C反应蛋白对心肌细胞基质金属蛋白酶-10表达调控的实验研究
发布时间：2018-08-19 21:21
【摘要】：背景：基质金属蛋白酶-10(Matrix metallopeptidase 10, MMP-10),又称基质分解素-2 (stromelysin 2,SL-2),是基质金属蛋白酶家族的成员之一,属于锌离子依赖的蛋白水解酶,其生物学功能主要是参与正常生理和疾病过程的细胞外基质(Extracellular matrix, ECM)的重构。有关MMP-10在心脏病心室重构中的作用及其信号转导通路目前还不清楚。我们在先前的研究中发现,心力衰竭患者的心肌组织的MMP-10水平与左室舒张末期直径呈正相关关系,提示MMP-10可能与心室重构关系密切。我们还发现在大鼠心肌梗死后早期,心肌MMP-10的表达水平即可明显升高,而心梗后早期为炎症期。因此,我们提出假说,即炎症因子可能促进MMP-10的表达。为了验证这一假说,我们观察了一些心梗后发生反应的主要炎症因子对心肌细胞MMP-10表达的影响。预实验的结果显示,C反应蛋白(C-reactive protein, CRP)对心肌细胞MMP-10的表达上调作用最为明显。而CRP作为重要的炎性细胞因子,可直接参与心梗的整个病理生理过程。在心梗后的早期,可促进参与心室重构的分子的产生,从而促进心梗后由炎症期向增殖期的转化,间接地促进心室重构。由此可见,MMP-10可能在炎症引起的心梗后心室重构过程中起着重要作用。本课题在此基础上重点研究和阐明CRP促进心肌细胞MMP-10表达的信号转导通路及相关的调控机制。 方法：(1)细胞培养：1-3d SD大鼠,取其心脏,用胰蛋白酶消化法原代培养乳鼠心肌细胞。(2)CRP对心肌细胞MMP-10表达的影响：用CRP刺激心肌细胞,收集细胞培养上清,用酪蛋白酶谱法观察心肌细胞培养上清中MMP-10的活性变化；提取细胞总RNA,应用荧光实时定量RT-PCR技术观察心肌细胞中MMP-10的mRNA表达变化：提取细胞总蛋白,用蛋白免疫印迹杂交技术观察心肌细胞中MMP-10的蛋白表达变化。(3)参与CRP诱导MMP-10表达变化的相关信号通路：应用特异的信号通路抑制剂,用蛋白免疫印迹杂交技术检测MMP-10的蛋白表达变化,并使用特异的信号分子的磷酸化抗体,用蛋白免疫印迹杂交技术检测其信号通路的激活情况。(4)转录因子对MMP-10表达变化的作用：提取细胞核蛋白,应用滤板法检测转录因子的DNA结合激活情况,并使用相应转录因子的siRNA,进一步分析转录因子对MMP-10的调节作用。 结果：(1)确定了CRP对MMP-10的上调作用。不同浓度CRP刺激心肌细胞24h,均与对照组比较,以5μg/ml CRP对细胞培养上清中MMP-10的活性增加的最高,比活为27076.34±138.78(mg·ml-1)-1(p0.01,n=3)；以5μg/ml CRP作用不同时间,刺激24h的细胞培养上清的MMP-10活性增加最高,比活为10897.51+136.56 (mg. ml-1)-1(p0.01,n=3):MMP-10 mRNA水平在12h达高峰(p0.01,n=3)；MMP-10的蛋白水平呈现时间依赖性,24h可达到对照组的3.18倍(p0.01,n=3)。(2)进一步确定了参与CRP诱导心肌MMP-10表达的信号转导通路。分别使用相应信号通路的特异性抑制剂,只有使用ERK1/2和JAK1的抑制剂能明显阻断CRP对MMP-10的表达上调。使用特异性磷酸化抗体来检测各信号分了的磷酸化水平的激活效应,结果发现,CRP可激活c-Raf/MEK/ERK级联信号转导通路的各信号分子的磷酸化水平,而相应的非磷酸化水平均不改变。另外,CRP还可激活JAK1和STAT3的Ser727[STAT3(S727)]的磷酸化水平,而相应的非磷酸化水平均不改变。使用JAK1的抑制剂可以降低ERK1/2的磷酸化水平,抑制MMP-10的蛋白表达和活性；使用ERK1/2信号通路抑制剂可以阻断STAT3 (S727)磷酸化水平的升高,抑制MMP-10的蛋白表达和活性。因此,c-Raf/MEK/ERK级联信号转导通路和JAK1/ERK信号通路共同参与调节CRP对MMP-10的表达上调。(3)从核蛋白水平,明确转录因子AP-1和STAT3的结合活性。以5μg/ml CRP分别作用6h,12h和24h,AP-1的DNA结合活性,均有所增加,其中12h达高峰(p0.05,n=3)；STAT3的DNA结合活性,也均有所增加,其中12h达高峰(p0.01,n=3)。使用ERK1/2的抑制剂U0126既可以抑制AP-1的DNA结合活性(p0.05,n=3),也可以抑制STAT3的DNA结合活性(p0.01,n=3),从而明确了ERK1/2与转录因了AP-1和STAT3之间的关联。(4)使用siRNA技术,进一步明确转录因子AP-1和STAT3在CRP诱导MMP-10上调过程中的调节作用。分别转染AP-1siRNA和STAT3siRNA干扰24h后,转录因了AP-1和STAT3的结合活性以及MMP-10的活性和表达均可被抑制。 结论：(1)明确了CRP促进心肌细胞MMP-10表达上调的作用。(2)首次详细地阐明了心肌细胞MMP-10表达调控的信号转导通路：CRP通过激活c-Raf/MEK/ERK级联信号转导通路和JAK1/ERK信号通路,促进转录因子AP-1和STAT3入核与MMP-10的启动子区域相应的结合位点相结合,诱导心肌细胞MMP-10的表达上调。其中,ERK1/2在CRP诱导心肌细胞MMP-10上调的过程中,是关键的交点,对MMP-10的表达调控起到至关重要的作用。鉴于MMP-10在心室重构过程中发挥的重要作用,阐明MMP-10在心肌细胞中的信号通路,可以帮助我们有效地对MMP-10的活性和表达进行控制,从而改善甚至逆转心肌梗死和心力衰竭引起的心室重构及心脏病的预后。
