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目的探讨肿瘤坏死因子样凋亡微弱诱导剂(TWEAK)调控大鼠心肌成纤维细胞(CFs)表达基质金属蛋白酶2(MMP2)及Ⅰ型胶原的作用机制。方法胰酶消化法分离培养新生大鼠原代CFs,利用Western blotting法检测磷酸化ERK1/2(p-ERK1/2)蛋白的表达水平,从而确定重组人TWEAK(rh TWEAK)及ERK1/2通路抑制剂PD98059对CFs的最佳干预浓度及干预时间。采用实时荧光定量PCR法(RT-PCR)以及Western blotting法检测干预后MMP2及Ⅰ型胶原的mRNA与蛋白表达水平,采用四甲基偶氮唑蓝(MTT)法检测不同处理对细胞增殖的影响。结果 100μg/L TWEAK干预CFs显著上调p-ERK1/2的蛋白表达、上调MMP2及Ⅰ型胶原mRNA与蛋白的表达水平,同时显著促进细胞增殖。抑制剂PD98059阻断ERK1/2通路后显著抑制MMP2与Ⅰ型胶原的mRNA及蛋白表达,抑制CFs的增殖。结论 TWEAK通过ERK1/2通路促进CFs表达MMP2及Ⅰ型胶原。
Objective To investigate the mechanism of tumor necrosis factor-like weak inducers (TWEAK) regulating the expression of matrix metalloproteinase 2 (MMP2) and type Ⅰ collagen in rat cardiac fibroblasts (CFs). Methods The primary CFs of neonatal rats were isolated and cultured by trypsin digestion. The expression of phosphorylated ERK1 / 2 (p-ERK1 / 2) protein was detected by Western blotting, and the recombinant human TWEAK and ERK1 / 2 pathway Inhibitor PD98059 on CFs best intervention concentration and intervention time. The mRNA and protein expressions of MMP2 and collagen Ⅰ were detected by real-time fluorescence quantitative PCR (RT-PCR) and Western blotting. The effects of different treatments on cell proliferation were detected by MTT assay . Results CFE at 100μg / L TWEAK significantly up-regulated the protein expression of p-ERK1 / 2, up-regulated the mRNA and protein expression of MMP2 and type Ⅰ collagen, and significantly promoted cell proliferation. Inhibitory effect of PD98059 on ERK1 / 2 pathway significantly inhibited the mRNA and protein expression of MMP2 and type Ⅰ collagen, and inhibited the proliferation of CFs. Conclusion TWEAK can promote the expression of MMP2 and type Ⅰ collagen in CFs via ERK1 / 2 pathway.