重组人Elafin转染气道上皮细胞对脂多糖致气道黏液高分泌的影响  

Effects of recombinant human elafin gene transfection on lipopolysaccharide-induced airway mucous hypersecretion

作　　者：徐晓艳 元东明 周向东 

机构地区：[1]山东诸城市人民医院呼吸内科,262200

出　　处：《中华医学杂志》2011年第5期340-344,共5页National Medical Journal of China

基　　金：基金项目：国家自然科学基金（30770951）

摘　　要：目的 探讨重组人自细胞弹性蛋白酶抑制因子Elafin对脂多糖（LPS）致气道黏液高分泌的影响.方法 通过构建人Elafin重组质粒pEGFP-C1-Elafin,并转染人人气道黏液上皮细胞系NCI-H292,将其与多形核粒细胞（PMN）共孵育,并用LPS刺激24 h,采用底物检测法、酶联免疫吸附测定（ELISA）和RT-PCR分别检测培养细胞上清中性粒细胞弹性蛋白酶（NE）活性、黏蛋白（MUC）5AC蛋白含量和细胞中MUC5AC mRNA水平.结果 LPS各浓度均可明显提高转染空载体组培养细胞上清液的NE活性、MUC5AC含最及细胞中MUC5AC mRNA水平,与空白对照组相比,差异有统计学意义（P＜0.01） 并且在0.5～25 mg/L终浓度范围内呈剂量依赖关系.转染Elafin组与转染空载体组在不同浓度LPS（终浓度分别为0.5 mg/L和5.0 mg/L）干预时各个指标相比较差异有统计学意义（P＜0.05）,但在LPS终浓度为25 mg/L组各指标与转染空载体组相比较差异无统计学意义（P＞0.05）.结论 转染重组人Elafin可抑制一定浓度范围LPS引起的炎症终效应因子NE活性及其诱导的黏液高分泌,但不能抑制高浓度LPS的作用.Objective To explore the effects of recombinant human Elafin gene transfection on lipopolysaccharide （LPS）-induced airway mucous hypersecretion. Methods An eukaryotic expression vector pEGFP-C1-Elafin was first constructed and then transfected into NCI-H292 cell. After co-incubation with polymorphonuclear granulocyte （PMN） plus LPS stimulation for 24 hour, the vital force unit of neutrophil elastase （NE） and the content of mucin （MUC） protein in culture media and the level of MUC 5AC mRNA in culture cells were detected with substrate detection technique, enzyme-linked immunosorbent assay （ELISA） and reverse transcription-polymerase chain reaction （RT-PCR） respectively. Results All parameters in control group showed a low level（0. 13 ±0. 03, 0. 28 ±0. 06, 0. 29 ±0. 04 respectively）. And transfectiing NCI-H292 cell with pEGFP-C1 at different levels of LPS, all parameters were obviously different （all P 〈0. 01 ） and they all showed a dose-dependent relationship. After LPS stimulation （0. 5, 5 mg/L）,as compared with the transfection of NCI-H292 cell with pEGFP-C1-Elafin, the vital force unit of NE and the content of MUC5AC protein in culture media and the level of MUC 5AC mRNA in transfecting pEGFP-C1NCI-H292 cell decreased obviously （all P 〈 0. 05 ）. But after LPS stimulation （25 mg/L）, as compared with the transfection of NCI-H292 cell with pEGFP-C1-Elafin, no parameter of transfecting pEGFP-C1 NCI-H292 cell had any obvious alteration （0. 67 ±0. 06, 0. 64 ±0. 08, 0. 67 ±0. 07 respectively, all P 〉0. 05）.Conclusion The transfection of recombinant human elafin gene into NCI-H292 cell may inhibit the force of inflammatory telo-effective factor NE induced by a certain level of LPS and airway mucous hypersecretion.But it can not inhibit the effects induced by a high level of LPS.

关 键 词：ELAFIN 脂多糖类 白细胞弹性蛋白酶 粘蛋白类 

分 类 号：R363[医药卫生—病理学]