[Abstract]:BACKGROUND: Matrix metalloproteinase-10 (MMP-10), also known as stromelysin-2 (SL-2), is a member of the matrix metalloproteinase family. It belongs to zinc ion-dependent proteolytic enzymes. Its biological functions are mainly extracellular matrix involved in normal physiological and disease processes. The role of MMP-10 in cardiac ventricular remodeling and its signal transduction pathways are still unknown. We have found a positive correlation between MMP-10 levels and left ventricular end-diastolic diameter in patients with heart failure, suggesting that MMP-10 may be closely related to ventricular remodeling. To test this hypothesis, we observed the effect of some major inflammatory factors on the expression of MMP-10 in myocardial cells. Preliminary results show that C-reactive protein (CRP) plays the most significant role in the up-regulation of MMP-10 expression in myocardial cells. CRP, as an important inflammatory cytokine, can directly participate in the whole pathophysiological process of MI. In the early stage after MI, CRP can promote the production of molecules involved in ventricular remodeling and thus promote MI. It can be concluded that MMP-10 may play an important role in the process of ventricular remodeling after myocardial infarction induced by inflammation.
Methods: (1) Cell culture: 1-3 days SD rat hearts were taken and primary cultured with trypsin digestion method. (2) Effect of CRP on the expression of MMP-10 in neonatal rat cardiomyocytes: CRP stimulated cardiomyocytes, cell culture supernatants were collected, and the activity of MMP-10 in the supernatants of cardiomyocytes was observed by casein spectroscopy. A. Real-time quantitative RT-PCR was used to observe the expression of MMP-10 mRNA in cardiomyocytes. Total protein was extracted and the expression of MMP-10 protein in cardiomyocytes was observed by Western blot hybridization. (3) Signal pathways involved in CRP-induced MMP-10 expression: specific signal pathway inhibitors were used, and proteins were used. The expression of MMP-10 protein was detected by Western blot hybridization, and the activation of its signal pathway was detected by immunoblot hybridization with phosphorylated antibody of specific signal molecule. (4) The effect of transcription factors on the expression of MMP-10: nuclear protein was extracted and DNA binding activation of transcription factors was detected by filter plate method. In addition, the transcription factor siRNA was used to further analyze the regulatory effect of transcription factors on MMP-10.
