局部给药的慢病毒介导的小干扰RNA抑制磨损颗粒诱导的炎症

[Locally administered lentivirus-mediated siRNA inhibits wear debris-induced inflammation].

作者信息

Peng Xiao-chun, Zhang Xian-long, Tao Kun, Cheng Tao, Zhu Jun-feng, Zeng Bing-fang

机构信息

Department of Orthopaedics, the Sixth Affiliated People's Hospital of Shanghai Jiaotong University, Shanghai 200233, China.

出版信息

Zhonghua Wai Ke Za Zhi. 2009 Mar 1;47(5):377-80.

PMID:19595019

Abstract

OBJECTIVE

To determine the safety and efficacy of local administration of lentivirus-mediated small interfering RNA (siRNA) targeting tumor necrosis factor-alpha (TNF-alpha) in murine air pouch model.

METHODS

From May 2007 to April 2008 a siRNA targeting TNF-alpha and a missense siRNA were designed, and recombine lentivirus which coexpressed the green fluorescent protein (GFP) as a marker gene was constructed. Air pouches were established and stimulated by Ti-6Al-4V particles. Pouches were divided into 3 groups randomly. Lentivirus-mediated siRNA targeting TNF-alpha (TNF-alpha group) or lentivirus-mediated missense siRNA (MS group), or virus-free saline (control group) were injected into pouches respectively. Pouch membrane, peripheral blood, heart, liver, spleen, kidney, lung and brain were harvested at 28 d after transfection, and assayed for markers of inflammation using histological, molecular, immunological techniques and Xenogen in vivo imaging system (IVIS) 50 vivo bioluminescent assay system.

RESULTS

Xenogen IVIS 50 vivo image revealed strong expression of GFP localized in pouch areas and no expression in other parts of mice both in TNF-alpha group and MS group at 4 weeks after transfection, while no expression of GFP was found in control group. By RT-PCR and ELISA, the mRNA and protein levels of TNF-alpha in TNF-alpha group decreased by 81.6% and 82.6% respectively compared to control group (P < 0.01), and decreased by 78.9% and 84.0% respectively compared to MS group (P < 0.01), whereas TNF-alpha level in peripheral blood, heart, liver, spleen, kidney, lung and brain remained invariant (P > 0.05). Less inflammatory responses (thinner pouch membrane and decreased cellular infiltration) were observed in TNF-alpha group.

CONCLUSION

Efficient local delivery of lentivirus-mediated siRNA targeting TNF-alpha into modified murine air pouch can inhibit debris-induced inflammation effectively, with no systemic adverse effects.

摘要

目的

在小鼠气袋模型中确定局部给予慢病毒介导的靶向肿瘤坏死因子-α（TNF-α）的小干扰RNA（siRNA）的安全性和有效性。

方法

2007年5月至2008年4月，设计了一种靶向TNF-α的siRNA和一种错义siRNA，并构建了共表达绿色荧光蛋白（GFP）作为标记基因的重组慢病毒。建立气袋并用Ti-6Al-4V颗粒刺激。将气袋随机分为3组。分别向气袋中注射慢病毒介导的靶向TNF-α的siRNA（TNF-α组）或慢病毒介导的错义siRNA（MS组），或无病毒生理盐水（对照组）。转染后28天收集气袋膜、外周血、心脏、肝脏、脾脏、肾脏、肺和脑，并用组织学、分子学、免疫学技术和Xenogen体内成像系统（IVIS）50活体生物发光测定系统检测炎症标志物。

结果

Xenogen IVIS 50活体成像显示，转染后4周，TNF-α组和MS组小鼠气袋区域均有强烈的GFP表达，小鼠其他部位无表达，而对照组未发现GFP表达。通过RT-PCR和ELISA检测，TNF-α组TNF-α的mRNA和蛋白水平与对照组相比分别下降了81.6%和82.6%（P<0.01），与MS组相比分别下降了78.9%和84.0%（P<0.01），而外周血、心脏、肝脏、脾脏、肾脏、肺和脑中的TNF-α水平保持不变（P>0.05）。TNF-α组观察到较少的炎症反应（气袋膜变薄和细胞浸润减少）。