C反應(yīng)蛋白對(duì)心肌細(xì)胞基質(zhì)金屬蛋白酶-10表達(dá)調(diào)控的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-08-19 21:21
【摘要】：背景：基質(zhì)金屬蛋白酶-10(Matrix metallopeptidase 10, MMP-10),又稱基質(zhì)分解素-2 (stromelysin 2,SL-2),是基質(zhì)金屬蛋白酶家族的成員之一,屬于鋅離子依賴的蛋白水解酶,其生物學(xué)功能主要是參與正常生理和疾病過程的細(xì)胞外基質(zhì)(Extracellular matrix, ECM)的重構(gòu)。有關(guān)MMP-10在心臟病心室重構(gòu)中的作用及其信號(hào)轉(zhuǎn)導(dǎo)通路目前還不清楚。我們?cè)谙惹暗难芯恐邪l(fā)現(xiàn),心力衰竭患者的心肌組織的MMP-10水平與左室舒張末期直徑呈正相關(guān)關(guān)系,提示MMP-10可能與心室重構(gòu)關(guān)系密切。我們還發(fā)現(xiàn)在大鼠心肌梗死后早期,心肌MMP-10的表達(dá)水平即可明顯升高,而心梗后早期為炎癥期。因此,我們提出假說(shuō),即炎癥因子可能促進(jìn)MMP-10的表達(dá)。為了驗(yàn)證這一假說(shuō),我們觀察了一些心梗后發(fā)生反應(yīng)的主要炎癥因子對(duì)心肌細(xì)胞MMP-10表達(dá)的影響。預(yù)實(shí)驗(yàn)的結(jié)果顯示,C反應(yīng)蛋白(C-reactive protein, CRP)對(duì)心肌細(xì)胞MMP-10的表達(dá)上調(diào)作用最為明顯。而CRP作為重要的炎性細(xì)胞因子,可直接參與心梗的整個(gè)病理生理過程。在心梗后的早期,可促進(jìn)參與心室重構(gòu)的分子的產(chǎn)生,從而促進(jìn)心梗后由炎癥期向增殖期的轉(zhuǎn)化,間接地促進(jìn)心室重構(gòu)。由此可見,MMP-10可能在炎癥引起的心梗后心室重構(gòu)過程中起著重要作用。本課題在此基礎(chǔ)上重點(diǎn)研究和闡明CRP促進(jìn)心肌細(xì)胞MMP-10表達(dá)的信號(hào)轉(zhuǎn)導(dǎo)通路及相關(guān)的調(diào)控機(jī)制。 方法：(1)細(xì)胞培養(yǎng)：1-3d SD大鼠,取其心臟,用胰蛋白酶消化法原代培養(yǎng)乳鼠心肌細(xì)胞。(2)CRP對(duì)心肌細(xì)胞MMP-10表達(dá)的影響：用CRP刺激心肌細(xì)胞,收集細(xì)胞培養(yǎng)上清,用酪蛋白酶譜法觀察心肌細(xì)胞培養(yǎng)上清中MMP-10的活性變化；提取細(xì)胞總RNA,應(yīng)用熒光實(shí)時(shí)定量RT-PCR技術(shù)觀察心肌細(xì)胞中MMP-10的mRNA表達(dá)變化：提取細(xì)胞總蛋白,用蛋白免疫印跡雜交技術(shù)觀察心肌細(xì)胞中MMP-10的蛋白表達(dá)變化。(3)參與CRP誘導(dǎo)MMP-10表達(dá)變化的相關(guān)信號(hào)通路：應(yīng)用特異的信號(hào)通路抑制劑,用蛋白免疫印跡雜交技術(shù)檢測(cè)MMP-10的蛋白表達(dá)變化,并使用特異的信號(hào)分子的磷酸化抗體,用蛋白免疫印跡雜交技術(shù)檢測(cè)其信號(hào)通路的激活情況。(4)轉(zhuǎn)錄因子對(duì)MMP-10表達(dá)變化的作用：提取細(xì)胞核蛋白,應(yīng)用濾板法檢測(cè)轉(zhuǎn)錄因子的DNA結(jié)合激活情況,并使用相應(yīng)轉(zhuǎn)錄因子的siRNA,進(jìn)一步分析轉(zhuǎn)錄因子對(duì)MMP-10的調(diào)節(jié)作用。 結(jié)果：(1)確定了CRP對(duì)MMP-10的上調(diào)作用。不同濃度CRP刺激心肌細(xì)胞24h,均與對(duì)照組比較,以5μg/ml CRP對(duì)細(xì)胞培養(yǎng)上清中MMP-10的活性增加的最高,比活為27076.34±138.78(mg·ml-1)-1(p0.01,n=3)；以5μg/ml CRP作用不同時(shí)間,刺激24h的細(xì)胞培養(yǎng)上清的MMP-10活性增加最高,比活為10897.51+136.56 (mg. ml-1)-1(p0.01,n=3):MMP-10 mRNA水平在12h達(dá)高峰(p0.01,n=3)；MMP-10的蛋白水平呈現(xiàn)時(shí)間依賴性,24h可達(dá)到對(duì)照組的3.18倍(p0.01,n=3)。