您的位置：山东大学 -> 科技期刊社 -> 《山东大学学报（医学版）》

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

PDF (PC)

360

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
[1] Sylvie S, Peggy J, Jens VP, et al. A multiparameter approach to monitor disease activity in collagen-induced arthritis[J]. Arthritis Res Ther, 2010, 12(4):160.
[2] Ben Hassine H, Zemni R, Bouagina E, et al. Lack of association between PADI4 polymorphisms and rheumatoid arthritis in the Tunisian population[J]. Joint Bone Spine, 2012, 79(3):329-330.
[3] 张玲玲,沈玉先,魏伟.类风湿关节炎的关节滑膜病理生理机制[J].生理科学进展, 2003, 34(2):144-146.
[4] Trentham DE, Townes AS, Kang AH. Autoimmunity to typeⅡcollagen:an experimental model of arthritis[J]. J Exp Med, 1977, 146(3):857-868.
[5] Holmdahl R, Mo J, Nordling C, et al. Collagen induced arthritis:an experimental model for rheumatoid arthritis with involvement of both DTH and immune complex mediated mechanisms[J]. Clin Exp Rheumatol, 1989, 7(Suppl 3):51-55.
[6] 张敏娜.类风湿关节炎的实验动物模型及其评价[J].实验动物科学, 2011, 28(4):57-59.
[7] Lin H, Ah Kioon MD, Lalou C, et al. Protective role of systemic furin in immune response-induced arthritis[J]. Arthritis Rheum, 2012, 64(9):2878-2886.
[8] Wang F, Wang L, Jiang H, et al. Inhibition of PCSK6 may play a protective role in the development of rheumatoid arthritis[J]. J Rheumatol, 2015, 42(2):161-169.
[9] Seidah NG, Khatib AM, Prat A. The proprotein convertases and their implication in sterol and/or lipid metabolism[J]. Biol Chem, 2006, 387(7):871-877.
[10] 徐华,马韵,龙喜带.前蛋白转化酶与恶性肿瘤[J].肿瘤防治研究, 2013, 40(1):105-110.
[11] Mains RE, Berard CA, Denault JB, et al. PACE4:a subtilisin-like endoprotease with unique properties[J]. Biochem J, 1997, 321(Pt 3):587-593.
[12] Nour N, Mayer G, Mort JS, et al. The cysteine-rich domain of the secreted proprotein convertases PC5A and PACE4 function as a cell surface anchor and interacts with tissue inhibitors of metalloproteinases[J]. Mol Biol Cell, 2005, 16(11):5215-5226.
[13] 李建平,王晓滨,陈常忠,等.偶合基本氨基酸裂解酶4基因多态性与高血压的相关性研究[J].中华心血管病杂志, 2004, 32(9):781-784. LI Jianping, WANG Xiaobin, CHEN Changzhong, et al. The association of a polymorphism in the paired basic amino acid cleaving enzyme 4 gene and hypertension[J]. Chin J Cardiol, 2004, 32(9):781-784.
[14] Malfait AM, Seymour AB, Gao F, et al. A role for PACE4 in osteoarthritis pain:evidence from human genetic association and null mutant phenotype[J]. Ann Rheum Dis, 2012, 71(6):1042-1048.
[15] 刘元刚,刘树滔,饶平凡.类风湿关节炎啮齿动物模型的研究进展[J].中国实验动物学报, 2007, 15(6):470-473. LIU Yuangang, LIU Shutao, RAO Pingfan. Recent advances in research on rodent models of rheumatoid arthritis[J]. Acta Lab Anim Sci Sin, 2007, 15(6):470-473.
[16] 许金鑫,张育,张学增,等.金雀异黄素对CIA大鼠炎症及细胞因子影响的研究[J].实用临床医药杂志, 2010, 14(19):1-5. XU Jinxin, ZHANG Yu, ZHANG Xuezeng, et al. Effect of Gen on the inflammation and cytokines of rats with collagen type Ⅱ induced arthritis[J]. Journal of Clinical Medicine in Practice, 2010, 14(19):1-5.
