Ag85B慢病毒抑制人支气管上皮细胞TSLP及其受体的表达

发布时间：2018-05-20 22:21

本文选题：胸腺基质淋巴细胞生成素 + 胸腺基质淋巴细胞生成素受体　；参考：《中国病理生理杂志》2014年11期

【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(P0.05)。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。
[Abstract]:Aim: to investigate the effects of mycobacterium tuberculosis antigen 85BnAg85B) lentivirus on the expression of thymic stromal lymphopoietin (TSLP) and its receptor TSLPR in normal human bronchial epithelial cells (NHBECs), and to explore the mechanism of Ag85B inhibiting airway inflammation in asthma. Methods: the recombinant plasmid of Ag85B gene was transferred into 293T cells to construct lentivirus vector p LVXIRES-Neo-EGFP-Ag85B(Ag85B lentivirus carrying Ag85B and EGFP reporter genes. HEK293 cells were infected with identified Ag85B lentivirus, the titer was calculated and the infection efficiency was observed. Ag85B lentivirus was infected with Lipofectamine? 2000 in vitro. Empty virus infection and blank control group. Real-time PCR and Western blotting were used to detect the expression of Ag85BtSLP, TSLPR mRNA and protein. Results: the Ag85B lentivirus infected NHBEC system was successfully constructed. After 48 h of NHBEC infection, strong green fluorescent protein expression was observed under fluorescence microscope, and the cells grew well. The titer of the virus was 2 脳 1011TU / L, and the infection efficiency was over 90%. Real-time PCR and Western blotting showed that Ag85B lentivirus was successfully infected with NHBECs, and the corresponding Ag85B mRNA and protein expression could be seen. Compared with empty virus infection and blank control group, the expression of TSLP mRNA in Ag85B lentivirus infection group showed a downward trend. The protein expression levels of TSLPR mRNA, TSLP and TSLPR were significantly decreased in Ag85B lentivirus infection group. Conclusion NHBEC infection with NHBEC can inhibit the expression of TSLP and TSLPR in NHBEC, suggesting that Ag85B may inhibit airway inflammation of asthma by blocking TSLP-TSLPR pathway.
【作者单位】：南方医科大学研究生学院;广东省人民医院广东省医学科学院呼吸内科;广东省人民医院广东省医学科学院医学研究中心;
【基金】：广州市科技计划(No.2014J4100040) 广东省自然科学基金资助项目(No.S2011010003664)
【分类号】：R562.2

【参考文献】