中文摘要：

目的：构建哺乳动物细胞表达的SPLUNC1重组蛋白，通过体外实验验证其杀菌／抑菌功能，并进一步研究其机制是否与SPLUNC1蛋白结合细菌脂多糖有关，从而为其将来临床应用奠定基础。方法：将SPLUNC1克隆入pCMV-tag4A哺乳动物表达载体，稳定转染鼻咽癌HNE1细胞株；收集细胞培养上清液，用其处理从患者上呼吸道分离的绿脓杆菌，比较转染SPLUNC1基因的细胞培养上清与转染空白载体的细胞培养上清对细菌克隆形成率的影响。同时将脂多糖包被96孔板，与SPLUNC1蛋白共同孵育，ELISA法检测SPLUNC1是否与细菌脂多糖结合；利用FITC标记的脂多糖干预转染SPLUNC1基因及空白栽体的细胞，观察细胞内脂多糖与SPLUNC1的结合作用。结果：转染SPLUNC1基因的细胞培养上清能显著抑制绿脓杆菌在LB软琼脂板上形成集落；体外试验表明，SPLUNC1能与细菌脂多糖结合，但结合效率低：HNE1细胞经转染SPLUNC1后，细菌脂多糖摄取明显增加。结论：重组SPLUNC1蛋白能经转染细胞分泌到培养上清液中，具有结合细菌脂多糖，杀灭或抑制上呼吸道绿脓杆菌的功能，从而可能具有重要的临床应用价值。

英文摘要：

Objective To express the recombinant SPLUNC1 protein in HNE1 cells and to study its function of bactericidal and binding to lipopolysaccharide （LPS）. Methods Full length of SPLUNC1 gene was cloned into pCMV-tag4A vector and stably transfected into HNE1 cell lines, the supernatant of cell cultures was collected. After being treated with the supernatant, the Pseudomonas aeruginosa was seeded to LB soft agar plate, and the bacteria clones were counted and analyzed. For in vitro LPS binding assay, LPS was coated to 96-well plates. We incubated in the plate with SPLUNC1 protein, and detected the binded SPLUNC1 protein by ELISA. Incubating the FITC-LPS with the SPLUNC1 stably transfected or control cells, the intracellular intensity of fluorescence was observed under the fluorescence microscope. Results SPLUNC 1 inhibited the bacteria clone formation obviously. Although the binding efficiency of LPS and SPLUNC1 in vitro was very low, more FITC-LPS entered into the SPLUNC1 stably transfected cells. Conclusion SPLUNC1 can inhibit the growth of Pseudomonas aeruginosa and bind LPS, and play an important defensive role in innate immunity of the upper airway.