【作者】 杨帆；

【导师】 王向晖；

【作者基本信息】 华东师范大学 ， 生物物理学， 2018， 硕士

【摘要】 前期研究发现0.4 mT工频磁场(Power Frequency Magnetic Field,PFMF)辐照可以通过激活表皮生长因子受体(Epidermal Growth Factor Receptor,EGFR)信号通路来诱发人羊膜成纤维细胞(Human amniotic epithelial,FL)迁移的应激反应,但尚不清楚此反应是否也涉及到与迁移密切相关的另一个细胞骨架组成—黏着斑信号通路的激活。本文先从与黏着斑相关的EGFR信号通路出发,利用免疫荧光染色和western-blotting方法检测了0.4 mT PFMF对FL细胞内黏着斑信号系统中的调控蛋白FAK、Paxillin的分布、含量和部分关键蛋白活性的影响;并通过细胞划痕实验验证了该磁场通过激活EGFR相关的黏着斑信号通路参与了PFMF激活细胞的迁移反应。实验结果显示,相较于假辐照组,PFMF辐照,1)导致黏着斑总数量上升,并诱导信号蛋白FAK与Paxillin更集中地分布在细胞边缘处,并且新生的黏着斑与新出现的片状伪足有共生关系;2)导致Paxillin的总含量增加,FAK蛋白总含量无显著性变化,但其Y397磷酸化水平明显上调;3)PFMF引发的这些变化均与EGFR的配体表皮生长因子(Epidermal Growth Factor,EGF)对细胞迁移机制产生的效果类似;4)分别用PD153035(PD)抑制EGFR的活性,或用FAK Inhibitor 14(FI)抑制Y397-FAK磷酸化,PFMF或EGF引起的上述现象均消失;5)细胞划痕实验结果表明,PFMF与EGF均促进了细胞的迁移,但当Y397-FAK被FI抑制后,PFMF对细胞迁移的促进效果消失。接着从另一条与黏着斑相关的Integrin信号通路出发,初步探究PFMF对Integrin信号通路中的Integrinβ1、Talin、Vinculin的分布和蛋白含量的影响,并通过细胞划痕实验验证了该磁场通过激活Integrin相关的黏着斑信号通路参与了PFMF激活细胞的迁移反应。结果显示,相较于假辐照组,PFMF辐照,1)导致Integrin信号通路中的Integrinβ1、Talin、Vinculin趋向边缘化且含量增加;2)用Integrin广谱抑制剂GLPG0187(G)抑制Integrin后,PFMF引起的黏着斑蛋白边缘化且含量增加的现象均消失;3)细胞划痕实验结果表明,当Integrin被G抑制后,PFMF对细胞迁移的促进效果全部消失。上述结果表明,一方面0.4 mT工频磁场可以通过EGFR信号通路调控黏着斑蛋白FAK、Paxillin的分布和含量以及FAK激活状态的变化,另一方面也可能同时通过Integrin信号通路来调控相关黏着斑蛋白Integrinβ1、Talin和Vinculin的分布和含量的变化,从而激活黏着斑信号通路,在细胞前端协助片状伪足的延展,促进细胞的黏着,参与到PFMF诱发的细胞迁移的分子机制中。更多还原

【Abstract】 In the previous researches,we have found that the 0.4 mT power frequency magnetic fields(PFMF)promoted the cytoskeleton reconstruction and motility of human amniotic epithelial(FL)cells by activating the epidermal growth factor receptor(EGFR)signaling pathway.However,it is not clear whether this reaction also involves the activation of another cytoskeleton component-the focal adhesion signaling pathway that is also closely involved in the cell migration.This paper starts from EGFR signaling pathway which is related with focal adhesion.The distributions,expression and activities of significant regulatory proteins in focal adhesion signaling pathway such as FAK,Paxillin were detected by using immunofluorescence and western blotting methods.Cell migration was evaluated to verify how PFMF affects the reaction of cell motility through activating FAK.The results showed that,compared to the sham group,PFMF exposure induced 1)the total amount of focal adhesion spots increased,the significant signaling protein FAK and Paxillin were more concentrated at the edges of cells and had symbiotic relationship with flake pseudopodia;2)the total content of Paxillin increased;the FAK protein level had no significant change,but its Y397 phosphorylation level increased significantly.3)These changes caused by PFMF were similar to that of EGFR ligand epidermal growth factor(EGF)did.4)inhibitions by either PD153035(PD)to inhibit the activity of EGFR or FAK Inhibitor 14(FI)to inhibit the phosphorylation of Y397-FAK,all results mentioned above induced by either PFMF or EGF disappeared;5)Cell scratch experimental results showed that similar to the function of EGF,PFMF also promoted cell migration,but the promoting effects of PFMF on cell migration was almost entirely disappeared when Y397-FAK was inhibited by FI.Then this paper makes a preliminary study of effects of PFMF on another focal adhesion signaling pathway-Integrin.The distributions,contents of significant regulatory proteins in Integrin signaling pathway such as Integrinβ1、Talin、Vinculin were detected.Cell migration assay was performed to verify how PFMF affects the reaction of cell motility through Integrin.The results show that,compared to the sham group,PFMF exposure induced 1)signaling proteins in integrin pathway such as Integrinβ1,Talin and Vinculin were also found concentrated at the cell edges and increased.2)the phenomena of focal adhesion proteins distributing at cell edges and increasing contents which were induced by PFMF disappeared when using GLPG0187(G)to inhibite Integrin;3)Cell scratch experimental results showed that the promoting effects of PFMF on cell migration were entirely disappeared when Integrin was inhibited by G.The above results suggested that 0.4 mT PFMF regulates the formation and distribution of FAK,Paxillin focal adhesion proteins by activating EGFR and probably through Integrin simultaneously to regulate the distributions and contents of Integrinβ1,Talin and Vinculin to activate Integrin focal adhesion signaling pathway,thus promoting cell adhesion and migration,so as to participate in the stress reaction mechanism of PFMF–induced cell motility along with the reactions of the actin cytoskeleton.更多还原