组织型激肽释放酶通过表皮生长因子受体促进神经突生长的机制研究
发布时间：2018-10-24 07:57
【摘要】：激肽释放酶—激肽系统是体内重要的调节系统,参与了脑缺血损伤的病理生理过程。组织型激肽释放酶(tissue kallikrein, TK)是激肽释放酶—激肽系统的核心成员,可以从抑制炎症反应、对抗氧化应激、减少细胞凋亡、促进血管新生、诱导神经再生等环节发挥脑缺血后神经血管的保护和修复作用。我们课题组前期的研究发现,TK可以促进神经元生长,可能对脑血管病后遗症的神经修复治疗具有一定帮助。因此,本研究以人类SH-SY5Y细胞系和小鼠大脑皮层原代神经元为离体细胞模型,将表皮生长因子受体(epidermal growth factor receptor, EGFR)、丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)和flotillin-2等蛋白作为研究靶点,对TK促进神经突生长的信号转导机制进行探索。第一章组织型激肽释放酶通过表皮生长因子受体激活细胞外调节蛋白激酶1/2目的：探讨TK激活EGFR、MAPK的信号转导机制。方法：本研究以人类SH-SY5Y细胞系作为体外神经元模型,采用western blot检测TK诱导的EGFR、细胞外调节蛋白激酶1/2 (extracellular regulated protein kinase 1/2, ERK1/2)、p38丝裂原活化蛋白激酶(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；采用免疫荧光法检测TK干预后EGFR的细胞内定位；本实验应用RNA干扰技术下调细胞EGFR后,对TK诱导的ERK1/2、p38、JNK等MAPK蛋白的磷酸化水平进行检测；并应用抑制剂PD98059阻断ERK1/2后,对TK诱导的EGFR磷酸化水平进行检测。结果：①较低浓度TK (0.0625μM、0.125μM)干预并不能提高EGFR的磷酸化水平,而较高浓度TK (0.25μM、0.5μM和1.0μM)则可以显著激活EGFR的磷酸化；②TK激活后,EGFR大量聚集在细胞核周围,发生了定位改变；③TK可以激活ERK1/2、p38磷酸化,两者分别在TK干预的5min和15min达到高峰,而JNK磷酸化水平并无显著变化；④下调SH-SY5Y细胞EGFR的蛋白水平后,TK诱导的ERK1/2磷酸化水平显著降低,而p38磷酸化水平无显著变化；⑤在PD98059预处理有效阻断TK诱导ERK1/2磷酸化的同时,TK诱导的EGFR磷酸化水平并无显著变化。结论：TK可以激活SH-SY5Y细胞的EGFR磷酸化,并促进EGFR向细胞核周围发生转位；TK可以激活ERK1/2、p38等信号蛋白磷酸化；TK通过EGFR调节下游的ERK1/2通路。第二章 组织型激肽释放酶通过flotillin-2激活表皮生长因子受体通路目的：探讨TK激活flotillin-2、EGFR、ERK1/2的信号转导机制。方法：本研究以人类SH-SY5Y细胞系作为体外神经元模型,应用相应抑制剂或RNA干扰技术分别阻断缓激肽B1受体、B2受体、flotillin-2、EGFR、ERK1/2等蛋白后,采用western blot检测TK干预后EGFR和ERK1/2磷酸化水平的变化；采用免疫共沉淀检测TK激活状态下flotillin-2和EGFR蛋白的结合关系；采用免疫荧光法检测TK干预后flotillin-2和EGFR在细胞内的定位。结果：①TK可以显著提高SH-SY5Y细胞EGFR和ERK1/2的磷酸化水平；②阻断缓激肽B1受体、B2受体,TK诱导的EGFR、ERK1/2磷酸化水平无显著改变；③阻断flotillin-2,TK诱导的EGFR、ERK1/2磷酸化水平显著降低；④阻断EGFR,TK诱导的ERK1/2磷酸化水平显著降低；⑤阻断ERK1/2,TK诱导的EGFR磷酸化水平无显著改变；⑥使用flotillin-2抗体进行的免疫共沉淀实验发现,TK激活后EGFR和磷酸化EGFR均呈现两条蛋白条带；⑦免疫荧光检测发现,正常状态下,flotillin-2和EGFR在细胞膜和细胞质内均有表达,当TK干预后,flotillin-2和EGFR大量聚集在细胞核周围,并且两者共定位。结论：TK不依赖缓激肽受体激活flotillin-2—EGFR—ERK1/2信号通路；flotillin-2和EGFR相互结合形成复合物,在TK的作用下,flotillin-2和EGFR的结合构象发生了改变；TK可以促进flotillin-2—EGFR复合物向细胞核周围转位。第三章 组织型激肽释放酶通过flotillin-2、表皮生长因子受体、细胞外调节蛋白激酶1/2介导神经突生长目的：探讨TK通过flotillin-2、EGFR、ERK1/2等蛋白介导神经突生长的机制。方法：本研究以小鼠大脑皮层原代神经元为体外细胞模型,应用相应抑制剂或RNA干扰技术分别阻断缓激肽B1受体、B2受体、flotillin-2、EGFR、ERK1/2等蛋白后给予TK干预,采用免疫荧光染色结合Image J软件对神经突的数量和平均突起长度进行检测。结果：①TK可以显著提高神经突的数量和平均突起长度；②阻断缓激肽B1受体、B2受体,神经突的数量和平均突起长度与TK组相比无显著统计学差异；③阻断flotillin-2后给予TK干预,神经突的生长趋势受到显著抑制；④阻断EGFR后,TK诱导的神经突的生长趋势被显著抑制；⑤阻断ERK1/2后给予TK干预,神经突的数量和平均突起长度与TK组相比显著降低。结论：TK不依赖缓激肽受体介导神经突生长；TK通过flotillin-2、EGFR、ERK1/2促进神经突生长。
[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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743
本文编号：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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743
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