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山东大学学报（医学版） ›› 2015, Vol. 53 ›› Issue (12): 20-26.doi: 10.6040/j.issn.1671-7554.0.2014.917

• 基础医学 • 上一篇    下一篇

siRNA沉默PCSK6基因对胶原诱导性关节炎的影响

姜慧钰1,2,3,4, 王林1,2,3, 潘继红1,2,3   

  1. 1. 山东省医药生物技术研究中心, 山东济南 250062;
    2. 国家卫生部生物技术药物重点实验室, 山东济南 250062;
    3. 山东省罕少见病重点实验室, 山东济南 250062;
    4. 济南大学山东省医学科学院医学与生命科学学院, 山东济南 250200
  • 收稿日期:2014-12-08 出版日期:2015-12-10 发布日期:2015-12-10
  • 通讯作者: 潘继红。E-mail:pjh933@suhu.com E-mail:pjh933@suhu.com
  • 基金资助:
    国家自然科学基金(81102275);山东省自然科学基金(ZR2011CQ028);国家重点基础研究发展计划(2010CB529105);山东省科技发展计划(2012GSF12115)

Effects of PCSK6 silence by siRNA on FLS in CIA

JIANG Huiyu1,2,3,4, WANG Lin1,2,3, PAN Jihong1,2,3   

  1. 1. Shandong Medicinal Biotechnology Center, Jinan 250062, Shandong, China;
    2. Key Laboratory for Biotech-Drugs Ministry of Health, Jinan 250062, Shandong, China;
    3. Key Laboratory for Rare & Uncommon Diseases of Shandong Province, Jinan 250062, Shandong, China;
    4. School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan 250200, Shandong, China
  • Received:2014-12-08 Online:2015-12-10 Published:2015-12-10

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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-qPCR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),G0/G1期的细胞数目增多(P<0.05),PCSK6下游基因CXCL9、MMP2、MMP9、NOSTRIN、HIF-1α、MPZL2、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。

关键词: PCSK6, siRNA, 胶原诱导性关节炎, 类风湿关节炎, 成纤维样滑膜细胞

Abstract: 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-qPCR 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 flow cytomertry. Expressions of genes related to proliferation, invasion, migration and inflammation were detected with RT-qPCR. 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). Flow cytometry revealed a G0/G1 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.

Key words: Collagen-induced arthritis, Rheumatoid arthritis, PCSK6, siRNA, Fibroblast-like synoviocytes

中图分类号: 

  • R593.22
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