組織型激肽釋放酶通過表皮生長因子受體促進神經(jīng)突生長的機制研究
發(fā)布時間：2018-10-24 07:57
【摘要】：激肽釋放酶—激肽系統(tǒng)是體內(nèi)重要的調(diào)節(jié)系統(tǒng),參與了腦缺血損傷的病理生理過程。組織型激肽釋放酶(tissue kallikrein, TK)是激肽釋放酶—激肽系統(tǒng)的核心成員,可以從抑制炎癥反應(yīng)、對抗氧化應(yīng)激、減少細胞凋亡、促進血管新生、誘導(dǎo)神經(jīng)再生等環(huán)節(jié)發(fā)揮腦缺血后神經(jīng)血管的保護和修復(fù)作用。我們課題組前期的研究發(fā)現(xiàn),TK可以促進神經(jīng)元生長,可能對腦血管病后遺癥的神經(jīng)修復(fù)治療具有一定幫助。因此,本研究以人類SH-SY5Y細胞系和小鼠大腦皮層原代神經(jīng)元為離體細胞模型,將表皮生長因子受體(epidermal growth factor receptor, EGFR)、絲裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)和flotillin-2等蛋白作為研究靶點,對TK促進神經(jīng)突生長的信號轉(zhuǎn)導(dǎo)機制進行探索。第一章組織型激肽釋放酶通過表皮生長因子受體激活細胞外調(diào)節(jié)蛋白激酶1/2目的：探討TK激活EGFR、MAPK的信號轉(zhuǎn)導(dǎo)機制。方法：本研究以人類SH-SY5Y細胞系作為體外神經(jīng)元模型,采用western blot檢測TK誘導(dǎo)的EGFR、細胞外調(diào)節(jié)蛋白激酶1/2 (extracellular regulated protein kinase 1/2, ERK1/2)、p38絲裂原活化蛋白激酶(p38 mitogen-activated protein kinase, p38)、c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)等蛋白的磷酸化水平；采用免疫熒光法檢測TK干預(yù)后EGFR的細胞內(nèi)定位；本實驗應(yīng)用RNA干擾技術(shù)下調(diào)細胞EGFR后,對TK誘導(dǎo)的ERK1/2、p38、JNK等MAPK蛋白的磷酸化水平進行檢測；并應(yīng)用抑制劑PD98059阻斷ERK1/2后,對TK誘導(dǎo)的EGFR磷酸化水平進行檢測。結(jié)果：①較低濃度TK (0.0625μM、0.125μM)干預(yù)并不能提高EGFR的磷酸化水平,而較高濃度TK (0.25μM、0.5μM和1.0μM)則可以顯著激活EGFR的磷酸化；②TK激活后,EGFR大量聚集在細胞核周圍,發(fā)生了定位改變；③TK可以激活ERK1/2、p38磷酸化,兩者分別在TK干預(yù)的5min和15min達到高峰,而JNK磷酸化水平并無顯著變化；④下調(diào)SH-SY5Y細胞EGFR的蛋白水平后,TK誘導(dǎo)的ERK1/2磷酸化水平顯著降低,而p38磷酸化水平無顯著變化；⑤在PD98059預(yù)處理有效阻斷TK誘導(dǎo)ERK1/2磷酸化的同時,TK誘導(dǎo)的EGFR磷酸化水平并無顯著變化。結(jié)論：TK可以激活SH-SY5Y細胞的EGFR磷酸化,并促進EGFR向細胞核周圍發(fā)生轉(zhuǎn)位；TK可以激活ERK1/2、p38等信號蛋白磷酸化；TK通過EGFR調(diào)節(jié)下游的ERK1/2通路。第二章 組織型激肽釋放酶通過flotillin-2激活表皮生長因子受體通路目的：探討TK激活flotillin-2、EGFR、ERK1/2的信號轉(zhuǎn)導(dǎo)機制。方法：本研究以人類SH-SY5Y細胞系作為體外神經(jīng)元模型,應(yīng)用相應(yīng)抑制劑或RNA干擾技術(shù)分別阻斷緩激肽B1受體、B2受體、flotillin-2、EGFR、ERK1/2等蛋白后,采用western blot檢測TK干預(yù)后EGFR和ERK1/2磷酸化水平的變化；采用免疫共沉淀檢測TK激活狀態(tài)下flotillin-2和EGFR蛋白的結(jié)合關(guān)系；采用免疫熒光法檢測TK干預(yù)后flotillin-2和EGFR在細胞內(nèi)的定位。結(jié)果：①TK可以顯著提高SH-SY5Y細胞EGFR和ERK1/2的磷酸化水平；②阻斷緩激肽B1受體、B2受體,TK誘導(dǎo)的EGFR、ERK1/2磷酸化水平無顯著改變；③阻斷flotillin-2,TK誘導(dǎo)的EGFR、ERK1/2磷酸化水平顯著降低；④阻斷EGFR,TK誘導(dǎo)的ERK1/2磷酸化水平顯著降低；⑤阻斷ERK1/2,TK誘導(dǎo)的EGFR磷酸化水平無顯著改變；⑥使用flotillin-2抗體進行的免疫共沉淀實驗發(fā)現(xiàn),TK激活后EGFR和磷酸化EGFR均呈現(xiàn)兩條蛋白條帶；⑦免疫熒光檢測發(fā)現(xiàn),正常狀態(tài)下,flotillin-2和EGFR在細胞膜和細胞質(zhì)內(nèi)均有表達,當TK干預(yù)后,flotillin-2和EGFR大量聚集在細胞核周圍,并且兩者共定位。結(jié)論：TK不依賴緩激肽受體激活flotillin-2—EGFR—ERK1/2信號通路；flotillin-2和EGFR相互結(jié)合形成復(fù)合物,在TK的作用下,flotillin-2和EGFR的結(jié)合構(gòu)象發(fā)生了改變；TK可以促進flotillin-2—EGFR復(fù)合物向細胞核周圍轉(zhuǎn)位。第三章 組織型激肽釋放酶通過flotillin-2、表皮生長因子受體、細胞外調(diào)節(jié)蛋白激酶1/2介導(dǎo)神經(jīng)突生長目的：探討TK通過flotillin-2、EGFR、ERK1/2等蛋白介導(dǎo)神經(jīng)突生長的機制。方法：本研究以小鼠大腦皮層原代神經(jīng)元為體外細胞模型,應(yīng)用相應(yīng)抑制劑或RNA干擾技術(shù)分別阻斷緩激肽B1受體、B2受體、flotillin-2、EGFR、ERK1/2等蛋白后給予TK干預(yù),采用免疫熒光染色結(jié)合Image J軟件對神經(jīng)突的數(shù)量和平均突起長度進行檢測。結(jié)果：①TK可以顯著提高神經(jīng)突的數(shù)量和平均突起長度；②阻斷緩激肽B1受體、B2受體,神經(jīng)突的數(shù)量和平均突起長度與TK組相比無顯著統(tǒng)計學差異；③阻斷flotillin-2后給予TK干預(yù),神經(jīng)突的生長趨勢受到顯著抑制；④阻斷EGFR后,TK誘導(dǎo)的神經(jīng)突的生長趨勢被顯著抑制；⑤阻斷ERK1/2后給予TK干預(yù),神經(jīng)突的數(shù)量和平均突起長度與TK組相比顯著降低。結(jié)論：TK不依賴緩激肽受體介導(dǎo)神經(jīng)突生長；TK通過flotillin-2、EGFR、ERK1/2促進神經(jīng)突生長。
