转化生长因子β1对肌成纤维细胞膜型基质金属蛋白酶1的影响  

Effects of siRNA Knockdown of Smad3 on TGF-β1 Mediated Inhibition of MT1-MMP in C2C12 Cells

作　　者：朱萍[1] 邓长柏[1] 

机构地区：[1]广州市第八人民医院,广州510060

出　　处：《热带医学杂志》2009年第9期1015-1018,共4页Journal of Tropical Medicine

摘　　要：目的观察转化生长因子β1(TGF-β1)对鼠肌成纤维细胞(C2C12细胞)分泌膜型基质金属蛋白酶1(MT1-MMP)的影响,探讨其内在机制。方法用不同浓度的TGF-β1(0、1、5、10ng/mL)干预C2C12细胞5h,200pmol/L siRNA-smad3转染C2C12细胞24h,用Western blot和Northern blot方法测定MT1-MMP及其mRNA表达。结果0、1、5、10ng/mLTGF-β1干预C2C12细胞5h,Western blot半定量检测MT1-MMP的结果分别为2.86±0.28、2.05±0.31、1.60±0.21、1.02±0.24,F=224.9,P<0.01;Northern blot定性结果显示,mRNA条带显示逐渐变弱。5ng/mLTGF-β1干预C2C12细胞4、12、24h,Western blot半定量检测结果分别为1.02±0.29、0.80±0.21、0.4±0.12,各时间点值比较,均P<0.05。Northern blot定性结果显示,mRNA条带显示随时间延长而逐渐变弱。200pmol/L siRNA-smad3转染C2C12细胞24h,再用5ng/mLTGF-β1干预C2C12细胞5h,半定量Western blot检测结果显示:内对照组0.82±0.12(siRNA-control+5ng/mLTGF-β1)、空白对照组1.61±0.21(siRNA-control)、实验组1.84±0.23(siRNA-Smad3+5ng/mLTGF-β1),内对照组和实验组比较差异有统计学意义(P<0.05);Northern blot定性结果显示:MT1-MMPmRNA表达在实验组和空白对照组较强,而内对照组较弱。结论TGF-β1抑制鼠C2C12细胞分泌MT1-MMP,呈剂量和时间依赖性;siRNA阻断Smad3表达可消除TGF-β1对MT1-MMP的抑制作用。Objective To evaluate the effects of Smad3 knockdown on TGF-β1 mediated suppression of MT1- MMP in C2C12 cells. Methods C2C12 cells were treated with various concentrations of TGF-β1（0,1,5 and 10 ng/mL） for 4 to 24 hrs. The expressions of MT1-MMPs proteins and their mRNA in C2C12 cells were measured with Western blot and Northern blot methods, respectively. In the transfection experiment, C2C12 cells were transfected with smad3 siRNA （200 pmol/L） for 24 hrs. Transfected cells were then treated with 5 ng/mL TGF-β1 for 5 hrs and the expressions of MT1-MMPs and their mRNA were measured. Results Results from the semi-quantitiative Western blotting analysis showed that the expression level of MT1-MMP at 5 h after TGF-β1 treatment was 2.86±0.28 （untreated）, 2.05±0.31 （1 ng/mL）, 1.60±0.21 （5 ng/mL） and 1.02±0.24 （10 ng/mL）. Dose dependent reduced in mRNA expression was also confirmed by quantitative Northern blot analysis. Protein expressions of MT1-MMPs were time dependently decreased in cells treated with 5 ng/mL of TGF-β1, and the expression value at 4 h, 12 h and 24 h was 1.02±0.29, 0.80±0.21 and 0.4±0.12, respectively （all P〈0.05）. In the smad3 knockdown study, the protein expression level in cells receiving the control siRNA （with TGF-β1 treatment）, control siRNA only and smad3 siRNA （with TGF-β1 treatment） was 0.82±0.12, 1.61±0.21 and 1.84±0.23, respectively. Conclusion TGF-β1 is effective in inhibiting MT1-MMP protein synthesis and mRNA expression in C2C12 cells in a dose and time-dependent manner. TGF-βI mediated inhibition can be prevented by knockdown of Smad3.

关 键 词：成纤维细胞 SMAD3基因 转化生长因子Β1 RNA干扰 基质金属蛋白酶 

分 类 号：Q556.9[生物学—生物化学]