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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 investigate the effect of transforming growth factor-β1 (TGF-β1) on the secretion of membrane-type matrix metalloproteinase-1 (MT1-MMP) in murine myofibroblasts (C2C12) cells and to explore its underlying mechanism. Methods C2C12 cells were treated with different concentrations of TGF-β1 (0, 1, 5 and 10 ng / mL) for 5 h and transfected into C2C12 cells with 200 pmol / L siRNA-smad3 for 24 h. MT1-MMP and its mRNA expression. RESULTS: After treated with 0, 1, 5 and 10ng / mL TGF-β1 for 5h, the results of semi-quantitative detection of MT1-MMP by Western blot were 2.86 ± 0.28,2.05 ± 0.31,1.60 ± 0.21,1.02 ± 0.24 and F = 224.9 respectively <0.01; Northern blot qualitative results showed that the mRNA bands showed weaker. C2C12 cells were treated with 5ng / mL TGF-β1 for 4, 12 and 24 hours respectively. The results of Western-blot were 1.02 ± 0.29, 0.80 ± 0.21 and 0.4 ± 0.12, respectively, all P <0.05. Northern blot qualitative results showed that the mRNA bands showed a gradual weakening with time. C2C12 cells were transfected with 200 pmol / L siRNA-smad3 for 24 h and then treated with 5 ng / mL TGF-β1 for 5 h. Western blot results showed that: 0.82 ± 0.12 (siRNA-control + 5 ng / mL TGF- There was significant difference between the control group and the experimental group (1.61 ± 0.21in siRNA-control group and 1.84 ± 0.23in siRNA-Smad3 + 5ng / mL TGF-β1 group) (P <0.05) The results showed that: MT1-MMP mRNA expression in the experimental group and the blank control group stronger, while the control group weaker. Conclusion TGF-β1 inhibits the secretion of MT1-MMP from C2C12 cells in a dose- and time-dependent manner. SiRNA blocking Smad3 expression abolishes the inhibitory effect of TGF-β1 on MT1-MMP.