论文部分内容阅读
目的阐明细菌内毒素细菌脂多糖(lipopolysaccharide,LPS)是否影响紧密连接蛋白claudin-11的表达及其分子机制。方法表达claudin-11基因的原代培养人皮肤成纤维细胞,加入纯化的LPS或核转录因子κB(NF-κB)特异性抑制剂MG132或p38丝裂原活化蛋白激酶(p38MAPK)抑制剂SB203580后,利用逆转录-聚合酶链反应(RT-PCR)和蛋白免疫印迹方法检测claudin-11基因mRNA和蛋白质表达水平变化,利用细胞免疫荧光染色法检测NF-κB核转移。结果LPS细胞膜受体TLR4在人皮肤成纤维细胞表达;2μg/ml LPS能提高claudin-11基因mRNA和蛋白质的表达水平。进一步研究发现:虽然LPS能使NF-κB从细胞质转移到细胞核,但LPS引起claudin-11基因表达增加不能被NF-κB特异性抑制剂MG132抑制,而能被p38丝裂原活化蛋白激酶(p38MAPK)抑制剂SB203580明显抑制。结论细菌内毒素LPS影响claudin-11基因的表达可能是通过p38 MAPK通路来调节的。
Objective To elucidate whether bacterial endotoxin bacterial lipopolysaccharide (LPS) affects the expression of claudin-11 and its molecular mechanism. Methods Primary cultured human dermal fibroblast cells expressing claudin-11 gene were treated with purified LPS or MG132, a specific inhibitor of nuclear factor kappaB (NF-κB), or SB203580, an inhibitor of p38 mitogen-activated protein kinase The mRNA and protein expression levels of claudin-11 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The nuclear translocation of NF-κB was detected by immunofluorescence staining. Results LPS cell membrane receptor TLR4 was expressed in human dermal fibroblasts; 2μg / ml LPS increased the expression of claudin-11 mRNA and protein. Further study found that: although LPS can transfer NF-κB from the cytoplasm to the nucleus, the increase of claudin-11 gene expression induced by LPS can not be inhibited by NF-κB specific inhibitor MG132, but can be activated by p38 MAPK ) Inhibitor SB203580 significantly inhibited. Conclusion Bacterial endotoxin LPS may affect the expression of claudin-11 gene through the p38 MAPK pathway.