中文摘要：

目的探讨siRNA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。方法采用牛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siRNA oligo-433干扰剂、siRNA oligo-688干扰剂、siRNA oligo-2506干扰剂特异性抑制PCSK6在CIA FLS中的表达,阴性siRNA为阴性对照组,瞬时转染24、36 h后,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表达后对细胞增殖、侵袭、迁移、细胞周期和相关炎性因子分泌的影响。结果转染特异性siRNA-PCSK6后,各干扰组与阴性对照组相比,siRNA oligo-2506干扰剂转染24 h时,PCSK6 mRNA水平抑制效果显著（P〈0.001）,CIA FLS的增殖受到抑制（P〈0.001）,细胞的侵袭和迁移能力下降（P〈0.001）,肿瘤坏死因子α（TNF-α）和白细胞介素-1β（IL-1β）分泌降低（P〈0.001）,G_0/G_1期的细胞数目增多（P〈0.05）,PCSK6下游基因CXCL9、MMP2、MMP9、NOSTRIN、HIF-1α、M PZL2、IGF2、PADI4表达均明显下降（P〈0.05）。结论 PCSK6基因沉默对CIA FLS的生物学行为具有一定作用,为进一步研究CIA发病机制奠定基础。

英文摘要：

Objective To explore the effects of PCSK6 silence by siRNA on the biological behaviors of fibroblast-like synoviocytes（ FLS） in collagen-induced arthritis（ CIA）. Methods Rat CIA models were induced with bovine collagen type II. Cultured FLS from CIA models were treated with anti-PCSK6 siRNA,including siRNA oligo-433,siRNA oligo-688 and siRNA oligo-2506. A negative siRNA was used as the negative control. The inhibiting effects were detected with RT-q PCR after 24 h and 36 h. The proliferation,invasion,migration capacity,secretion of inflammation factors and cell cycle were determined with MTT,transwell,wound healing,ELISA and flowcytomertry.Expressions of genes related to proliferation,invasion,migration and inflammation were detected with RT-q PCR. Results Compared with negative control,the inhibiting effect was remarkable after anti-PCSK6 siRNA（ siRNA oligo-2506） was transfected into FLS at 24 h（ P〈0. 001）. PCSK6-siRNA treatment significantly decreased cell proliferation（ P〈0. 001）,invasion（ P〈0. 001） and migration（ P〈0. 001） of FLS in CIA,as well as secretion of tumor necrosis factor alpha（ TNF-α） and IL-1β（ P〈0. 001）. Flowcytometry revealed a G_0/ G_1 arrest of FLS（ P〈0. 05）. Expression of genes,including CXCL9,MMP-2,MMP-9,NOSTRIN,HIF-1α,MPZL2,IGF-2 and PADI4 all decreased significantly（ P〈0. 05）. Conclusion Inhibition of PCSK6 may play a protective role in the biological behaviors of FLS in CIA,which may provide information for further study of the pathogenesis of CIA.