重组人β防御素2在膀胱上皮细胞中的表达及活性检测  

Expression of recombinant human β-defensin 2 in human bladder epithelial cells and its antimicrobial activity

作　　者：赵俊丽[1] 王志平[2] 王俭勤[1] 

机构地区：[1]兰州大学第二医院肾病内科,兰州730030 [2]兰州大学第二医院泌尿研究所,兰州730030

出　　处：《山东大学学报（医学版）》2011年第4期43-46,51,共5页Journal of Shandong University:Health Sciences

基　　金：甘肃省自然科学基金资助项目(0803RJZA007);兰州大学中央高校基本科研业务费专项资金(lzujbky-2010-149)

摘　　要：目的探讨重组人β防御素2(hBD2)真核表达载体在人膀胱上皮细胞中的表达,并观察重组hBD2的体外抗菌活性。方法采用脂质体转染法将hBD2重组真核表达质粒pCAGG-hBD2导入无血清培养的人膀胱上皮细胞株T24细胞,利用RT-PCR法、Western blotting分别在核酸水平及蛋白质水平检测hBD2的表达。ELISA测定培养上清中hBD2的浓度,菌落计数法检测上清对尿路致病性大肠杆菌(UTI89)和克雷白杆菌(TOP52)的临床分离株的抗菌效果。结果 RT-PCR和Western blotting结果显示,转染后hBD2可在T24细胞中有效地表达。上清中hBD2的含量为(36.5±3.2)ng/106个细胞,菌落计数法显示,hBD2对UTI89和TOP52临床分离株有显著的杀菌作用。结论 hBD2重组真核表达载体脂质体法转染人膀胱上皮细胞后,可高效表达具抗菌活性的基因重组hBD2。Objective To examine expression of humanβ-defensin 2（hBD2） in human bladder epithelial cells（T24 cells）,and to observe the antimicrobial activity of recombinant hBD2 in vitro.Methods By using the cationic Liposome-TransFastTM Transfection Reagent,the plasmid vector carrying the hBD2 gene（pCAGG-hBD2） was transferred into T24 cells cultured in serum-free medium.Expression of hBD2 mRNA in T24 cells was determined by reverse transcription-polymerase chain reaction（RT-PCR）.HBD2 peptide expression was detected by Western blot and concentration of recombinant hBD2 in supernatants was measured by ELISA.Antimicrobial activity of recombinant hBD2 was assessed by the colony-forming unit（CFU） assay.Results hBD2 could be effectively expressed in T24 cells,and the concentration of recombinant hBD2 in supernatants was（36.5±3.2）ng/106 cells.Recombinant hBD2 in supernatants showed antimicrobial activity.Conclusion The recombinant eukaryotic expressing vector carrying the hBD-2 gene could be effectively expressed in human bladder epithelial cells,and recombinant hBD2 in supernatants has an obvious antimicrobial effect on some bacterial lines.

关 键 词：人Β防御素2 基因转染 人膀胱上皮细胞 

分 类 号：R392.11[医药卫生—免疫学] R691.3[医药卫生—基础医学]