Elafin重组腺病毒载体在气道上皮细胞中表达的时相性变化研究

Changes of the Expression of Ad-Elafin in Human Primary Airway Epithelial Cell

为研究elafin重组腺病毒表达载体Ad～elafin转染气道上皮细胞的特性，探讨其在上皮细胞中表达的时相性变化，运用①应用原代培养技术培养人气道上皮细胞；②Ad—elafin表达载体转染气道上皮细胞；③在转染后不同的时相中，采用荧光显微镜观察报告基因GFP的表达，ELISA法单克隆抗体检测细胞上清液elafin蛋白的表达，Northern膜杂交检测elafin基因mRNA的表达量等方法进行研究．发现：①原代培养人气道上皮细胞约5d即可作为靶细胞。②转染Ad—elafin24h时，荧光显微镜下可见上皮细胞内有少量的GFP表达，随着时间的延长，细胞内荧光逐渐增加，96h时GFP荧光阳性的细胞约为80％。③转染24～96h，实验组细胞上清液elafin的含量高于对照组（p〈0．01）；同时elafinmRNA的水平高于对照组（P〈0．05，0．01），且elafin表达及elafinmRNA水平在一定时间内呈时间依赖性关系。结论：Elafin重组腺病毒表达载体Ad—elafin成功转染人气道上皮细胞，目的基因不仅能在上皮细胞中表达，而且elafin蛋白具有细胞外分泌作用．

Objective To realize the characteristics of primary airway epithelial cells transfected with Ad-elafin, and to research of the expression of Ad-elafin in these cells. Methods The primary airway epithelial cells was employed in this study, and then was transfected with Ad-elafin. The GFP expression in airway epithelial cells was observed by fluorescence microscopy, the authors have examined the effect of human elafin on the transcription and secretion protein with the light density integral of Northern blot hybridization membrane from cellular RNA and ELISA techniques. Results. The GFP expression was observed in airway epithelial cells transfected with Ad-elafin for 24 hours, and almost 80% cells has been expressed GFP for 96 hours,the concentrations ranging from （2.2±0.4） ng. mL^-1 and （8.8±1.7） ng. mL^-1resulted in a significant increase in the secreted elafin Protein as measured in the cell supernatant, and also the light density integral of Northern blot hybridization membrane ranging from 0.42±0.05 and 9.86±0.09 resulted in a significant increase in the transcription elafin gene as analyzed in cellular mRNA. Moreover the elafin protein secreted and mRNA expressed in a time-dependent manner. Conclusions. The human elafin gene can be expressed and elafin protein can be secreted in the human primary airway epithelial cell with transfecting Ad-elafin.