CAJ | 학술논문

目的研究Toll样受体7(TLR7)的配体Gardiquimod对HepG2细胞中血管内皮生长因子( VEGF)和基质金属蛋白酶抑制剂1(TIMP1) mRNA表达的影响,并分析其激活的信号通路。方法HepG2细胞经不同时间的Gardiquimod(2μg/ml)处理后,提取细胞总 RNA 和总蛋白, RT-PCR 分析VEGF和TIMP1转录水平的变化；Western blot分析丝裂原蛋白激活激酶-胞外信号调节激酶( MAPKs-ERK1/2)信号通路,并通过MAPKs-ERK1/2特异性阻断剂PD98059阻断相应的信号途径,进而分析两者的相关性。结果HepG2细胞中表达 TLR7,但较外周血单个核细胞( PBMC )较少。Gardiquimod刺激HepG210 min后下调细胞中VEGF mRNA的表达,而刺激细胞30 min后TIMP1 mRNA的表达有明显的上调；Western blot结果显示,细胞中的ERK1/2的磷酸化水平明显降低；并且ERK1/2的特异性抑制剂( PD98059)能有效抑制HepG2细胞中ERK1/2磷酸化作用。结论TLR7激活能够下调HepG2细胞中VEGF的表达,并与ERK1/2信号通路相关；同时 TLR7激活上调 TIMP1的表达,但与ERK1/2信号通路无相关性。
목적연구Toll양수체7(TLR7)적배체Gardiquimod대HepG2세포중혈관내피생장인자( VEGF)화기질금속단백매억제제1(TIMP1) mRNA표체적영향,병분석기격활적신호통로。방법HepG2세포경불동시간적Gardiquimod(2μg/ml)처리후,제취세포총 RNA 화총단백, RT-PCR 분석VEGF화TIMP1전록수평적변화；Western blot분석사렬원단백격활격매-포외신호조절격매( MAPKs-ERK1/2)신호통로,병통과MAPKs-ERK1/2특이성조단제PD98059조단상응적신호도경,진이분석량자적상관성。결과HepG2세포중표체 TLR7,단교외주혈단개핵세포( PBMC )교소。Gardiquimod자격HepG210 min후하조세포중VEGF mRNA적표체,이자격세포30 min후TIMP1 mRNA적표체유명현적상조；Western blot결과현시,세포중적ERK1/2적린산화수평명현강저；병차ERK1/2적특이성억제제( PD98059)능유효억제HepG2세포중ERK1/2린산화작용。결론TLR7격활능구하조HepG2세포중VEGF적표체,병여ERK1/2신호통로상관；동시 TLR7격활상조 TIMP1적표체,단여ERK1/2신호통로무상관성。
Objective To investigate the effect of Toll-like receptor 7 ( TLR7 ) ligand Gardiquimod on vascular en-dothelial growth factor (VEGF) mRNA and matrix metallo-proteinase inhibitor 1(TIMP1) mRNA in HepG2 cells, and to analyze their activiated signal transduction pathways. Methods Extracted total RNA and total protein of cul-tured HepG2 cells, which were treated with Gardiquimod (2 μg/ml) for different time. The mRNA expression of VEGF and TIMP1 was measured by Real-time PCR, and analyzed the ERK1/2 signal transduction pathway by u-sing Western blot. And to use the specific inhibitor of ERK1/2(PD98059) to block corresponding signaling path-ways, then the correlation was analyzed. Results TLR7 was expressed in HepG2 cells, but less than that in pe-ripheral blood mononuclear cells( PBMC) . After the HepG2 cells were treated with Gardiquimod for 10 min, the mRNA of VEGF in the cells was obviously reduced. However, when the cells were treated with Gardiquimod for 30 min, the mRNA of TIMP1 in HepG2 cells was obviously increased. Western blot results indicated that the phospho-rylation of ERK1/2 in the cells was obviously downregulated. What's more, the specific inhibitor of ERK1/2 ( PD98059) could effectively suppress the phosphorylation of ERK1/2 in HepG2 cells. Conclusion TLR7 activa-tion downregulates the expression of VEGF in HepG2 cells, and associates with ERK1/2 signal pathway;TLR7 ac-tivation raises the expression of TIMP1 at the same time, which is not related with ERK1/2 signal pathway.