摘要
为从基因转录水平阐明纤维连接蛋白(fibronectin,Fn)与整合素(integrins)结合反应对支气管上皮细胞(bronchial epithelialCells,BECs)的抗氧化保护机制,本文在先前的工作基础上用臭氧(ozone,O_3)攻击原代培养的免BEC,RT-PCR扩增过氧化氢酶(catalase,CAT)的cDNA,PCR产物经琼脂糖凝胶电泳后用凝胶成像系统进行灰度分析,反映CAT mRNA的原始表达丰度,观察Fn处理的影响及蛋白酪氨酸激酶抑制剂genistein和钙调素抑制剂W_7的作用。同时,将电泳展开的PCR产物电转移至尼龙膜上,用CAT特异性寡核苷酸探针杂交,证实PCR扩增产物为特异性目的基因的转录产物。结果证实:Fn(10μg/ml)处理可提高CAT表达(P<0.01),蛋白酪氨酸激酶抑制剂genistein可阻断Fn对CAT mRNA表达的增强效应(P<0.01);钙调素抑制剂W_7对Fn处理后CAT mRNA表达增强也有抑制作用。提示:Fn可提高BEC细胞内CAT编码基因的转录水平,其上游信号途径与整合素介导的酪氨酸磷酸化或Ca^(2+)-钙调素通路有关。
We have previously shown that the binding of integrins with extracellular matrix component fibronectin (Fn) can improve the
ability of bronchial epithelial cells (BECs) in resisting oxidant injury by up-regulating the activity of catalase and increasing the content of GSH.
However, the molecular mechanism or its signaling pathway of this protection is still unclear. In order to examine the intracellular signaling
mechanism activated by Fn-integrin binding reaction, the present study investigated the mRNA expression of catalase in primary cultured
rabbit BECs using RT-PCR based on a cell-injury model made with ozone exposure. The product bands of target gene CAT were checked with
Southern blot and oligonucleotide probe hybridization. The results showed that Fn (10μg/ml) promoted the catalase mRNA transcription (P
<0.01). This effect was abolished either by protein-tyrosine kinase inhibitor genistein or calmodulin inhibitor W_7 (P<0.01). These results indicate
that the promotion of catalase activity induced by Fn-integrin reaction is partly due to the elevation of catalase mRNA transcription, and
that its signalling are possibly relevant to tyrosine phosphorylation or calmodulin pathway.
出处
《生理学报》
CAS
CSCD
北大核心
2004年第3期365-368,共4页
Acta Physiologica Sinica
基金
This work was supported by the National Natural Science Foundation of China (No. 30270586)
