肿瘤坏死因子α基因沉默对无菌性炎症的抑制作用

Inhibition of aseptic inflammation by gene silencing of tumor necrosis factor-α

目的研究局部应用靶向肿瘤坏死因子α(TNF-α)的小干扰RNA(siRNA)对磨损颗粒诱导的无菌性炎症的抑制作用。方法构建重组慢病毒载体,设计靶向TNF-α的siRNA序列和错配序列各1条。建立小鼠气囊模型,气囊内注射聚甲基丙烯酸甲酯颗粒诱导无菌性炎症反应。将建模后的小鼠随机分为3组,每组12只,分别在小鼠气囊内注入TNF-αsiRNA(TNF-α组)、错配siRNA(MS组)和PBS(对照组);慢病毒转染后第14和28天每组各处死6只小鼠,取出气囊组织。采用Real-Time PCR法检测气囊中TNF-α、IL-1β和IL-6 mRNA的表达;ELISA法检测TNF-α蛋白的含量;组织学检测气囊壁厚度和炎症细胞计数;活体荧光成像定量分析绿色荧光蛋白(GFP)荧光的强度和分布。结果慢病毒介导TNF-αsiRNA的局部应用显著降低了TNF-α组中TNF-α、IL-1和IL-6 mRNA的表达(P<0.01),也降低了TNF-α蛋白的含量(P<0.01);TNF-α组炎症反应(囊壁厚度和细胞浸润)显著减弱(P<0.05或P<0.01)。活体荧光成像显示,GFP的表达局限于气囊局部,TNF-α组和MS组荧光量比对照组升高5倍左右。结论局部应用TNF-αsiRNA可有效抑制磨损颗粒诱导的无菌性炎症,且无全身性不良作用。

Objective To investigate the inhibitory effect of local application of small interfering RNA（siRNA） targeting tumor necrosis factor-α（TNF-α） on wear debris-induced aseptic inflammation. Methods Recombinant lentivirus vector was constructed,and a siRNA targeting TNF-α and a missense siRNA were designed.Air pouches were established and stimulated by polymethylmethacrylate（PMMA） particles for aseptic inflammation.Mice were randomly divided into 3 groups,with 12 mice in each group,and TNF-α siRNA（TNF-α group）,missense siRNA（MS group） and PBS（control group） were locally injected into pouches respectively.Mice in each group were sacrificed on the 14th day and 28th day after lentivirus transfection,with 6 rats on each day,and pouch homogenates were obtained.The expression of TNF-α,IL-1β and IL-6 mRNA in pouches was detected by Real-Time PCR,the content of TNF-α protein was determined by ELISA,the thickness of pouch membrane was measured by histological analysis,inflammatory cell counting was performed,and intensity and distribution of green fluorescent protein（GFP） were quantitatively analysed by in vivo bioluminescence imaging. Results The transfection of lentivirus-mediated TNF-α siRNA in vivo significantly decreased the expression of TNF-α,IL-1 and IL-6 mRNA and the content of TNF-α protein in TNF-α group（P0.01）.Histological analysis revealed less inflammatory responses（thinner pouch membrane and decreased cellular infiltration） in TNF-α group（P0.05 or P0.01）.In vivo bioluminescence imaging indicated the expression of GFP in pouches was locally restrained,and the fluorescence quantity in TNF-α group and MS group was about 5 times higher that in control group. Conclusion Local application of TNF-α siRNA may effectively inhibit wear debris-induced aseptic inflammation,with no systemic adverse effects.