PRP对RA-FLS细胞迁移和侵袭的影响

发布时间：2018-06-23 19:18

本文选题：富血小板血浆 + 类风湿关节炎滑膜成纤维细胞　；参考：《扬州大学》2015年硕士论文

【摘要】：研究背景和目的：富血小板血浆(platelet-rich plasma, PRP)是一种含有大量血小板和各种生长因子的自体血浆。当PPR被凝血酶、氯化钙激活后,PRP中的α颗粒经脱颗粒的方式,释放出大量的生长因子,包括血小板衍生生长因子(Platelet-derived growth factor, PDGF)、转化生长因子(Transforming growth factor-β, TGF-β)、血管内皮生长因了(Vascular endothelial growth factor, VEGF)和胰岛素样生长因子(Insulin-like growth factor, IGF)等,而这些生长因子在细胞的增殖、分化、骨质产生、软组织愈合和胶原合成等方面起着重要的作用。类风湿关节炎(Rheumatoid arthritis, RA)是一类以炎性细胞浸润和滑膜增生为特点,最终导致关节的破坏和畸形的慢性自身免疫性疾病。滑膜样成纤维细胞(Fibroblast-like synovial cells, FLS)在RA的发生和发展过程中起着重要的作用,RA-FLS能够粘附细胞外基质,分泌金属基质蛋白酶,迁移、侵袭至关节软骨,造成关节的破坏。有研究报道血小板数目的增多和RA的疾病活动度相关,本文在主要目的是探讨PRP对RA-FLS迁移和侵袭的影响。方法：1.利用凝血酶和氯化钙活化PRP,离心后取上清用于实验；2.利用划痕实验、transwell细胞迁移和侵袭实验研究PRP对RA-FLS细胞迁移和侵袭的影响；3. 利用RA-FLS与I型胶原粘附实验研究PRP对RA-FLS与细胞外基质(extracellular matrix, ECM)粘附的影响；4.利用免疫荧光实验观察PRP对RA-FLS细胞肌动蛋白细胞骨架重排、丝状和片状伪足形成的影响；5. 利用RT-qPCR检测PRP作用后RA-FLS细胞中MMPs的mRNA的表达。结果：1.划痕实验显示PRP能够促进RA-FLS细胞的迁移；Transwell细胞迁移实验结果显示,相较于NC组,2%PRP和5%PRP组穿过小室的相对细胞数分别为2.02±0.78,2.59±0.90(P0.05)。2. Transwe 11细胞侵袭实验结果显示,相较于NC组,2%PRP和5%PRP组的细胞穿越Matrigel屏障的相对细胞数分别为1.50±0.22,2.68±0.35(P0.05)。3. RA-FLS细胞与I型胶原粘附实验显示,相较于NC组,2%PRP和5%PRP组的细胞粘附于I型胶原的相对细胞数分别为1.50±0.27,1.87±0.62(P0.05)。4.免疫荧光实验结果显示,PRP能够促进RA-FLS细胞骨架的重排,板状伪足和丝状伪足增多,张力纤维减少。5. RT-qPCR显示PRP能够促进RA-FLS上调MMP-1、MMP-3、MMP-10的mRNA的表达。结论：1.PRP促进RA-FLS细胞的迁移、侵袭和粘附；2.PRP可能通过调控RA-FLS细胞肌动蛋白骨架纤维的重排、上调MMP-1、MMP-3和MMP-10 mRNA的表达来实现其促进迁移、侵袭和黏附作用的；
[Abstract]:Background & AIM: platelet-rich plasma is a kind of autologous plasma containing lots of platelets and various growth factors. When PPR was activated by thrombin and calcium chloride, 伪 particles in PRP were degranulated, and a large number of growth factors were released. These include platelet-derived growth factor (PDGF), transforming growth factor- 尾 (TGF- 尾), vascular endothelial growth factor, growth factor (Vascular endothelial growth factor, VEGF) and insulin-like growth factor (IGF). Soft tissue healing and collagen synthesis play important roles. Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammatory cell infiltration and synovial hyperplasia, which ultimately leads to joint destruction and malformation. Fibroblast-like synovial cells (FLS) play an important role in the genesis and development of RA. RA-FLS can adhere to extracellular matrix, secrete metalloproteinases, migrate and invade articular cartilage, resulting in joint destruction. It has been reported that the increase of platelet count is related to the disease activity of RA. The main purpose of this study is to investigate the effects of PRP on RA-FLS migration and invasion. Method 1: 1. PRP was activated by thrombin and calcium chloride. The effect of PRP on the migration and invasion of RA-FLS cells was studied by scratch test. The effect of RA-FLS on the adhesion of RA-FLS to extracellular matrix (extracellular matrix,) was studied by using RA-FLS and type I collagen adhesion test. The effects of PRP on actin cytoskeleton rearrangement filamentous and lamellar pseudopodia formation in RA-FLS cells were observed by immunofluorescence assay. The mRNA expression of MMPs in RA-FLS cells treated with PRP was detected by RT-qPCR. The result is 1: 1. The results of scratch test showed that PRP could promote the migration of RA-FLS cells. The results showed that compared with NC group, the relative number of cells passing through the compartment of PRP group and 5PRP group was 2.02 卤0.78U 2.59 卤0.90 (P0.05) .2. Compared with NC group, the relative number of cells passing through Matrigel barrier was 1.50 卤0.222.68 卤0.35 (P0.05). The adhesion of RA-FLS cells to type I collagen was found to be 1.50 卤0.270.87 卤0.62 (P0.05) compared with NC group (1.50 卤0.27) and 5PRP group (P < 0.05). The results of immunofluorescence assay showed that PRP could promote the cytoskeleton rearrangement of RA-FLS cells, increase the number of platelike pseudopodia and filamentous pseudopodia, and decrease the tension fiber. RT-qPCR showed that PRP could up-regulate the expression of MMP-3 MMP-10 mRNA in RA-FLS. Conclusion: 1. PRP promotes the migration of RA-FLS cells. 2. PRP may promote the migration, invasion and adhesion of RA-FLS cells by regulating the rearrangement of actin skeleton fibers and up-regulating the expression of MMP-3 and MMP-10 mRNA in RA-FLS cells.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.22

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