[Abstract]:The peptide-releasing enzyme-releasing peptide system is an important regulatory system in vivo, and is involved in the pathophysiological process of cerebral ischemia injury. Tissue-type peptide release enzyme (TK) is a core member of peptide-releasing enzyme-free peptide system, which can play a role in the protection and repair of neurovascular after cerebral ischemia by inhibiting inflammatory reaction, resisting oxidative stress, reducing apoptosis, promoting angiogenesis, inducing nerve regeneration, and the like. We have found that TK can promote neuronal growth and may be helpful in the treatment of cerebral vascular sequelae. Therefore, the human SH-SY5Y cell line and the primary neurons of the cerebral cortex of the mouse were isolated from the somatic cell model, and the epidermal growth factor receptor (EGFR), primary activated protein kinase (MAPK) and flotillin-2 were used as research targets. To explore the signal transduction mechanism of TK promoting neurite growth. The first chapter is to investigate the signal transduction mechanism of TK-activated EGFR and MAPK through activation of extracellular regulated protein kinase 1/ 2 by epidermal growth factor receptor. Methods: Human SH-SY5Y cell line was used as an in vitro neuronal model, and western blot was used to detect TK-induced EGFR, extracellular regulated protein kinase 1/ 2, ERK1/ 2, p38 MAPK 1/ 2, c-Jun N-terminal kinase. The phosphorylation level of JNK and other proteins was detected by immunofluorescence assay; the intracellular localization of EGFR after TK intervention was detected by immunofluorescence; after the cell EGFR was down-regulated by RNA interference technique, the phosphorylation level of MAPK protein was detected by TK-induced ERK1/ 2, Jurkat, JNK and so on, and ERK1/ 2 was blocked by inhibitor PD98059. TK-induced EGFR phosphorylation was detected. Results: The phosphorylation of EGFR was not increased by the intervention of lower concentration of TK (0.0625. mu.M, 0.125. mu.M), while higher concentration of TK (0. 25. mu.M, 0.5. mu.M and 1. 0. mu.M) could significantly activate EGFR phosphorylation; after activation of HMTK, EGFR was heavily concentrated around the nucleus, Localization was changed; KTK could activate ERK1/ 2, p38 phosphorylation, both at 5min and 15min of TK intervention, while JNK phosphorylation was not significantly changed; after downregulation of the protein level of EGFR in SH-SY5Y cells, TK-induced ERK1/ 2 phosphorylation was significantly reduced, However, there was no significant change in tau phosphorylation level, but there was no significant change in the level of EGFR phosphorylation induced by TK at the same time as PD98059 pretreatment effectively blocked TK-induced ERK1/ 2 phosphorylation. Conclusion: TK can activate the EGFR phosphorylation of SH-SY5Y cells and promote the translocation of EGFR to the nucleus; TK can activate signal protein phosphorylation of ERK1/ 2 and ERK1; TK can regulate the downstream ERK1/ 2 pathway through EGFR. In chapter II, the pathway of epidermal growth factor receptor was activated by flotillin-2, and the signal transduction mechanism of TK-activated flotillin-2, EGFR, ERK1/ 2 was discussed. Methods: Human SH-SY5Y cell line