天津医药

• 实验研究 •    

TNF-α siRNA表达载体的构建及其对胶原性关节炎大鼠TNF-α和IL-1的影响

庞春艳1,霍建新2,吕凤凤2,常志芳2,王永福2   

  1. 1. 内蒙古包头医学院风湿免疫研究所
    2. 内蒙古包头包头医学院风湿免疫研究所
  • 收稿日期:2012-11-29 修回日期:2013-05-08 出版日期:2013-10-15 发布日期:2013-10-15
  • 通讯作者: 王永福

Construction of TNF-αsiRNA Expression Vector and its Therapeutic Effect on TNF-αand IL-1in Type Ⅱ Collagen Induced Arthritis Rat

PANG Chun yan,HUO Jian xin,Lü Feng feng,CHANG Zhi fang,WANG Yong fu   

  1. Department of Rheumatology of Baotou Medical College
  • Received:2012-11-29 Revised:2013-05-08 Published:2013-10-15 Online:2013-10-15
  • Contact: WANG Yong fu

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摘要：  【摘要】  目的  观察肿瘤坏死因子（TNF）-α的小干扰RNA（siRNA）真核表达载体对胶原性关节炎（CIA）大鼠的治疗作用。  方法  构建TNF-α的siRNA真核表达载体并注射CIA大鼠，随机分为模型组、空载体组、TNF-α-siRNA1组和TNF-α-siRNA2组，每组6只，各组分别尾静脉注射磷酸盐缓冲液（PBS）、pGFP-V-RS空载体、TNF-α-siRNA1真核表达载体和TNF-α-siRNA2真核表达载体。另设6只未造CIA模型只注射PBS的大鼠为空白组。于注射后1、5、9和13d采血，ELISA检测血清中IL-1的变化；注射后13d处死大鼠，RT-PCR检测TNF-αmRNA的表达水平。  结果  注射第1、5、9和13天模型组大鼠血清中IL-1的表达水平高于空白组（P<0.05），注射第1、5和9天TNF-α-siRNA1组和TNF-α-siRNA2组IL-1的表达水平低于模型组和空载体组（P<0.05）；注射后第13天模型组大鼠全血中TNF-αmRNA的表达水平明显高于空白组（P<0.05），TNF-α-siRNA1组和TNF-α-siRNA2组mRNA表达水平低于模型组和空载体（P<0.05）。  结论  TNF-α的特异性siRNA能抑制TNF-αmRNA和IL-1的表达水平，从而为类风湿关节炎的基因治疗提供实验依据。

关键词: 肿瘤坏死因子-α, RNA, 小干扰, 白细胞介素1, 关节炎, 实验性, 疾病模型, 动物

Abstract:

[Abstract]   Objective   To investigate the therapeutic effect of tumor necrosis factor (TNF)-αsiRNA on typeⅡ colla?
gen induced arthritis (CIA) in rats.  Methods   The expression vectors of siRNA against TNF-αgene were constructed successfully and were injected by tail veil into CIA rats. Twenty-four CIA rats were randomly divided into4groups including model group, empty vector group, TNF-α-siRNA1group and TNF-α-siRNA2group. CIA rats were injected with the same dose of phosphate buffered sodium (PBS) and pGFP-V-RS vector respectively in model group and empty vector group, while TNF-α-siRNA1group and TNF-α-siRNA2group were injected with TNF-α-siRNA1eukaryotic expression vector andTNF-α-siRNA2eukaryotic expression vector respectively. Another6rats, which were not established CIA model, were in-jected with PBS (blank control group). The serum expression levels of IL-1were detected by ELISA on day1, 5, 9and13after injection. The expression level of TNF-αmRNA was detected by reverse transcriptase polymerase chain reaction (RTPCR) on day13.   Results   The expression level of IL-1was significantly higher on day1, 5, 9and13in model group than that of blank group (P<0.05). The expression levels of IL-1were significantly lower on day1, 5and9in TNF-α-siRNA1group and TNF-α- siRNA2group than that of model group and blank group (P<0.05). The expression level of TNF-αmRNA was significantly higher on day13in model group than that of blank group (P<0.05). The expression levels of TNF-αmRNA were significantly lower in TNF-α-siRNA1group and TNF-α-siRNA2group than those of model group and empty vector group (P<0.05).   Conclusion   TNF-αspecific siRNA can suppress the levels of TNF-αmRNA and IL-1, which provides experimental basis for gene therapy of rheumatoid arthritis.

Key words: tumor necrosis factor-α, RNA, Small interfering, interleukin-1, arthritis, experimental, disease models, 动物