页
Chinese Journal of Cancer