局部应用小干扰RNA抑制磨损颗粒诱导无菌性炎症

目的 评估小鼠气囊模型中局部应用慢病毒介导靶向肿瘤坏死因子α(TNF-α)的小干扰RNA(siRNA)抑制无菌性炎症的有效性和安全性.方法 2007年5月至2008年4月针对TNF-α基因设计干扰序列和错配序列各1条,构建共表达绿色荧光蛋白(GFP)的重组慢病毒.应用BALB/e小鼠建立气囊模型,气囊内注射钛合金颗粒刺激无菌性炎症.随机分为3组,气囊内分别注射慢病毒介导靶向TNF-α的siRNA(TNF-α组)、慢病毒介导错配siRNA(MS组)和牛理盐水(对照组).4周后处死,取气囊和外周血、心、肝、脾、肺、肾、脑组织,进行组织形态学和分子生物学检测,处死前行体内活体实时成像(IVIS)检测.结果 IVIS显示注射重组慢病毒4周后GFP稳定表达并局限于气囊局部.RT-PCR和ELISA结果显示TNF.ot组气囊内TNF-α mRNA和蛋白水平相比对照组分别下降81.6%和82.6%(P<0.01),相比MS组分别下降78.9%和84.0%(P<0.01),但3组间外周血、心、肝、脾、肺、肾、脑组织中TNF-α表达无显著差异(P>0.05).TNF-α组炎性反应(囊壁厚度和细胞浸润)显著减弱(P<0.05).结论 气囊内局部应用慢病毒介导TNF-α siRNA可有效抑制磨损颗粒诱导的无菌性炎症,且无明显全身性副作用.

Locally administered lentivirus-mediated siRNA inhibits wear debris-induced inflammation

Objective To determine the safety and efficacy of local administration of lentivirus-mediated small interfering RNA (siRNA) targeting tumor necrosis factor-st (TNF-α) in routine air pouch model. Methods From May 2007 to April 2008 a siRNA targeting TNF-α and a missense siRNA were designed, and recombinate lentivirus which coexpressed the green fluorescent protein (GFP) as a marker gene was constructed. Air pouches were established and stimulated by Ti-6A1-4V particles. Pouches were divided into 3 groups randomly. Lentivirus-mediated siRNA targeting TNF-α ( TNF-α 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-α 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-α in TNF-α 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-α 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-α group. Conclusion Efficient local delivery of lentivirus-mediated siRNA targeting TNF-a into modified murine air pouch can inhibit debris-induced inflammation effectively, with no systemic adverse effects.