乙型肝炎病毒X蛋白对MMPs及TIMPs的影响  

Effects of hepatitis B virus X protein on matrix metalloproteinases and tissue inhibitors of metalloproteinases

作　　者：柏世玉[1] 黄清玲[1] 李晖[1] 王林[1] 郑瑾[1] 林建银[1] 林旭[1] 

机构地区：[1]福建医科大学分子医学研究中心福建省高校感染与肿瘤重点实验室,福建福州350004

出　　处：《中国病原生物学杂志》2007年第4期241-246,共6页Journal of Pathogen Biology

基　　金：全国优秀博士学位论文作者专项资金资助项目(No200359);福建省重大科技基金资助项目(No2002F005);福建省高校创新团队培育计划资助项目(NoFMU-RT001)

摘　　要：目的全面分析乙型肝炎病毒X蛋白(Hepatitis B virus X protein,HBx)对基质金属蛋白酶(Matrix Metallo-proteinases,MMPs)及组织金属蛋白酶抑制物(Tissue Inhibitors of Metalloproteinases,TIMPs)的影响,探讨其在肝细胞癌的侵袭转移中的可能作用。方法PCR扩增HBVX基因并克隆入真核表达载体pcDNA3.1/HisC,重组载体及空载体分别以Lipofectamine2000转染HepG2细胞并以800μg/mlG418筛选抗性细胞克隆。以Western blot检测抗性细胞HBx表达。抽提细胞总RNA,半定量RT-PCR检测MMPs及TIMPs。收集细胞培养液上清,以明胶酶谱检测MMP2及MMP9活性,反相明胶酶谱检测TIMPs活性。结果构建了HBx重组载体pcDNA3.1-XB。该重组载体及对照空载体转染HepG2细胞后,G418筛选,分别获得抗性细胞克隆HepG2-XB及HepG2-HIS,前者经Western blot证实可表达HBx。半定量RT-PCR显示HBx可促进MMP2、7、13、14、16、17、19、23、24及TIMP1、4基因的转录,抑制MMP1、3、8、9、10、11、12、15、20及TIMP2、3基因的转录。明胶酶谱检测显示HBx可促进酶原MMP2(Pro-MMP2)及活性MMP9(Active-MMP9)表达,抑制酶原MMP9(Pro-MMP9)表达;反相明胶酶谱显示HBx可促进TIMP1、TIMP4的表达,同时抑制TIMP2及糖基化TIMP3的表达。结论HBx蛋白对MMPs、TIMPs转录表达的影响是多方面的,但这种影响在HBx促进肝癌细胞侵袭转移过程中的确切机制有待进一步阐明。Objective To comprehensively evaluate the effects of hepatitis B virus X protein （HBx） on the expressions of matrix metalloproteinases （MMPs） and tissue inhibitors of metalloproteinases （TIMPs）, then explore the potential role of HBx on invasion and metastasis of hepatocelluar carcinoma（HCC）. Methods The HBx gene was amplified by means of PCR, and cloned into the eukaryotic expression vector pcDNA3. 1/HisC. The recombinant plasmid or empty vector was separately transfected into HepG2 cells, and the drug-resistant colonies were screened by G418 at the final concentration of 800 μg/ml. Western blot analysis using anti-Xpress antibody was used to confirm the expression of HBx. Total RNA of cells were extracted, the transcriptional levels of MMPs and TIMPs were measured by semi-quantitative RT- PCR. The serum-free supernatants were harvested, the activities of MMP2 and MMP9 were measured by gelatin zymog- raphy, and the TIMPs were detected by reverse gelatin zymography. Results HBx gene recombinant plasmid, i. e. , pcDNA3. 1-XB, was successfully constructed. After transfection and G418 selection, two drug-resistant cell lines, HepG2-XB（harboring pcDNA3.1-XB） and HepG2-HIS（harboring empty vector pcDNA3.1/HisC）, were obtained separately, and the expression of HBx in HepG2-XB was confirmed by Western blot. Semi-quantitative RT-PCR showed that HBx could enhance the transcriptions of MMP2, MMP7, MMP13, MMP14, MMP16, MMP17, MMP19, MMP23, MMP24, TIMP1 and TIMP4, but inhibit that of MMP1, MMP3, MMP8, MMP9, MMP10, MMPll, MMP12, MMP15, MMP20, TIMP2 and TIMP3. Gelatin zymography revealed that HBx could promote the expression of Pro- MMP2 and the active-MMP9, but inhibit that of Pro-MMP9. Reverse gelatin zymography demonstrated that HBx could enhance the expressions of TIMP1 and TIMP4 but inhibit those of TIMP2 and glycosylated TIMP3 （gTIMP3）. Conclusion The effects of HBx on the expressions of MMPs and TIMPs are sophisticated, the exact mechanism of these effects attributing to the enhancement of th

关 键 词：乙型肝炎病毒 肝细胞癌 基质金属蛋白酶 组织金属蛋白酶抑制物 

分 类 号：R373.2[医药卫生—病原生物学]