was used as an in vitro neuronal model, and the changes of EGFR and ERK1/ 2 phosphorylation level after TK intervention were detected by western blot. The binding relationship between flotllin-2 and EGFR protein was detected by immune co-precipitation, and the localization of flotillin-2 and EGFR in cells after TK intervention was detected by immunofluorescence. Results: The phosphorylation level of EGFR and ERK1/ 2 in SH-SY5Y cells could be significantly improved by HMTK, but there was no significant change in EGFR and ERK1/ 2 phosphorylation levels induced by cyclin B1 receptor, B2 receptor and TK, and the EGFR and ERK1/ 2 phosphorylation levels induced by flotillin-2 and TK were significantly decreased. The level of ERK1/ 2 phosphorylation induced by TK was significantly decreased, but there was no significant change in the level of EGFR phosphorylation induced by ERK1/ 2 and TK. The immune co-precipitation experiment using flotillin-2 antibody showed that both EGFR and phosphorylated EGFR presented two protein bands after TK activation. In normal state, flotillin-2 and EGFR were expressed in both cell membranes and cytoplasm, and after TK intervention, flotillin-2 and EGFR were clustered around the nucleus, and both were co-located. Conclusion: TK doesn't depend on slow kinin receptor to activate flotillin-2 and EGFR-ERK1/ 2 signaling pathway; flotillin-2 and EGFR bind to form complex, and the binding conformation of flotillin-2 and EGFR is changed under the action of TK; TK can promote the translocation of flotillin-2 and EGFR complex to the nucleus. The third chapter is to investigate the mechanism of TK through flotillin-2, epidermal growth factor receptor and extracellular regulated protein kinase 1/ 2 to mediate neurite growth: To investigate the mechanism of TK through flotillin-2, EGFR, ERK1/ 2 and other proteins to mediate neurite growth. Methods: The primary neurons of cerebral cortex of mouse were used as in vitro cell model, and TK intervention was given by using the corresponding inhibitor or RNA interference technique to block the protein of kinin B1 receptor, B2 receptor, flotillin-2, EGFR, ERK1/ 2, respectively. The number and average protrusion length of neurite were detected by immunofluorescence staining and Image J software. Results: KTK could significantly increase the number of neurite and the length of neurite, but the number and average neurite length of Bradykinin B1 receptor, B2 receptor and neurite were not significantly different from that of TK group, and TK intervention was given after flotillin-2 was blocked. The tendency of neurite growth was inhibited significantly; after blocking of EGFR, the growth tendency of TK-induced neurite outgrowth was inhibited significantly; the number of neurite and the average neurite length were significantly decreased compared with TK group after the blockade of ERK1/ 2. Conclusion: TK does not depend on slow kallikrein to mediate neurite outgrowth; TK promotes neurite outgrowth through flotillin-2, EGFR, ERK1/ 2.
【學位授予單位】：復(fù)旦大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R743
本文編號：2290789