(2)進(jìn)一步確定了參與CRP誘導(dǎo)心肌MMP-10表達(dá)的信號(hào)轉(zhuǎn)導(dǎo)通路。分別使用相應(yīng)信號(hào)通路的特異性抑制劑,只有使用ERK1/2和JAK1的抑制劑能明顯阻斷CRP對(duì)MMP-10的表達(dá)上調(diào)。使用特異性磷酸化抗體來(lái)檢測(cè)各信號(hào)分了的磷酸化水平的激活效應(yīng),結(jié)果發(fā)現(xiàn),CRP可激活c-Raf/MEK/ERK級(jí)聯(lián)信號(hào)轉(zhuǎn)導(dǎo)通路的各信號(hào)分子的磷酸化水平,而相應(yīng)的非磷酸化水平均不改變。另外,CRP還可激活JAK1和STAT3的Ser727[STAT3(S727)]的磷酸化水平,而相應(yīng)的非磷酸化水平均不改變。使用JAK1的抑制劑可以降低ERK1/2的磷酸化水平,抑制MMP-10的蛋白表達(dá)和活性；使用ERK1/2信號(hào)通路抑制劑可以阻斷STAT3 (S727)磷酸化水平的升高,抑制MMP-10的蛋白表達(dá)和活性。因此,c-Raf/MEK/ERK級(jí)聯(lián)信號(hào)轉(zhuǎn)導(dǎo)通路和JAK1/ERK信號(hào)通路共同參與調(diào)節(jié)CRP對(duì)MMP-10的表達(dá)上調(diào)。(3)從核蛋白水平,明確轉(zhuǎn)錄因子AP-1和STAT3的結(jié)合活性。以5μg/ml CRP分別作用6h,12h和24h,AP-1的DNA結(jié)合活性,均有所增加,其中12h達(dá)高峰(p0.05,n=3)；STAT3的DNA結(jié)合活性,也均有所增加,其中12h達(dá)高峰(p0.01,n=3)。使用ERK1/2的抑制劑U0126既可以抑制AP-1的DNA結(jié)合活性(p0.05,n=3),也可以抑制STAT3的DNA結(jié)合活性(p0.01,n=3),從而明確了ERK1/2與轉(zhuǎn)錄因了AP-1和STAT3之間的關(guān)聯(lián)。(4)使用siRNA技術(shù),進(jìn)一步明確轉(zhuǎn)錄因子AP-1和STAT3在CRP誘導(dǎo)MMP-10上調(diào)過程中的調(diào)節(jié)作用。分別轉(zhuǎn)染AP-1siRNA和STAT3siRNA干擾24h后,轉(zhuǎn)錄因了AP-1和STAT3的結(jié)合活性以及MMP-10的活性和表達(dá)均可被抑制。 結(jié)論：(1)明確了CRP促進(jìn)心肌細(xì)胞MMP-10表達(dá)上調(diào)的作用。(2)首次詳細(xì)地闡明了心肌細(xì)胞MMP-10表達(dá)調(diào)控的信號(hào)轉(zhuǎn)導(dǎo)通路：CRP通過激活c-Raf/MEK/ERK級(jí)聯(lián)信號(hào)轉(zhuǎn)導(dǎo)通路和JAK1/ERK信號(hào)通路,促進(jìn)轉(zhuǎn)錄因子AP-1和STAT3入核與MMP-10的啟動(dòng)子區(qū)域相應(yīng)的結(jié)合位點(diǎn)相結(jié)合,誘導(dǎo)心肌細(xì)胞MMP-10的表達(dá)上調(diào)。其中,ERK1/2在CRP誘導(dǎo)心肌細(xì)胞MMP-10上調(diào)的過程中,是關(guān)鍵的交點(diǎn),對(duì)MMP-10的表達(dá)調(diào)控起到至關(guān)重要的作用。鑒于MMP-10在心室重構(gòu)過程中發(fā)揮的重要作用,闡明MMP-10在心肌細(xì)胞中的信號(hào)通路,可以幫助我們有效地對(duì)MMP-10的活性和表達(dá)進(jìn)行控制,從而改善甚至逆轉(zhuǎn)心肌梗死和心力衰竭引起的心室重構(gòu)及心臟病的預(yù)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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1
本文編號(hào)：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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 魏英杰;胡盛壽;李君;張曉玲;崔傳玨;黃銀霞;沈雅;黃潔;張浩;;MMP-7、MMP-10和TIMP-4在心力衰竭心室重構(gòu)中的表達(dá)[J];中國(guó)病理生理雜志;2009年03期
，本文編號(hào)：2192858
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