小干扰RNA干扰核因子-κB蛋白表达抑制同种异体气管移植排斥反应的研究

Blockage of the nuclear factor-κB expression by short interference RNA inhibited the rejection of an allogeneic tracheal graft

目的 观察抑制气管移植物核因子-κB （NF-κB）蛋白表达对同种异体气管移植急性排斥反应的影响.方法 取50只BALB/c和C57BL/6小鼠分为3组：同基因移植组（A组）、脂质体对照组（B组）和小干扰RNA （siRNA）干扰组（C组）.体外建立siRNA干扰NF-κB蛋白低表达支气管移植物以及气管移植动物模型,最后比较各组移植后第7、14和28天排斥反应的程度.反转录-聚合酶链反应（RT-PCR）和Western blot检测NF-κB基因和蛋白的表达水平,苏木素-伊红（HE）染色检测炎性细胞浸润、气管通畅度和上皮细胞存活率.结果 与A组比较,气管移植后第14天,B组支气管大量炎性细胞浸润,管腔出现纤维阻塞,通畅度为（62.0±16.7）％,支气管上皮细胞存活率为（11.0±24.6）％;而C组少量炎性细胞浸润,支气管管腔通畅度为100％ （P ＜0.05）,C组支气管上皮细胞存活率为（78.80±4.41）％（P＜0.05）;气管移植后第21天,B、C两组管腔通畅度及上皮细胞存活率均为0.结论 siRNA沉默支气管上皮细胞NF-κB蛋白表达可通过抑制急性排斥反应的水平,延长气管管腔的通畅时间和上皮细胞存活时间,从而延缓闭塞性细支气管炎的发展.

Objective To investigate the effects of nuclear factor-κB （NF-κB） on chronic allograft rejection after trachea transplantation.Methods Fifty BALB/c and C57BL/6 nude mice were divided into 3 groups：transplantation group,lipidosome negative control group,and small interfering RNA （siRNA） group.With the establishment of low-expressed NF-κB transplants,degrees of rejection reaction were compared among these groups at 7,14 and 28 days after transplantation respectively.Reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting were used to detect the expression of NF-κB transcriptionally and post-transcriptionally.The tracheal epithelium survival rate and inflammatory cell infiltration rate were quantified by H＆amp;E staining.Results At 14th day after trachea transplantation,negative control group showed severe infiltration of inflammatory cells,the patency rate was （62.0 ± 16.7） %,and survival rate of tracheal epithelia was （11.0 ± 24.6） %,and siRNA-treated group showed intact trachea epithelium,mild infiltration of inflammatory cells,the patency rate was 100% （P 〈 0.05）,and survival rate of tracheal epithelia was （78.80 ± 4.41） % （P 〈 0.05）.However,at 21 st day after transplantation,both negative control group and siRNA-treated group demonstrated 0% in both patency rate and survival rate.Conclusion The knock-down of NF-κB expression in the tracheal epithelia can inhibit acute rejection reaction and can alleviate the development of occlusive bronchiolitis.