防御素5和LL37真核重组质粒构建及转染人阴道上皮细胞的研究

Construction of Recombinant Eukaryotic Plasmids of HD5 and LL37 and Their Transfection and Expression in Human Vaginal Epithelial Cells

目的:构建防御素5(HD5)和LL37的真核重组质粒并瞬时转染人阴道上皮细胞,以期探讨阴道上皮细胞抵抗微生物感染的机制。方法:(1)从人阴道上皮细胞中提取总RNA,经逆转录-聚合酶链反应(RT-PCR)扩增出HD5和LL37的cDNA,并将其插入真核表达载体pcDNA3.1(+)-EGFP中,构建重组质粒pcDNA3.1(+)/HD5-EGFP和pcDNA3.1(+)/LL37-EGFP。(2)经组织块法原代培养人阴道上皮细胞并传代,将2种质粒分别或联合转染人阴道上皮细胞,转染6、12、24和48 h后,采用荧光显微镜检测细胞转染情况,ELISA方法测定细胞培养上清液中HD5及LL37的表达情况。结果:成功构建了pcDNA3.1(+)/HD5-EGFP和pcDNA3.1(+)/LL37-EGFP真核表达载体,实现了HD5和LL37在阴道上皮细胞中表达,细胞培养上清中有HD5和LL37蛋白分子表达,且在转染24 h时表达量最高。联合转染组的HD5和LL37水平高于单独转染组,单独转染组高于未转染组,差异均有统计学意义(均P<0.001)。LL37组和联合转染组的LL37水平,联合转染组的HD5水平均是6 h时分泌最低,24 h达高峰,然后呈下降趋势。HD5组的HD5水平随时间增加而呈增加趋势。结论:HD5和LL37成功转染入人阴道上皮细胞并成功表达,为研究重组HD5和LL37的抗菌功能及阴道上皮细胞先天免疫机制奠定了基础。

Objective： To construct eukaryotic expression vectors of human defensin 5 （HD5） and LL37 and transiently transfeet primary human vaginal epithelial cells, and the mechanism of vaginal epithelial ceils against microbial infection thereof. Methods： （1） Total RNA was extracted from human vaginal epithelial cells. The cDNA encoding the HD5 and LL37 was amplified through RT-PCR, which was inserted into pcDNA3.1（＋）-EGFP, a expression vector. （2）The vaginal epithelial cells were cultured primarily by serum-free keratinoeyte medium and tissue piece culture method. These two kinds of recom- binant eukaryotic plasmids were separately transfected or co-transferred into vaginal epithelial ceils. After 6, 12, 24 and 48 h the transfection efficacy was observed by fluorescence microscope. The supernatant of every group was collected to determine ex- pressions of HD5 and LL37 by ELISA method. Results： This study constructed pcDNA3.1（＋）/HDS-EGFP and pcDNA3.1（＋）/ LL37-EGFP eukaryotie expression vector, and achieved the expression of HD5 and LL37 in vaginal epithelial cells. Expressions of HD5 and LL37 proteins were found in the cell culture supernatant. The highest expressions of HD5 and LL37 protein were found 24 h after transfeetion. The levels of HD5 and LL37 were significantly higher in co-transfection group than those of single transfection group, and levels were significantly higher in single transfection group than those of untransfected group （P 〈 0.001）. Except for the untransfected group, the lowest secretion was at 6 h and reached the peak at 24 h, and then contin- ued the downward trend. Conclusion： HD5 and LL37 were successfully transfected into human vaginal epithelial cells, which was the basis for the further study the antibacterial function of recombinant HD5 and LL37and vaginal epithelial cell innate immune system.