[Abstract]:The peptide-releasing enzyme-releasing peptide system is an important regulatory system in vivo, and is involved in the pathophysiological process of cerebral ischemia injury. Tissue-type peptide release enzyme (TK) is a core member of peptide-releasing enzyme-free peptide system, which can play a role in the protection and repair of neurovascular after cerebral ischemia by inhibiting inflammatory reaction, resisting oxidative stress, reducing apoptosis, promoting angiogenesis, inducing nerve regeneration, and the like. We have found that TK can promote neuronal growth and may be helpful in the treatment of cerebral vascular sequelae. Therefore, the human SH-SY5Y cell line and the primary neurons of the cerebral cortex of the mouse were isolated from the somatic cell model, and the epidermal growth factor receptor (EGFR), primary activated protein kinase (MAPK) and flotillin-2 were used as research targets. To explore the signal transduction mechanism of TK promoting neurite growth. The first chapter is to investigate the signal transduction mechanism of TK-activated EGFR and MAPK through activation of extracellular regulated protein kinase 1/ 2 by epidermal growth factor receptor. Methods: Human SH-SY5Y cell line was used as an in vitro neuronal model, and western blot was used to detect TK-induced EGFR, extracellular regulated protein kinase 1/ 2, ERK1/ 2, p38 MAPK 1/ 2, c-Jun N-terminal kinase. The phosphorylation level of JNK and other proteins was detected by immunofluorescence assay; the intracellular localization of EGFR after TK intervention was detected by immunofluorescence; after the cell EGFR was down-regulated by RNA interference technique, the phosphorylation level of MAPK protein was detected by TK-induced ERK1/ 2, Jurkat, JNK and so on, and ERK1/ 2 was blocked by inhibitor PD98059. TK-induced EGFR phosphorylation was detected. Results: The phosphorylation of EGFR was not increased by the intervention of lower concentration of TK (0.0625. mu.M, 0.125. mu.M), while higher concentration of TK (0. 25. mu.M, 0.5. mu.M and 1. 0. mu.M) could significantly activate EGFR phosphorylation; after activation of HMTK, EGFR was heavily concentrated around the nucleus, Localization was changed; KTK could activate ERK1/ 2, p38 phosphorylation, both at 5min and 15min of TK intervention, while JNK phosphorylation was not significantly changed; after downregulation of the protein level of EGFR in SH-SY5Y cells, TK-induced ERK1/ 2 phosphorylation was significantly reduced, However, there was no significant change in tau phosphorylation level, but there was no significant change in the level of EGFR phosphorylation induced by TK at the same time as PD98059 pretreatment effectively blocked TK-induced ERK1/ 2 phosphorylation. Conclusion: TK can activate the EGFR phosphorylation of SH-SY5Y cells and promote the translocation of EGFR to the nucleus; TK can activate signal protein phosphorylation of ERK1/ 2 and ERK1; TK can regulate the downstream ERK1/ 2 pathway through EGFR. In chapter II, the pathway of epidermal growth factor receptor was activated by flotillin-2, and the signal transduction mechanism of TK-activated flotillin-2, EGFR, ERK1/ 2 was discussed. Methods: Human