Results: (1) The up-regulation effect of CRP on MMP-10 was determined. Different concentrations of CRP stimulated myocardial cells for 24 hours. Compared with the control group, the activity of MMP-10 in the supernatant of cell culture was increased by 5 ug/ml CRP, the specific activity of MMP-10 was 27076.34 (+ 138.78) mg (+ ml-1) -1 (p0.01, n = 3), and 5 ug/ml CRP stimulated the supernatant of cell culture for 24 hours. The specific activity of MMP-10 was 10897.51+136.56 (mg.ml-1) -1 (p0.01, n=3): MMP-10 mRNA levels peaked at 12 hours (p0.01, n=3); MMP-10 protein levels showed a time-dependent manner, and reached 3.18 times (p0.01, n=3) of the control group at 24 hours. (2) The signal transduction pathways involved in CRP-induced MMP-10 expression were further identified. Only the inhibitors of ERK1/2 and JAK1 significantly blocked the up-regulation of MMP-10 expression by CRP. Specific phosphorylated antibodies were used to detect the activation effect of phosphorylation levels of signal fragments. The results showed that CRP could activate the phosphorylation levels of signal molecules in the c-Raf/MEK/ERK cascade signal transduction pathway. In addition, CRP can activate the phosphorylation levels of JAK1 and STAT3 Ser727 [STAT3 (S727)] and the corresponding non-phosphorylation levels remain unchanged. Therefore, c-Raf/MEK/ERK cascade signal transduction pathway and JAK1/ERK signaling pathway are involved in regulating the up-regulation of CRP on MMP-10 expression. (3) The binding activity of transcription factors AP-1 and STAT3 is determined from the nuclear protein level. The binding activity of transcription factors AP-1 and STAT3 is regulated by 5 ug/ml CRP for 6 h, 12 h and 24 h, respectively. The DNA binding activity of AP-1 and STAT 3 increased at 12h (p0.05, n = 3), and the DNA binding activity of STAT 3 increased at 12h (p0.01, n = 3). U0126, an inhibitor of ERK1/2, could inhibit both DNA binding activity of AP-1 (p0.05, n = 3) and DNA binding activity of STAT 3 (p0.01, n = 3). Transcriptions are related to AP-1 and STAT3. (4) Using siRNA technology, we further clarified the regulatory role of transcription factors AP-1 and STAT3 in CRP-induced MMP-10 up-regulation.
Conclusion: (1) The role of CRP in promoting the up-regulation of MMP-10 expression in cardiomyocytes was clarified. (2) The signal transduction pathway regulating the expression of MMP-10 in cardiomyocytes was clarified in detail for the first time: CRP promotes the entry of transcription factors AP-1 and STAT3 into the nucleus corresponding to the promoter region of MMP-10 by activating c-Raf/MEK/ERK cascade signal transduction pathway and JAK1/ERK signal pathway. Combining binding sites induces up-regulation of MMP-10 expression in cardiomyocytes. ERK1/2 is the key intersection in CRP-induced up-regulation of MMP-10 expression and plays an important role in regulation of MMP-10 expression. It can help us effectively control the activity and expression of MMP-10, thereby improving or even reversing ventricular remodeling and prognosis of heart disease caused by myocardial infarction and heart failure.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1
本文编号：2192858
[Abstract]:BACKGROUND: Matrix metalloproteinase-10 (MMP-10), also known as stromelysin-2 (SL-2), is a member of the matrix metalloproteinase family. It belongs to zinc ion-dependent proteolytic enzymes. Its biological functions are mainly extracellular matrix involved in normal physiological and disease processes. The role of MMP-10 in cardiac ventricular remodeling and its signal transduction pathways are still unknown. We have found a positive correlation between MMP-10 levels and left ventricular end-diastolic diameter in patients with heart failure, suggesting that MMP-10 may be closely related to ventricular remodeling. To test this hypothesis, we observed the effect of some major inflammatory factors on the expression of MMP-10 in myocardial cells. Preliminary results show that C-reactive protein (CRP) plays the most significant role in the up-regulation of MMP-10 expression in myocardial cells. CRP, as an important inflammatory cytokine, can directly participate in the whole pathophysiological process of MI. In the early stage after MI, CRP can promote the production of molecules involved in ventricular remodeling and thus promote MI. It can be concluded that MMP-10 may play an important role in the process of ventricular remodeling after myocardial infarction induced by inflammation.
