论文部分内容阅读
目的:构建天然内生多肽Elafin真核表达载体,探讨其对气道粘液高分泌的影响。方法:抽提Elafin行RT-PCR获取Elafin cDNA,双酶切后将片段装载到pMD18-T载体上。以pMD18-T-Elafin为模板行PCR反应,产物胶回收并双酶切后定向克隆至pEGFP-N1上,转化,筛选,双酶切鉴定重组质粒。将pEGFP-N1-Elafin转染正常人支气管上皮细胞HBE16,给予脂多糖(LPS)刺激,Western blot检测细胞内Elafin蛋白的相对含量,RT-PCR检测各组Elafin mRNA和粘蛋白(MUC)5AC mRNA表达水平,荧光素酶报告基因检测系统测定核转录因子-κB(NFκ-B)的活性;ELISA法分析各组细胞MUC5AC蛋白的相对含量。结果:成功构建Elafin真核表达载体,转染重组Elafin的HBE16细胞成功表达Elafin蛋白。LPS刺激可增强NF-κB的活性,该活性在转染重组Elafin后显著降低;MUC5AC蛋白含量及mRNA水平在LPS刺激后也显著升高,在转染重组Elafin后二者的表达水平明显降低。结论:Elafin真核表达载体成功构建,初步发现Elafin可通过降低NFκ-B的活性来下调MUC5AC的表达,为进一步深入研究其对气道粘液高分泌的调节机制奠定了基础。
Objective: To construct eukaryotic expression vector of Elafin, a natural endogenous peptide, and explore its effect on airway mucus hypersecretion. Methods: Elafin cDNA was extracted by RT-PCR and double-digested to load the fragment into pMD18-T vector. The PCR reaction was carried out using pMD18-T-Elafin as a template. The product gel was recovered and double-digested and cloned into pEGFP-N1. The recombinant plasmids were transformed, selected and double-digested. The normal human bronchial epithelial cells HBE16 were transfected with pEGFP-N1-Elafin, and stimulated by lipopolysaccharide (LPS). The relative content of Elafin protein in the cells was detected by Western blot. The expression of Elafin mRNA and mucin (MUC) 5AC mRNA The expression level of NF-κB was detected by luciferase reporter assay system. The relative content of MUC5AC protein in each group was analyzed by ELISA. Results: Elafin eukaryotic expression vector was successfully constructed and Elafin protein was successfully expressed in HBe16 cells transfected with recombinant Elafin. LPS stimulation enhanced the activity of NF-κB, which was significantly decreased after transfected with recombinant Elafin. MUC5AC protein content and mRNA level were also significantly increased after LPS stimulation. The expression of MUC5AC protein was significantly decreased after transfected with recombinant Elafin. CONCLUSION: Elafin eukaryotic expression vector was successfully constructed. It was found that Elafin down-regulated the expression of MUC5AC by decreasing the activity of NFκ-B, which laid the foundation for further study of its regulatory mechanism on airway mucus hypersecretion.