呼吸道合胞病毒感染诱导人支气管上皮细胞表达胸腺基质淋巴生成素的信号转导机制探讨

Signal transduction in respiratory syncytial virus infection-induced thymic stromal lymphopoietin expression in human epithelial cells

目的探讨哮喘发病过程中呼吸道合胞病毒(RSV)感染通过诱导支气管上皮细胞表达胸腺基质淋巴生成素(TSLP)来加重哮喘症状的具体信号转导机制。方法构建RSV病毒F蛋白真核表达载体pcDNA3-F,转染体外培养的人气管上皮细胞株CRL-9483,同时转染Smad7表达载体pcDNA3/Smad7或者添加p38、ERK1/2、JNK、JAK/STAT通路选择性阻断剂,24h后利用RT-PCR观察各组细胞中TSLP mRNA量的改变情况,观察哪些抑制剂可以下调TSLP mRNA水平。对TSLP显著下调的实验组扩大细胞培养规模,提取细胞浆蛋白后,利用Western Blotting在TSLP蛋白水平进行观察。结果体外培养的CRL-9483细胞在转染空载体pcDNA3情况下基本不表达TSLP蛋白(TSLP/GAPDHPCR产物相对灰度0.10±0.03),F蛋白的转染可以明显促进其mRNA水平的增高(0.42±0.20,P=0.024),这种增高可以部分被p38抑制剂(0.14±0.04)和JNK抑制剂(0.23±0.07)所阻断,与F蛋白转染组相比P值分别为0.036和0.048,而Smad7阻断剂(0.60±0.25)、ERK1/2抑制剂(0.45±0.23)以及JAK/STAT1阻断剂(0.44±0.25)无此阻断作用,与单纯F蛋白转染组相比P>0.05。利用Western Blotting分析发现与单独RSV病毒F蛋白表达组(TSLP/GAPDH蛋白条带相对灰度8.13±2.20)相比,p38抑制剂(3.67±1.18)和JNK抑制剂(1.48±0.77)也可以导致TSLP蛋白水平的下降。结论RSV感染可以通过其F蛋白来刺激气道上皮细胞表达TSLP,p38和JNK信号通路参与到该过程中,这可能是RSV感染诱导或加重哮喘气道炎症的一个重要原因。

Objective To explore the mechanism of signal transduction in respiratory syncytial virus（RSV）-induced expression of thymic stromal lymphopoietin（TSLP）in bronchial epithelial cells.Methods The eukaryotic expression vector of RSV F protein,pcDNA3-F,was constructed and transfected into in vitro cultured human bronchial epithelial cell line CRL-9483,which was also transfected with Smad7 expression vector pcDNA3/Smad7 or exposed to p38,ERK1/2,JNK,and JAK/STAT1 inhibitors.The mRNA levels of TSLP and the housekeeping GAPDH gene were analyzed 24 h later with semi-quantitative RT-PCR.In cells with downregulated TSLP mRNA expression due to the addition of the signal inhibitors,cytoplasm TSLP or GAPDH protein levels were further analyzed using Western blotting.Results Virtually no TSLP mRNA expression was detected by RT-PCR in cultured CRL-9483 cells transfected with pcDNA3 exclusively（TSLP/GAPDH relative total gray scale of 0.10±0.03）,while cell transfection with pcDNA3-F resulted in significantly increased TSLP mRNA level（0.42±0.20,P=0.024）.In the presence of F protein expression,both p38 and JNK inhibitors could downregulate TSLP mRNA levels（0.14±0.04,P=0.036;0.23±0.07,P=0.048,respectively）,while TGF-β-Smad inhibiting protein Smad7（0.60±0.25）,ERK1/2 inhibitor（0.45±0.23）,and JAK/STAT1inhibitor（0.44±0.25）failed to block TSLP expression（P〉0.05）.Western blotting showed that p38 inhibitor（TSLP/GAPDH relative total gray scale=3.67±1.18,P=0.018）and JNK inhibitor（1.48±0.77,P=0.004）also downregulated the protein levels of TSLP as compared with pcDNA3-F transfection group（8.13±2.20）.Conclusions RSV F protein can stimulate TSLP expression in human bronchial epithelial cells mediated partially by p38 and JNK signal pathways.