中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (24): 4393-4397.doi: 10.3969/j.issn.1673-8225.2012.24.005

• 骨组织构建 bone tissue construction • 上一篇    下一篇

骨关节炎疾病中白细胞介素17协同肿瘤坏死因子α调节诱导型一氧化氮合酶的表达

李永忠，闵 华，刘 超，吴 煌   

  1. 宜昌市第二人民医院骨科，湖北省宜昌市 443000
  • 收稿日期:2011-10-30 修回日期:2011-12-10 出版日期:2012-06-10 发布日期:2013-11-05
  • 作者简介:李永忠，男，1968年生，湖北省宜昌市人，1990年三峡大学毕业，副主任医师，主要从事骨关节外科研究。 liuchao8668@139.com
  • 基金资助:

    2009~2010年度湖北省卫生厅科研项目(JX4B67)

Interleukin-17 and tumor necrosis factor alpha regulate the expression of inducible nitric oxide synthase in rheumatoid arthritis

Li Yong-zhong, Min Hua, Liu Chao, Wu Huang   

  1. Department of Orthopedics, Second People's Hospital of Yichang, Yichang 443000, Hubei Province, China
  • Received:2011-10-30 Revised:2011-12-10 Online:2012-06-10 Published:2013-11-05
  • About author:Li Yong-zhong, Associate chief physician, Department of Orthopedics, Second People's Hospital of Yichang, Yichang 443000, Hubei Province, China liuchao8668@139.com

PDF

450

摘要：

背景：白细胞介素17是由新近被证实的CD4+辅助性T细胞亚型Thl7细胞产生的促炎性细胞因子，表达在T细胞上，其受体表达在大多数细胞和组织上。
目的：探讨人白细胞介素17 通过与其受体(白细胞介素17受体A，白细胞介素17受体C)结合，在与肿瘤坏死因子α的共同作用下对滑膜组织诱导型一氧化氮合酶启动子的表达和对一氧化氮合成的影响。
方法：提取人膝关节关节软骨及滑膜组织中mRNA，同时构建白细胞介素17受体A和白细胞介素17受体C的干扰RNA表达质粒，转染至293T，在G418筛选压力下建立白细胞介素17受体表达缺陷型细胞模型。将含有诱导型一氧化氮合酶启动子的重组真核质粒分别转染至野生型293T和白细胞介素17受体A表达缺陷型293T细胞，在外源性白细胞介素17和肿瘤坏死因子α的共同诱导下，通过检测荧光素酶含量和一氧化氮的浓度来观察诱导型一氧化氮合酶启动子的表达和诱导型一氧化氮合酶的活性。
结果与结论：①成功获得含有人诱导型一氧化氮合酶启动子全长cDNA的真核表达质粒pGL3-iNOSp-luciferase。② 建立了人白细胞介素17受体A表达缺陷型细胞模型。③在白细胞介素17受体A表达缺陷型细胞中，诱导型一氧化氮合酶启动子和诱导型一氧化氮合酶活性显著降低。④成功构建了人白细胞介素17受体C的特异性shRNA真核表达载体shRNA-IL-17RC。表明白细胞介素17通过其受体白细胞介素17受体A与诱导型一氧化氮合酶启动子的结合，显著促进了细胞诱导型一氧化氮合酶基因的表达和一氧化氮产量。

关键词: 白细胞介素17受体A, 诱导型一氧化氮合酶, shRNA, 骨关节炎, 肿瘤坏死因子α

Abstract:

BACKGROUND: Recently, interleukin-17 (IL-17) is proved to be a pro-inflammatory cytokine, which produced by CD4+helper T cells subtype Th 17 cells. IL-17 is expressed in T cells, and IL-17 receptor is expressed in subtotal cells and tissues.
OBJECTIVE: To explore the synergetic effects of IL-17 binding IL-17 receptor (IL-17RA, IL-17RC) and tumor necrosis factor α on expression of inducible nitric oxide synthase (iNOS) promoter in the synovium as well as synthesis of nitric oxide.
METHODS: mRNA of human knee joint cartilage and synovial tissue was extracted. RNA interference expression vector of IL-17RA and IL-17RC were constructed together, and transfected into 293T cells. IL-17 receptor-deficient expression cell model was established under the G418 selection pressure. The recombinant eukaryotic plasmids containing iNOS promoter were transfected into wild type 293T cells and 293T cells deficient in IL-17RA expression respectively. Through the synergetic induction of IL-17 and tumor necrosis factor α, expression of iNOS promoter and activity of iNOS were observed by detection of luciferase and nitric oxide concentrations.
RESULTS AND CONCLUSION: ①Full-length cDNA from human knee joint cartilage and synovial tissue iNOS promoter and eukaryotic expression plasmids pGL3-iNOS-p luciferase were obtained successfully. ②Cell model of IL-17RA expression deficiency was established. ③In IL-17RA-deficient expression cells, the expression of iNOS promoter and activity of iNOS were significantly reduced. ④IL-17RC specific shRNA eukaryotic expression vector of shRNA-IL-17RC was successfully constructed. These findings suggest that IL-17 via its receptor IL-17RC cooperated with iNOS promoter to obviously promote gene expression of iNOS and nitric oxide production.

中图分类号: