PCSK6对类风湿关节炎和胶原诱导性关节炎滑膜细胞生物学的作用及分子机制研究

发布时间：2018-06-04 17:26

本文选题：PCSK6 + SiRNA　；参考：《济南大学》2015年硕士论文

【摘要】：目的1.探讨si RNA沉默PCSK6基因对胶原诱导性关节炎(CIA)成纤维样滑膜细胞(FLS)生物学行为的影响。2.探讨外源性PCSK6重组蛋白对类风湿关节炎(RA)成纤维样滑膜细胞生物学行为的影响及其分子机制。方法1.采用牛Ⅱ型胶原诱导大鼠CIA模型,取膝关节滑膜组织原代培养FLS;采用人工合成的大鼠P CSK6特异si RNA特异性抑制PCSK6在CIA FLS中的表达,以negative si RNA为阴性对照组,瞬时转染24 h、36 h,采用RT-q PCR法检测抑制效率,同时检测PCSK6基因沉默后各相关基因的表达;采用MTT、Transwell、细胞划痕、流式细胞术、ELISA等方法检测干扰PCSK6表达后对大鼠CIA FLS增殖、侵袭、迁移、细胞周期和相关炎性因子分泌的影响。2.采用PCSK6重组蛋白诱导RA FLS,采用MTT、Transwell、细胞划痕、流式细胞术、E LISA等方法检测PCSK6重组蛋白对RA FLS增殖、侵袭、迁移、细胞周期和凋亡以及相关炎性因子分泌的影响;采用R T-q PCR法检测PCSK6重组蛋白刺激后各相关基因的表达;采用W estern blot法检测PCSK6重组蛋白对基质金属蛋白酶M MP9以及ERK、STAT3、Wnt3a、Nodal、m TOR、NF-κB p65、NF-κB p105/p50信号转导通路的影响。采用通路抑制剂P D98059、Stattic、PDTC分别抑制ERK、STAT3、NF-κB p65通路,与PCSK6重组蛋白共同作用于RA FLS,利用MTT、Transwell、细胞划痕、流式细胞术、E LISA等方法检测ERK、STAT3、NF-κB p65通路抑制剂对PCSK6重组蛋白诱导的R A FLS增殖、侵袭、迁移、细胞周期和凋亡以及相关炎性因子分泌的影响。结果1.转染特异性si RNA-PCSK6后,干扰组与阴性对照组相比,PCSK6 m RNA水平明显被抑制(P0.001),CIA FLS的增殖受到抑制(P0.001),细胞的侵袭和迁移能力下降(P0.001),肿瘤坏死因子α(TNF-α)和白细胞介素-1β(IL-1β)分泌降低(P0.001),G0/G1期的细胞数目增多(P0.05),PCSK6下游基因CXCL9、MMP2、MMP9、NOSTRIN、HIF-1α、MPZL2、IGF2、PADI4表达均明显下降(P0.05)。2.PCSK6重组蛋白诱导RA FLS后,与空白对照组相比,150ng/m L PCSK6重组蛋白促进效果最显著,能使RA FLS的增殖速率显著提高(P0.01),细胞的迁移和侵袭能力加强(P0.001),IL-1α、IL-1β、IL-17、IL-6和TNF-α分泌均升高(P0.05),S期和G2/M期的细胞数增多(P0.01),细胞晚期凋亡率减少(P0.01),PCSK6下游基因C10ORF116、CXCL9、CXCL10、HIF-1α、IL-6、IL-10、IL-26、MMP9、NOSTRIN、TNF-α表达均明显升高(P0.001),MMP9前体和成熟的蛋白量均升高,诱导6h时可显著激活FLS中的ERK、STAT3、NF-κB p65信号转导通路。加入通路抑制剂P D98059、Stattic和PDTC后,150ng/m L PCSK6重组蛋白诱导的R A FLS的增殖(P0.05)、迁移(P0.001)、侵袭(P0.001)、炎性因子(IL-1α、IL-1β、IL-17、IL-6和TNF-α)分泌(P0.05)均受到抑制;通路抑制剂Stattic和PDTC可以逆转PCSK6重组蛋白对细胞周期的诱导(P0.05);通路抑制剂PDTC可以逆转PCSK6重组蛋白对F LS的细胞凋亡的影响(P0.05)。结论1.内源性PCSK6基因沉默对CIA FLS的生物学行为具有一定抑制作用,为进一步研究C IA发病机制奠定基础。2.外源性PCSK6重组蛋白对RA FLS的增殖、迁移、侵袭和炎症因子分泌等生物学行为具有一定促进作用;P CSK6通过参与ERK、STAT3、NF-κB p65信号转导通路来调控RA FLS的生物学行为。PCSK6可以作为研究RA发病机制、药物筛选以及免疫治疗的新靶点。
[Abstract]:Objective 1. to investigate the effect of Si RNA silencing of PCSK6 gene on the biological behavior of collagen induced arthritis (CIA) fibroid synovial cells (FLS).2. to explore the effect of exogenous PCSK6 recombinant protein on the biological behavior of rheumatoid arthritis (RA) fibroid synovial cells and its molecular mechanism. Method 1. using bovine type II collagen induced CIA model in rats The knee joint synovial tissue was cultured for FLS, and the P CSK6 specific Si RNA was used to inhibit the expression of PCSK6 in CIA FLS, and negative Si RNA was used as a negative control group. The inhibitory efficiency was detected by the transient transfection of 24 h, 36, and the expression of the related genes