Gen通过p38通路影响RA-FLS迁移及侵袭的研究

发布时间：2018-05-08 15:26

本文选题：类风湿关节炎 + 成纤维细胞样滑膜细胞　；参考：《扬州大学》2016年硕士论文

【摘要】：目的类风湿关节炎(Rheumatoid Arthritis, RA)是以滑膜炎为其基本病理改变的慢性全身性自身免疫性疾病。位于RA患者滑膜衬里层的成纤维细胞样滑膜细胞(Fibroblast-like synoviocytes, FLS)具有类似于肿瘤细胞的与细胞外基质(Extracellular matrix, ECM)的黏附、迁移和侵袭的特征,在RA的发生和发展过程中起着重要的作用。课题组先前的研究显示金雀异黄素(genistein, Gen)能够抑制CIA大鼠FLS的增殖,抑制炎症因子(TNF-α IL-1β)的分泌,并有控制CIA大鼠的关节炎症、抑制CIA大鼠关节破坏的作用。且相关文献报道显示Gen可以抑制肿瘤细胞与ECM黏附及迁移、侵袭。故本实验旨在探究Gen对RA-FLS与ECM的黏附、迁移和侵袭的影响及其可能存在的机制。方法1、以人类风湿关节炎成纤维细胞样滑膜细胞株(MH7A)作为研究对象,应用CCK8细胞活力实验研究不同浓度Gen对RA-FLS细胞活力的影响,选取对RA-FLS细胞活力无明显影响的浓度进行研究。2、通过RA-FLS与ECM类似物(Collagen Ⅰ、Fibronectin、Matrigel)的黏附,研究Gen对RA-FLS与ECM黏附的影响。3、通过细胞划痕实验、transwell细胞迁移实验研究Gen对RA-FLS迁移的影响；4、通过transwell细胞侵袭实验研究Gen对RA-FLS侵袭的影响。5、通过罗丹明标记的鬼笔环肽对RA-FLS的F-actin细胞骨架系统进行染色,观察Gen对RA-FLS肌动蛋白纤维的分布、丝状和片状伪足形成的影响。6、应用Western Blot检测相关信号通路蛋白及MMP-2的表达水平以探究其中可能存在的机制。研究结果1、细胞活力实验的结果表明Gen在20μM、40μM、60 μM组测得的细胞活力相较于空白对照组分别为98.30±2.90%、95.70±5.65%和92.70±7.01%,对RA-FLS的细胞活力均无明显影响(p0.05),故选取20 μM、40μM、 60 μM浓度的Gen进行进一步实验。2、与ECM类似物的黏附实验结果表明,与对照组相比,RA-FLS在20 μM、40 μM、60μM组与Collagen I的黏附率分别为97.43±1.18%、88.92±4.23%及78.25±4.90%,与Fibronectin的黏附率分别为95.18±2.00%、89.35±2.35%、78.43±3.27%,与Matrigel的黏附率分别为96.35±2.13%、84.96±3.21%、75.82±1.38%；Gen在40μM、60μM对RA-FLS与Collagen Ⅰ、Fibronectin、Matrigel的黏附均有明显的抑制作用(p0.05),且在60μM时均较40μM时明显(p0.05),而20μM时对其黏附无明显影响(p0.05)。3、细胞划痕实验结果表明,Gen在20μM,40 μM,60μM均可以抑制伤痕的愈合,且随着浓度的增加抑制作用越强；在transwell细胞迁移实验中, RA-FLS在20μM、40μM60μM的迁移率相较于空白对照组分别为97.10±5.16%、77.97±5.44%及51.95±5.26%；40μM、60μM的Gen对RA-FLS的迁移均有明显的抑制作用(p0.05),且60 μM时更为明显(p0.05),但201μM时对其迁移无明显影响(p0.05)。4、Transwell细胞侵袭实验结果表明,RA-FLS在20 μM,40 μM,60 μM的细胞侵袭数目相较于空白对照组分别为94.79±5.24%、64.54±10.13%及51.32±12.36%；40μM、60μM对RA-FLS的侵袭均有明显的抑制作用(p0.05),且60 μM更为明显(p0.05),而20 μM对其侵袭无明显影响(p0.05)。5、免疫荧光染色实验的结果显示Gen能够使RA-FLS肌动蛋白细胞骨架重排,使张力纤维数目增多,片状伪足和丝状伪足的数量减少,且随着浓度的增加这种作用越强,在60μM浓度最明显。6、 Western blot的结果显示,与空白对照组相比Gen在20μM,40μM.60μM对RA-FLS中p-JNK、JNK、p-ERK、ERK水平无明显影响(p0.05)；Gen在20μM浓度对RA-FLS的p-p38水平无明显影响(p0.05),但在40 μM,60μM浓度可以下调RA-FLS中p-p38水平(p0.05),而这些浓度对细胞中的p38水平则无明显影响(p0.05)；并且Gen在这些浓度对RA-FLS中MMP-2的表达水平亦无明显影响(p0.05)。结论1、Gen在40 μM,60μM浓度时可以抑制RA-FLS与ECM类似物(Collagen Ⅰ、 Fibronectin、Matrigel)的黏附、迁移和侵袭,在20μM浓度时对RA-FLS与ECM类似物(Collagen Ⅰ、Fibronectin、Matrigel)的黏附、迁移和侵袭无明显影响。2、Gen可能通过下调p-p38水平调控RA-FLS中F-actin细胞骨架纤维的重构,使张力纤维数目增多,片状伪足和丝状伪足的形成减少,从而实现其抑制RA-FLS与ECM类似物(Collagen Ⅰ、Fibronectin、Matrigel)的黏附、迁移和侵袭的作用。
