【摘要】 目的:研究结核分枝杆菌抗原85B(Ag85B)慢病毒对正常人支气管上皮细胞(NHBEC)表达胸腺基质淋巴细胞生成素(thymic stromal lymphopoietin,TSLP)和其受体TSLPR的影响,探究Ag85B抑制哮喘气道炎症的机制。方法:将Ag85B基因重组质粒转入293T细胞,构建携带Ag85B和EGFP报告基因的慢病毒载体p LVXIRES-Neo-EGFP-Ag85B(Ag85B慢病毒)。用鉴定后的Ag85B慢病毒感染HEK293细胞,计算滴度并观察感染效率。利用Lipofectamine? 2000使Ag85B慢病毒感染体外培养NHBEC,设立空病毒感染和空白对照组。Real-time PCR和Western blotting检测Ag85B、TSLP和TSLPR mRNA和蛋白表达水平。结果:成功构建Ag85B慢病毒感染NHBEC体系,Ag85B慢病毒感染NHBEC 48 h后,荧光显微镜下可见强绿色荧光蛋白表达,细胞生长状态良好,病毒滴度为2×1011TU/L,感染效率达到90%以上。Real-time PCR和Western blotting结果显示Ag85B慢病毒成功感染NHBEC,可见相应Ag85B mRNA和蛋白表达。与空病毒感染和空白对照组比较,Ag85B慢病毒感染组TSLP mRNA表达有下降趋势,TSLPR mRNA与TSLP和TSLPR的蛋白表达水平均显著降低(P<0.05)。结论:Ag85B慢病毒感染NHBEC可以抑制NHBEC的TSLP和TSLPR的表达,提示Ag85B可能通过靶向阻滞TSLP-TSLPR途径抑制哮喘气道炎症。更多还原

【Abstract】 AIM: To investigate the effect of Mycobacterium tubeculosis antigen( Ag85B) lentivirus on the expression of thymic stromal lymphopoietin( TSLP) and TSLP receptor( TSLPR) in cultured normal human bronchial epithelial cells( NHBEC) in vitro. METHODS: Ag85 B gene was subcloned into 293 T cells with recombinant plasmid to yield the Ag85 B lentivirus. The NHBEC were cultured and transfected with Ag85 B lentivirus,NS control and empty lentivirus control using Lipofectamine? 2000. The expression of Ag85 B,TSLP and TSLPR in NHBEC at mRNA and protein levels was confirmed by real-time PCR and Western blotting. RESULTS: Ag85 B lentivirus was constructed with a viral titer of2 × 1011 TU / L and the Ag85 B expression was detected. As compared with control group and empty lentivirus group,the mRNA expression of TSLP in Ag85 B lentivirus group was found to slightly decreased,and the mRNA expression of TSLPR and the protein expression of TSLP and TSLPR were significantly decreased in Ag85 B lentivirus group( P < 0. 05). CONCLUSION: The constructed Ag85 B lentivirus antagonizes the expression of TSLP and TSLPR in cultured NHBEC,suggesting that Ag85 B inhibits the initiation of asthmatic inflammation by blockage of TSLP-TSLPR pathway.更多还原