人小肠黏膜防御素5mRNA的表达特点及意义

Characteristics of HD-5 mRNA expression in human small intestinal mucosa and its significance

目的 检测人小肠黏膜防御素 5基因 (Alphadefensin 5 ,DEFA5)的表达 ,为从分子水平上探讨小肠黏膜抗菌防御机制提供实验依据。方法 从 2例新鲜尸体和 2例右半结肠癌患者的回肠部位取小肠黏膜提取总RNA ,通过RT PCR扩增出DEFA5的两个外显子序列 ,比较其个体间的差异 ;沿小肠轴间隔 30cm取新鲜尸体小肠黏膜提取总RNA ,仍通过RT PCR扩增编码HD 5的阅读框序列 ,比较HD 5沿肠轴的表达趋势。结果 DEFA5在个体间的表达是一致的 ,通过RT PCR获得的外显子序列大小为 4 5 1bp ,且碱基序列与GenBank中的DEFA5标准序列相一致 ;通过PCR获得的编码HD 5的阅读框序列为 30 3bp ,其中 ,酶切位点 12bp ,保护性碱基 6bp ,终止子 3bp ,编码HD 5的序列 2 82bp(94AA) ;沿着空肠到回肠的肠轴方向 ,DEFA5在空回肠黏膜均有表达 ,至回肠末端最强 ,并有逐渐增强的趋势。结论 DEFA5基因的表达在个体间是保守的 ,而且沿着空肠到回肠的肠轴方向在mRNA水平的表达有逐渐增加的趋势 ,提示HD 5作为天然免疫物质在小肠黏膜屏障功能的维护中起着十分重要的作用。此外 ,本实验获得的HD 5阅读框序列为进一步重组表达HD 5奠定了基础。

Objective To investigate the expression of human defensin5 (HD-5) mRNA in the small intestinal mucosa of human being. Methods Total RNA was isolated from the ileal mucosa of 2 fresh corpses and 2 patients with cancer in the right hemicolon. Two exons of DEFA 5 (HD-5 cDNA) were gained using reverse transcriptional polymerase chain reaction (RT-PCR), and the inter-individual discrepancy of them was observed. The total RNA from different point (30 cm interval) along the axis of small intestinal was extracted, and the open reading frame of HD-5 was obtained using RT-PCR. Then the expression tendency of HD-5 mRNA was observed. The amplification products of RT-PCR were electrophoresed and the images were analyzed by UVI software. Results The expression of DEFA 5 in the four individuals was identical, while the base sequence of DEFA 5 (451 bp) from them were the same with that from GenBank. The open reading frame of DEFA 5 obtained by PCR was composed of 303 bp (restrictive site 12 bp, protective base 6 bp, terminator 3 bp, and coding sequence of HD-5 282 bp). The expressions of HD-5 cDNA were found at each of 13 points and had the tendency to increase gradually along the axis of small intestine from jejunum to ileum, and reached the highest level at the distal end of ileum. Conclusion The expression of DEFA 5 in each of the individuals is conservative and has the tendency to increase gradually along the axis of small intestine. Therefore, as a innate immunological substance, HD-5 plays a very important role in the maintenance of small intestinal mucosal barrier. In addition, the open reading frame of HD-5 obtained in the experiment gives the basis for recombinant HD-5