[17] Lee A, Qiao Y, Grigoriev G, et al. TNFα induces sustained signaling and a prolonged and unremitting inflammatory response in synovial fibroblasts[J]. Arthritis Rheum, 2013, 65(4):928-938.
[18] Feldmann M, Maini RN. Lasker Clinical Medical Research Award. TNF defined as a therapeutic target for rheumatoid arthritis and other autoimmune diseases[J]. Nat Med, 2003, 9(10):1245-1250.
[19] Joosten LA, Helsen MM, van de Loo FA, et al. Anticytokine treatment of established type Ⅱ collagen-induced arthritis in DBA/1 mice:a comparative study using anti-TNFalpha, anti-IL-1alpha/beta and IL-1Ra[J]. Arthritis Rheum, 2008, 58(2 suppl):110-122.
[20] Kuan WP, Tam LS, Wong CK, et al. CXCL9 and CXCL10 as sensitive markers of disease activity in patients with rheumatoid arthritis[J]. J Rheumatol, 2010, 37(2):257-264.
[21] Pardo A, Selman M. Matrix metalloproteases in aberrant fibrotic tissue remodeling[J]. Proc Am Thorac Soc, 2006, 3(4):383-388.
[22] Kovacevic I, Hu J, Siehoff-Icking A, et al. The F-BAR protein NOSTRIN participates in FGF signal transduction and vascular development[J]. EMBO J, 2012, 31(15):3309-3322.
[23] Hollander AP, Corke KP, Freemont AJ, et al. Expression of hypoxia-inducible factor 1alpha by macrophages in the rheumatoid synovium:implications for targeting of therapeutic genes to the inflamed joint[J]. Arthritis Rheum, 2001, 44(7):1540-1544.
[1] 李健,徐冰,闫新峰,徐万菊,常晓天. 筛选TXNDC5与胰岛素相关信号通路关键基因的探讨[J]. 山东大学学报（医学版）, 2017, 55(3): 88-93.
[2] 夏传友,李路超,李孝峰,颜克强, 张念昭,范医东,刘承. 组蛋白甲基化酶SMYD3在前列腺癌组织的表达及对mTOR通路的影响[J]. 山东大学学报（医学版）, 2016, 54(5): 12-16.
[3] 赵琰,郑亚冰,闫新峰,张虎,常晓天. 筛选类风湿关节炎中糖代谢关键基因的探讨[J]. 山东大学学报（医学版）, 2016, 54(3): 30-35.
[4] 郑加田，朱凯，孙红胜，付敏，潘正论. IL-18和MCP-1基因多态性与类风湿关节炎易感性关系的Meta分析[J]. 山东大学学报（医学版）, 2014, 52(6): 98-104.
[5] 丁玲1,2， 巩会平1，杜贻萌1，王欣1，张岩3，王旭平2，鹿庆华1. SIRT3 siRNA对ox-LDL刺激的人主动脉内皮细胞ICAM-1、E-selectin表达的影响[J]. 山东大学学报（医学版）, 2014, 52(4): 43-48.
[6] 王介忠1，韩波1，高聆2，杨文巍1，刘奉琴1，吕建利1，赵立健1 . CD40siRNA对EAM大鼠的作用及其对CD4+CD25+Treg的影响[J]. 山东大学学报（医学版）, 2014, 52(1): 1-4.
[7] 李凤1，许菁2，郑加田1，张源潮1，孙红胜1，潘正论1. 山东地区汉族人群类风湿关节炎与CD14基因多态性的关联性[J]. 山东大学学报(医学版), 2013, 51(4): 87-91.
[8] 刘洪丽，张友忠，吕昌帅，丁佰娟，王颉. 核仁素表达下调对宫颈癌细胞Hela生物学行为的影响[J]. 山东大学学报(医学版), 2013, 51(11): 46-50.
[9] 梁婷婷，张萍，马德美，朱琳. 靶向HPV16-E5的siRNA对TRAIL诱导宫颈癌Caski细胞凋亡的影响[J]. 山东大学学报(医学版), 2013, 51(10): 24-28.
[10] 宋爱琴1，2，鞠秀丽1，孙立荣2，王玲珍2，李晓玲2，于洪升3. 慢病毒载体介导的siRNA抑制Jiyoye细胞c-myc基因表达的实验研究[J]. 山东大学学报(医学版), 2011, 49(4): 80-84.
[11] 张玉军，丁峰，王春晓，李兴福，遇晓. 99Tc-MDP对大鼠佐剂性关节炎PGE1和PGE2的影响[J]. 山东大学学报(医学版), 2010, 48(8): 50-53.
[12] 韦超1,吴围屏1,赵燕1,王林1,闫新峰2 ,常晓天1. 雌激素对胶原诱导关节炎动物模型发病过程的作用[J]. 山东大学学报(医学版), 2010, 48(8): 54-59.
[13] 葛勇鹏,杨清锐,张源潮. 类风湿关节炎与肿瘤坏死因子受体Ⅱ196位点多态性相关性的Meta分析[J]. 山东大学学报(医学版), 2010, 48(5): 79-84.
[14] 舒强,李栋,李兴福,刘花香 . 类风湿关节炎滑膜成纤维样细胞增殖特性的体外研究[J]. 山东大学学报(医学版), 2006, 44(11): 1095-1099.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
版权所有 © 2018 《山东大学学报(医学版)》编辑部　
地址:山东大学科技期刊社（济南市历城区山大南路27号山东大学中心校区明德楼B座721室） 电话: 0531-88366918 E-mail: xbyxb@sdu.edu.cn
本系统由北京玛格泰克科技发展有限公司设计开发