after the silence gene was detected. L, cell scratch, flow cytometry, ELISA and other methods were used to detect the proliferation, invasion, migration, cell cycle and related inflammatory factors of CIA FLS in rats after interference of PCSK6 expression..2. was used to induce RA FLS by PCSK6 recombinant protein, and MTT, Transwell, cell scratch, flow cytometry, E replication and other methods were used to detect the proliferation of the recombinant protein. Invasion, migration, cell cycle and apoptosis, and the effect of related inflammatory factors; the expression of the related genes were detected by the R T-q PCR method and the W estern blot method was used to detect the M MP9 and ERK of the matrix metalloproteinase. The pathway inhibitor P D98059, Stattic, and PDTC inhibit ERK, STAT3, NF- kappa B p65 pathway respectively, and the PCSK6 recombinant protein co acted on RA FLS. Results after 1. transfection of specific Si RNA-PCSK6, the level of PCSK6 m RNA was significantly inhibited (P0.001), the proliferation of CIA FLS was inhibited (P0.001), cell invasion and migration energy decreased (P0.001), tumor necrosis factor alpha (TNF- alpha) and white blood cells, compared with the negative control group. The secretion of -1 beta (IL-1 beta) decreased (P0.001), and the number of cells in the G0/G1 phase increased (P0.05), and the downstream genes of the PCSK6 were CXCL9, MMP2, MMP9, NOSTRIN, HIF-1 alpha. The migration and invasion ability of cells increased (P0.001), IL-1 alpha, IL-1 beta, IL-17, IL-6 and TNF- alpha increased (P0.05), and the number of cells in S and G2/M increased (P0.01). Significantly increased (P0.001), MMP9 precursor and mature protein content increased, and induced 6h to significantly activate ERK, STAT3, NF- kappa B p65 signal transduction pathway in FLS. Beta, IL-17, IL-6 and TNF- alpha) secretion (P0.05) were inhibited; pathway inhibitors Stattic and PDTC could reverse the cell cycle induction (P0.05) of the recombinant protein of PCSK6; the pathway inhibitor PDTC could reverse the effect of PCSK6 recombinant protein on the apoptosis of F LS. In order to further study the pathogenesis of C IA, the basis for the further study of the pathogenesis of C IA can promote the biological behavior of RA FLS, such as proliferation, migration, invasion and secretion of inflammatory factors, and P CSK6 can be used to regulate the biological behavior of ERK, STAT3, NF- kappa B. To study the pathogenesis of RA, drug screening and new targets of immunotherapy.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.22

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