【作者】 祝秉东；

【导师】 王伯瑶；

【作者基本信息】 四川大学 ， 病理学与病理生理学， 2004， 博士

【摘要】 人β-防御素-1(hBD-1)是一重要的抗菌肽分子,广泛表达于分泌腺和皮肤粘摸的上皮细胞中,具有重要的抗感染功能。长期以来hBD-1被认为固有表达于各种组织,不被脂多糖(LPS)等所诱导。然而近来越来越多的研究表明hBD-1mRNA的表达水平可以被某些微生物及炎性介质上调。我们实验室发现卡介苗胞壁蛋白组分能够刺激人肺腺上皮细胞SPC-A-1中hBD-1 mRNA的表达增强四倍左右。hBD-1基因已被克隆,有两个较小的外显子中间隔一6962 bp的内含子。和人β-防御素-2(hBD-2)等抗菌肽不同,hBD-1基因调控序列中未发现NF-κB位点。然而,对hBD-1基因启动子活性和转录调控机制尚缺乏系统的研究。本实验即针对这些问题展开研究,并着重探讨BCG增强hBD-1基因转录的调控机制。 首先,由于hBD-1基因的表达有组织特异性,我们运用RT-PCR和Northern杂交方法探讨了hBD-1基因在一些细胞株的表达情况。实验表明肺腺上皮细胞SPC-A-1、肺上皮细胞A549和单核细胞THP-1表达hBD-1 mRNA。而在脐静脉血管内皮细胞ECV304、纤维母细胞FB和Ad5DNA转化的胚肾细胞株293中未检测到hBD-1的表达。其中单核细胞THP-1表达hBD-1 mRNA是首次被发现。 其次,我们对hBD-1基因不同长度启动子的启动活性做了研究。我们将不同长度的hBD-1基因5′-端上游序列连接入无启动子的绿色荧光蛋白更多还原

【Abstract】 Human β-defensin-1 (hBD-1), an important antimicrobial peptide defending against microorganism infection, was widely expressed in secretory glands and mucosal epithelial cells. Previously it was considered that the expression of hBD-1 was constitutive, and could not be enhanced by inflammation stimulator such as lipopolysaccharide (LPS) and TNF-a etc. However, recently it was reported that hBD-1 mRNA expression could be up-regulated by some microorganisms or inflammatory factors. Our group found that the expression could be obviously enhanced in pulmonary gland epithelial SPC-A-1 cells upon the stimulation with bacille Calmette-Guerin (BCG) cell wall components. Sequencing of the hBD-1 gene revealed that there were two exons and one intron spanning 6962 bp. NF- k B motif, which played an important role in regulating the inducible expression of some antimicrobial peptides such as human β -defensin-2 (hBD-2) by LPS, had not been found in the regulatory region of hBD-1 gene. However, investigation of hBD-1 gene promoter activity as well as its transcriptional regulation mechanism of the induction expression had not been systematically studied. Therefore we didsome works on that issue. There were three parts in this thesis: first, examining constitutive expression of hBD-1 mRNA in various cell lines; second, investigating the hBD-1 gene promoter activity; lastly, exploring the transcriptional regulation mechanism of the up-regulation of hBD-1 gene in SPC-A-1 cells upon the stimulation with BCG cell wall components.The gene expression of hBD-1 was investigated in several cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR) and northern hybridization. It was found that Pulmonary gland epithelial cell SPC-A-1, pulmonary epithelial cell A549, and mononuclear cell THP-1 constitutively expressed hBD-1 mRNA, while there was no hBD-1 mRNA signal to have been detected in the umbilical vein endothelium cell ECV304, fibroblast FB and 293 cells.In order to determine the promoter activity of hBD-1 gene, several upstream sequences of that gene were cloned into a promoterless plasmid pEGFP-1, and these constructs were transfected into SPC-A-1 cells respectively. The green fluorescence protein (GFP) expression was detected by fluorescence inversion microscope and flow cytometry. Results indicated that the promoter activity of -69 bp region was lower than that of -575 and -314 bp, both of which had powerful promoter activity.Furthermore, experiments were performed to examine the stimulatory effect of BCG cell wall components on hBD-1 gene expression and analyze the response element in the 5’-flanking region of that gene. Firstly, BCG cell wall proteins were extracted by sodium dodecyl sulfate (SDS) and fractionated by Sephadex G-150 chromatography. Progressive deletions of 5’-flanking region of hBD-1 gene were produced by PCR and ligated into another promoterlesschloramphenicol acetyltransferase (CAT) expression plasmid to construct pCAT hBD-1 reporter plasmids. One of them, -575 pCAT hBD-1 construct was applied to determine the effective components of BCG cell wall proteins which could enhance the expression of hBD-1. Results showed that the cell wall components with a molecular weight of 18 kDa to 30 kDa could enhance the transcription of hBD-1 gene. Secondly, SPC-A-1 cells were co-transfected with a series of pCAT hBD-1 constructs and pSV-P-Galactosidase control vector (as an internal control to normalize transfection efficiency) and stimulated with effective BCG cell wall components (3 mg/L) at the 6 hour after the transfection. Following incubation with BCG components for 39 hours, CAT and P-Gal protein expression were determined by ELISA. The concentration of CAT was normalized by that of P-Gal to indicate the transcription activity of the regulatory sequences. Results showed that the -314 bp region of hBD-1 gene was responsible for the up-regulated expression by BCG cell wall components, and the -69 bp still remained half of the increasing tendency of the -314 bp. Thirdly, Using Matlnd and Matlnspector analysis programs, the -575 bp/+14 bp sequence of the hBD-1 gene was searched for potential transcription factor-binding sites in a Transfac transcription factor database (http://tansfac.gbf.de/TRANSFAC) . Multiple high homologous cis elements were found among the sequences from -314 bp to the first exon. They included CCAAT/Enhancer binding protein- ￡ (C/EBP P) in -50 bp region, activator protein-1 (AP-1) in -141 bp region, and CP2 cis element in -197 bp region. Fourthly, in order to determine whether the -69 bp sequences (especially C/EBPp motif) was essential for the induction of hBD-1 gene expression by BCG or not, the gel electrophoresis mobility shift assay (EMSA) was carriedout. Double-stranded oligonucleotide probe containing the C/EBPp binding element was incubated with nuclear extracts from SPC-A-1 cells treated with or without the active BCG cell wall proteins, and electrophoresis mobility shift was analyzed. The result showed that there was a slight difference between the stimulated and unstimulated cells, suggesting that the -69 bp sequences (especially C/EBPP motif) probably took part in the up-regulation of hBD-1 gene by BCG. In summary, BCG cell wall components (18 kDa to 30 kDa) can stimulate hBD-1 gene transcription in pulmonary gland epithelial cells. The sequence (-314 /+14 bp) containing C/EBP P , AP-1, and CP2 motif is responsible for the induction, and a more short sequence, the -69 bp is essential for the enhancement.更多还原