[Abstract]:Objective To investigate the effects of Gen on the adhesion , migration and invasion of RA - FLS and ECM , and to investigate the effects of Gen on the adhesion , migration and invasion of RA - FLS .
4 . The effects of Gen on the invasion of RA - FLS were investigated by transwell cell invasion . The results showed that the cell viability of RA - FLS was 98.30 卤 2.90 % , 95.70 卤 5.65 % and 92.70 卤 7.01 % , respectively , and the adhesion of RA - FLS was 95.18 卤 2 . 00 % , 89.35 卤 2.35 % , 78.43 卤 3.27 % , respectively , and the adhesion rate of RA - FLS was 96.35 卤 2.13 % , 84.96 卤 3.21 % , 75.82 卤 1.38 % , respectively .
The adhesion of Gen at 40 渭M , 60 渭M to RA - FLS was significantly inhibited by adhesion of RA - FLS ( p < 0.05 ) and 40 渭M at 60 渭M ( p < 0.05 ) . The results of cell scratch test showed that Gen 20 渭M , 40 渭M , 60 渭M inhibited wound healing and increased with increasing concentration .
The mobility of RA - FLS was 97.10 卤 5.16 % , 77.97 卤 5.44 % and 51.95 卤 5.26 % , respectively .
The results of invasion and invasion of RA - FLS were 94.79 卤 5.24 % , 64.54 卤 10.13 % and 51.32 卤 12.36 % , respectively .
The results showed that Gen was able to rearrange the cytoskeletal structure of RA - FLS . The results showed that Gen was able to rearrange the cytoskeletal structure of RA - FLS . The results of immunofluorescence staining showed that Gen was able to decrease the number of RA - FLS actin , and the stronger the concentration increased . The results of Western blot showed that Gen was 20 渭M , 40 渭M and 60 渭M in RA - FLS .
The level of p - p38 in RA - FLS was not significantly affected by Gen at 20 渭M ( p < 0.05 ) , but the level of p - p38 in RA - FLS could be downregulated at 40 渭M and 60 渭M ( p < 0.05 ) , while these concentrations did not significantly affect the level of p38 in the cells ( p . 05 ) ;
Conclusion 1 , Gen may inhibit the adhesion , migration and invasion of RA - FLS and ECM analogues in RA - FLS at concentrations of 40 渭M and 60 渭M . Gen may reduce the adhesion , migration and invasion of RA - FLS and ECM analogues ( . I , Fibronectin , Matrigel ) at the concentration of 20 . m

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R593.22

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