PRL-2基因转染对肝细胞基质金属蛋白酶及其抑制剂表达的影响  

Expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in hepatocellular cell lines transfected by PRL-2 gene

作　　者：程超[1] 郭爱林[2] 吴伟康[3] 罗红鹤[1] 钟佛添[1] 张萌[1] 

机构地区：[1]中山大学附属第一医院,广东广州510080 [2]广东省人民医院医学研究中心,广东广州510080 [3]中山大学基础医学院病理生理教研室,广东广州510080

出　　处：《中国病理生理杂志》2006年第11期2189-2193,共5页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30100218);广东省科技厅资助项目(No.2004B30301010)

摘　　要：目的:研究PRL-2基因增强肿瘤细胞侵袭及转移能力的机制。方法:采用脂质体转染的方法将PRL-2基因表达质粒转染至正常永生化肝细胞系CL1中,G418筛选阳性克隆。应用明胶酶谱法检测转染肝细胞分泌MMPs酶谱变化,W estern b lotting及RT-PCR检测转染肝细胞MMP-2、MMP-9、TIMP-1和TIMP-2的变化。以PRL-2磷酸酯酶特异性抑制剂处理转染细胞,观察抑制PRL-2活性对上述指标的影响。结果:经过8周G418筛选及RT-PCR和W estern b lotting鉴定,获得稳定表达PRL-2的细胞亚系PRL-2-CL1。转染后的CL1细胞分泌MMP-9、活性型MMP-9和MMP-2,均显著高于转染前CL1细胞的MMPs分泌(P<0.01);使用特异性抑制剂后,MMP-9、活性型MMP-9和MMP-2活性显著降低(P<0.01)。W estern b lotting及RT-PCR检测显示PRL-2-CL1细胞MMP2、MMP9蛋白及mRNA含量均较转染前显著升高(P<0.05),TIMP-2则显著降低(P<0.05);使用抑制剂后,可以逆转上述变化。结论:PRL-2在永生化肝细胞中获得稳定、高效表达,PRL-2基因增强肝细胞侵袭及转移能力与其提高细胞MMP2、MMP9表达,降低TIMP-2有关。AIM： To investigate the possible mechanism of PRL - 2 in invasive metastasis of tumors. METHODS ： The PRL - 2 vector was transfected into CL1 cell with lipofectamine reagent, the transfectants were selected by growth in the medium supplemented with G418. Zymographic analysis of metalloproteinases （MMPs） activity was performed, RTPCR was used to determine the mRNA levels of MMP-2, MMP - 9, TIMP - 1 and TIMP - 2, the protein levels of MMP - 2, MMP - 9, TIMP - 1 and TIMP - 2 were analyzed by Western blotting. The effects of the special inhibitor of PRL -2 on transfected cells were also observed. RESULTS： The stable cell line selected by G418 was identified by RT- PCR and Western blotting. More abundance of MMP - 2, MMP - 9 and its activated type were secreted by the CL - 1 - PRL - 2 cells than untransfected cells and transfected vector cells （ P 〈0. 01 ） , the special inhibitor of PRL - 2 could evidently decrease the secretion of MMPs by CL - 1 - PRL - 2 cells. PRL - 2 more highly expressed MMP - 2, MMP - 9 gene transcripts and reduced the mRNA level of TIMP - 2 in the CL - 1 - PRL - 2 cells than in the untransfected cells and transfected vector cells （ P 〈 0.05 ） , the special inhibitor of PRL - 2 could reverse the above results （ P 〈 0. 05 ）, CONCLUSION ： The stable cell line stably expressing human PRL -2 was established, The ability of PRL -2 to enhance invasive activity and induce metastatic tumor formation may be associated with the up - regulation of MMPs and down - regulation of TIMP - 2.

关 键 词：肝细胞 蛋白质酪氨酸磷酸酶 基因转染 基质金属蛋白酶 

分 类 号：R392.11[医药卫生—免疫学]