转染RECK基因对人骨肉瘤MG-63细胞MMP-2活化及侵袭能力的影响

Effects of RECK Gene Transfection on MMP-2 Activation and Invasive Ability of MG-63 Cells

目的观察转染RECK基因对人骨肉瘤细胞MG-63的MMP-2活化及细胞侵袭力的影响。方法以脂质体LipofectamineTM2000介导的方法将含RECK全长基因的真核重组表达质粒pcDNA3-RECK转染入MG-63细胞,RT-PCR、流式细胞术检测目的基因的表达,明胶酶谱法、Matrigel侵袭实验分别检测MG-63细胞MMP-2活化比例及细胞侵袭力变化;四甲基偶氮唑蓝(MTT)比色法和细胞生长曲线法观察转染质粒DNA对细胞的毒性作用。结果转染后RECK基因在MG-63细胞mRNA和蛋白水平分别有稳定高表达;明胶酶谱显示重组质粒转染组MMP-2的活化比例均明显低于正常对照组、空载质粒转染组(均P<0.01),Matrigel侵袭实验显示重组质粒转染组穿透Matrigel的细胞数目均明显低于正常对照组、空载质粒转染组(均P<0.05);而MTT法测重组质粒转染组细胞生长曲线与正常对照组、空载质粒转染组无明显差异(均P>0.05)。结论RECK基因过表达可显著减少骨肉瘤细胞MG-63的MMP-2活化及其侵袭能力,RECK基因可能成为肿瘤治疗的新靶点。

Objective To investigate the effect of RECK gene transfection on MMP-2 activation and invasive ability of human osteosarcoma cell line MG-63 cells. Methods The recombinant eukaryotic expression vector pcDNA3-RECK inserted by the full length cDNA encoding human RECK gene was stably transfected into MG-63 cells by LipofectamineTM 2000. RT-PCR and flow cytometry were used to assay RECK gene expression. MMP-2 activation and invasive ability of MG-63 cells were analyzed by gelatinase zymography and Matrigel invasion assay, respectively. The cytotoxicity of plasmid DNA transfection on MG-63 cells was determined by MTT assay and cell growth curve method. Results The stable and higher expression of RECK mRNA and protein was detected in MG-63 cells. Gelatinase zymography revealed that MMP-2 activation ratio in recombinant plasmid transfected group was obviously lower than in blank plasmid transfected group and normal control group （both P〈 0.01）. Matrigel invasion assay indicated that cell number invading through Matrigel was dramatically decreased in recombinant plasmid transfected group as compared with that in blank plasmid transfected group and normal control group （both P〈0.05）. MTT showed no significant difference was found in recombinant plasmid transfected group as compared with blank plasmid transfected group and normal control group in cell growth curve （both P〉0.05）. Conclusion Over-expression of RECK gene significantly inhibited MMP-2 activation and cell invasive ability of MG-63 cells, suggesting RECK may be a new target for osteosareoma treatment.