SH-SY5Y cell line was used as an in vitro neuronal model, and the changes of EGFR and ERK1/ 2 phosphorylation level after TK intervention were detected by western blot. The binding relationship between flotllin-2 and EGFR protein was detected by immune co-precipitation, and the localization of flotillin-2 and EGFR in cells after TK intervention was detected by immunofluorescence. Results: The phosphorylation level of EGFR and ERK1/ 2 in SH-SY5Y cells could be significantly improved by HMTK, but there was no significant change in EGFR and ERK1/ 2 phosphorylation levels induced by cyclin B1 receptor, B2 receptor and TK, and the EGFR and ERK1/ 2 phosphorylation levels induced by flotillin-2 and TK were significantly decreased. The level of ERK1/ 2 phosphorylation induced by TK was significantly decreased, but there was no significant change in the level of EGFR phosphorylation induced by ERK1/ 2 and TK. The immune co-precipitation experiment using flotillin-2 antibody showed that both EGFR and phosphorylated EGFR presented two protein bands after TK activation. In normal state, flotillin-2 and EGFR were expressed in both cell membranes and cytoplasm, and after TK intervention, flotillin-2 and EGFR were clustered around the nucleus, and both were co-located. Conclusion: TK doesn't depend on slow kinin receptor to activate flotillin-2 and EGFR-ERK1/ 2 signaling pathway; flotillin-2 and EGFR bind to form complex, and the binding conformation of flotillin-2 and EGFR is changed under the action of TK; TK can promote the translocation of flotillin-2 and EGFR complex to the nucleus. The third chapter is to investigate the mechanism of TK through flotillin-2, epidermal growth factor receptor and extracellular regulated protein kinase 1/ 2 to mediate neurite growth: To investigate the mechanism of TK through flotillin-2, EGFR, ERK1/ 2 and other proteins to mediate neurite growth. Methods: The primary neurons of cerebral cortex of mouse were used as in vitro cell model, and TK intervention was given by using the corresponding inhibitor or RNA interference technique to block the protein of kinin B1 receptor, B2 receptor, flotillin-2, EGFR, ERK1/ 2, respectively. The number and average protrusion length of neurite were detected by immunofluorescence staining and Image J software. Results: KTK could significantly increase the number of neurite and the length of neurite, but the number and average neurite length of Bradykinin B1 receptor, B2 receptor and neurite were not significantly different from that of TK group, and TK intervention was given after flotillin-2 was blocked. The tendency of neurite growth was inhibited significantly; after blocking of EGFR, the growth tendency of TK-induced neurite outgrowth was inhibited significantly; the number of neurite and the average neurite length were significantly decreased compared with TK group after the blockade of ERK1/ 2. Conclusion: TK does not depend on slow kallikrein to mediate neurite outgrowth; TK promotes neurite outgrowth through flotillin-2, EGFR, ERK1/ 2.
【學位授予單位】：復(fù)旦大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R743
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