调控RECK基因表达对早孕绒毛外滋养细胞MMP-2活化及细胞侵袭力的影响

目的观察RECK基因过表达对人早孕绒毛外滋养细胞系TEV-1的MMP-2活化比例及细胞侵袭力的影响。方法以脂质体Lipofectamine^TM 2000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TEV-1细胞，以获得稳定转染目的基因的TEV-1细胞株。RT-PCR、流式细胞术检测目的基因的表达；明胶酶谱法、Transwell侵袭小室实验分别检测TEV-1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质粒DNA转染的细胞毒性情况。结果目的基因RECK在TEV-1细胞mRNA和蛋白水平分别有独立表达；明胶酶谱显示重组质粒pcDNA3-RECK转染组活化的MMP-2比例均明显低于正常对照组、空载质粒转染组（均P〈0.05），Transwell侵袭实验显示重组质粒pcDNA3-RECK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〈0.01）；MTT法测重组质粒pcDNA3-RECK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。结论RECK过表达可显著减少绒毛外滋养细胞的MMP-2活化及其侵袭能力，可能参与了早孕绒毛外滋养细胞侵蚀机制。

Modulation of RECK Gene Expression and MMP-2 Activation and Invasive Ability of Extravillous Trophoblast of Early Pregnancy

Objective To investigate the effect of RECK gene overexpression on MMP-2 activation and invasive ability of extravillous trophoblast cell line TEV-1.Methods The recombinant eukaryotic expression vector pcDNA3-RECK inserted by the full length cDNA encoding human RECK gene was stably transfected into extravillous trophoblast cell line TEV-1 by LipofectamineTM 2000.The expression levels of RECK mRNA and protein in TEV-1 was detected by RT-PCR and flow cytometry respectively.MMP-2 activation and changes in invasive ability of the cells were analyzed by gelatinase zymography and transwell invasion chamber assay,respectively.The cytotoxicity of plasmid DNA transfection on TEV-1 cells was determined by using MTT assay and cell growth curve method.Results The expression of RECK mRNA and protein was detected in vitro.Gelatinase zymography showed MMP-2 activation ratio in recombinant plasmid pcDNA3-RECK group was obviously lower than that in blank plasmid pcDNA3 group and normal control group(both P<0.05).Transwell invasion assay revealed that cell number invading through Matrigel was dramatically decreased in recombinant plasmid pcDNA3-RECK group as compared with that in blank plasmid pcDNA3 group and normal control group(both P<0.01).MTT showed that no significant difference was found in cell proliferation between recombinant plasmid pcDNA3-RECK group and blank plasmid pcDNA3 group,normal control group.Conclusion Overexpression of RECK gene significantly inhibits MMP-2 activation and cell invasive ability of extravillous trophoblast,suggesting RECK is a probable mediator of trophoblast invasion of early pregnancy.