卡介苗增强人β-防御素-1基因在肺腺上皮细胞表达的分子调节机制

Preliminary Study of the Molecular Regulation of BCG-mediated Enhancement of hBD-1 Gene Expression in Human Pulmonary Gland Epithelial Cells

目的 研究人 β-防御素 - 1(h BD- 1)基因启动子结构及卡介苗胞壁蛋白增强其 m RNA在肺腺上皮细胞 (SPC- A- 1)中表达的转录调控机制。方法 h BD- 1基因 5′-端上游序列被连接入无启动子的 p EGFP- 1质粒和p CAT basic质粒 ,构建了系列 5′-端缺失的 p EGFP h BD- 1及 p CAT h BD- 1报告质粒。p EGFP h BD- 1质粒转染细胞后用荧光倒置显微镜观察绿色荧光表达强度。 p CAT h BD- 1报告质粒和 p SV- β- Galactosidase control质粒 (内对照 )共转染 SPC- A- 1细胞后 ,给予卡介苗胞壁蛋白活性组分刺激 ,用 EL ISA检测 CAT和 β- Gal浓度。结果 h BD-1基因上游 - 314段有较强的启动子活性 ,- 6 9段则启动活性减弱 ;BCG刺激后 ,即使上游序列缩短至 - 6 9位点时 ,报告基因 CAT相对表达量仍明显增高。该区域有一同源性极高的 C/ EBPβ(CCAAT/ Enhancer- binding proteinβ)结合元件。结论 h BD- 1基因上游 - 314 bp段具有较完整的启动子活性 ,其中含有 C/ EBPβ结合位点的 - 6 9/ +5 4 bp序列介导卡介苗对 h BD-

Objective To define the regulation mechanism of the enhancement expression of human β defensin 1 (hBD 1) stimulated by bacille Calmette Guerin (BCG) at transcription level. Methods A series of 5′ flanking deletions of hBD 1 gene were ligated into pEGFP 1 and pCAT basic vector. pEGFP hBD 1 recombinants were transfected into SPC A 1 cells and the green fluorescence protein (GFP) expression was observed by fluorescence microscopy. SPC A 1 cells were co transfected with pCAT hBD 1 constructs and pSV β Galactosidase control vector and were stimulated with effective BCG cell wall components. CAT and β Gal protein expressions were determined by ELISA. Results The promoting activity of the -69 region was lower than that of the region -575 and -314. After being stimulated with the active fraction of BCG cell wall proteins, the relative CAT expression of pCAT hBD 1/-69 still showed the increasing tendency of -2161 construct. Computer consensus match analysis indicated that the nucleotides between -63 bp to -50 bp was the potential binding site for C/EBPβ. Conclusion The nucleotides from -69 to +54 bp is essential to the enhancement of hBD 1 gene transcription by BCG.