Methods: (1) Cell culture: 1-3 days SD rat hearts were taken and primary cultured with trypsin digestion method. (2) Effect of CRP on the expression of MMP-10 in neonatal rat cardiomyocytes: CRP stimulated cardiomyocytes, cell culture supernatants were collected, and the activity of MMP-10 in the supernatants of cardiomyocytes was observed by casein spectroscopy. A. Real-time quantitative RT-PCR was used to observe the expression of MMP-10 mRNA in cardiomyocytes. Total protein was extracted and the expression of MMP-10 protein in cardiomyocytes was observed by Western blot hybridization. (3) Signal pathways involved in CRP-induced MMP-10 expression: specific signal pathway inhibitors were used, and proteins were used. The expression of MMP-10 protein was detected by Western blot hybridization, and the activation of its signal pathway was detected by immunoblot hybridization with phosphorylated antibody of specific signal molecule. (4) The effect of transcription factors on the expression of MMP-10: nuclear protein was extracted and DNA binding activation of transcription factors was detected by filter plate method. In addition, the transcription factor siRNA was used to further analyze the regulatory effect of transcription factors on MMP-10.
Results: (1) The up-regulation effect of CRP on MMP-10 was determined. Different concentrations of CRP stimulated myocardial cells for 24 hours. Compared with the control group, the activity of MMP-10 in the supernatant of cell culture was increased by 5 ug/ml CRP, the specific activity of MMP-10 was 27076.34 (+ 138.78) mg (+ ml-1) -1 (p0.01, n = 3), and 5 ug/ml CRP stimulated the supernatant of cell culture for 24 hours. The specific activity of MMP-10 was 10897.51+136.56 (mg.ml-1) -1 (p0.01, n=3): MMP-10 mRNA levels peaked at 12 hours (p0.01, n=3); MMP-10 protein levels showed a time-dependent manner, and reached 3.18 times (p0.01, n=3) of the control group at 24 hours. (2) The signal transduction pathways involved in CRP-induced MMP-10 expression were further identified. Only the inhibitors of ERK1/2 and JAK1 significantly blocked the up-regulation of MMP-10 expression by CRP. Specific phosphorylated antibodies were used to detect the activation effect of phosphorylation levels of signal fragments. The results showed that CRP could activate the phosphorylation levels of signal molecules in the c-Raf/MEK/ERK cascade signal transduction pathway. In addition, CRP can activate the phosphorylation levels of JAK1 and STAT3 Ser727 [STAT3 (S727)] and the corresponding non-phosphorylation levels remain unchanged. Therefore, c-Raf/MEK/ERK cascade signal transduction pathway and JAK1/ERK signaling pathway are involved in regulating the up-regulation of CRP on MMP-10 expression. (3) The binding activity of transcription factors AP-1 and STAT3 is determined from the nuclear protein level. The binding activity of transcription factors AP-1 and STAT3 is regulated by 5 ug/ml CRP for 6 h, 12 h and 24 h, respectively. The DNA binding activity of AP-1 and STAT 3 increased at 12h (p0.05, n = 3), and the DNA binding activity of STAT 3 increased at 12h (p0.01, n = 3). U0126, an inhibitor of ERK1/2, could inhibit both DNA binding activity of AP-1 (p0.05, n = 3) and DNA binding activity of STAT 3 (p0.01, n = 3). Transcriptions are related to AP-1 and STAT3. (4) Using siRNA technology, we further clarified the regulatory role of transcription factors AP-1 and STAT3 in CRP-induced MMP-10 up-regulation.
Conclusion: (1) The role of CRP in promoting the up-regulation of MMP-10 expression in cardiomyocytes was clarified. (2) The signal transduction pathway regulating the expression of MMP-10 in cardiomyocytes was clarified in detail for the first time: CRP promotes the entry of transcription factors AP-1 and STAT3 into the nucleus corresponding to the promoter region of MMP-10 by activating c-Raf/MEK/ERK cascade signal transduction pathway and JAK1/ERK signal pathway. Combining binding sites induces up-regulation of MMP-10 expression in cardiomyocytes. ERK1/2 is the key intersection in CRP-induced up-regulation of MMP-10 expression and plays an important role in regulation of MMP-10 expression. It can help us effectively control the activity and expression of MMP-10, thereby improving or even reversing ventricular remodeling and prognosis of heart disease caused by myocardial infarction and heart failure.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1
【参考文献】
相关期刊论文 前1条
1 魏英杰;胡盛寿;李君;张晓玲;崔传珏;黄银霞;沈雅;黄洁;张浩;;MMP-7、MMP-10和TIMP-4在心力衰竭心室重构中的表达[J];中国病理生理杂志;2009年03期
，本文编号：2192858
本文链接：https://www.wllwen.com/xiyixuelunwen/2192858.html
最近更新
教材专著
