雾化吸入肺内转染人β防御素2基因的实验研究  

Transfection of human ± defensin 2 gene into the lung by aerosol inhalation： experiment with rats

作　　者：雷撼[1] 钱桂生[1] 吴国明[1] 黄桂君[1] 

机构地区：[1]第三军医大学附属新桥医院全军呼吸内科研究所,重庆400037

出　　处：《中华医学杂志》2008年第20期1425-1428,共4页National Medical Journal of China

基　　金：国家自然科学基金资助项目（30270592）

摘　　要：目的探讨以雾化吸入方式向大鼠气管上皮细胞转染人β防御素2基因（hBD2）的可行性。方法薄膜法制备新型阳离子脂质体，并按不同重量比与pLXSN-hBD2重组质粒混合构成脂质体／质粒复合物。将筛选出的适当重量比的脂质体／质粒复合物以雾化吸入方式对大鼠进行肺内转染（实验组，n=11）；对照组（n=5）转染空白载体pLXSN。以PCR法和蛋白质印迹法检测雾化吸入后大鼠气管上皮细胞中hBD2的表达。结果雾化后不同重量比脂质体／质粒复合物的微囊结构均受到明显影响，以10：1者结构破坏最小，更适于雾化。PCR检测显示雾化吸入后2组大鼠气管上皮细胞基因组DNA中均整合入相应质粒。蛋白质印迹法检测显示实验组大鼠转染后2d气管上皮细胞hBD2蛋白表达量最高（4866．9±148．2），随时间延续而逐渐降低，21d表达量为3．2±1．5；而对照组无hBD2蛋白表达。结论新型阳离子脂质体雾化后具有转染活性，pLXSN-hBD2重组质粒能通过雾化吸入方式转染大鼠气管上皮细胞并获得表达，并且hBD2蛋白的表达能持续一段时间。Objective To investigate the feasibility to transfect human β defensin 2 （hBD2） gene into the lung so as to enhance the endogenous hBD2 expression to defend infections diseases. Methods Recombinant plasmid containing hBD2 gene, pLXSN-hBD2, was mixed with a new cationic liposome prepared by film at different weight ratios. Sixteen Wistar rats were divided into 2 groups： experimental group （ n = 11 ）, undergoing aerosol inhalation of liposome/pLXSN-hBD2 complex, and control group （ n = 5 ）, undergoing aerosol inhalation of blank vector pLXSN. The tracheae were taken out from 5 rats of the experiment and control groups each 2 days later, and from 2 rats of the experimental group 6, 15, and 21 days later respectively so as to obtain the epithelial cells of trachea. DNA was extracted from the tracheal epithelial cells and PCR was used to examine the transfection and integration of hBD2. Western blotting was used to detect the protein expression of hBD2. Results Aerosolization impacted obviously the mierocapsule structure of liposome/plasmid complexes, and there was the least structural destruction of complex at a ratio of 10：1 that suited for aerosolization best. After the inhalation the relevant plasmids were all successfully integrated into the epithelial cells in both groups. Protein expression of hBD2 was not detected in the control group and the hBD2 protein expression level 2 days after transfection of the experimental group was 4866.9±148.2, and then decreased gradually, and reached 3.2±1.5 twenty-one days after the transfection. Conclusion The recombinant plasmid pLXSN-hBD2 can be transfect into the airway epithelial cells via aerosol inhalation and the expression of hBD2 sustains for a period of time.

关 键 词：Β防御素 脂质体 转染 上皮细胞 吸入法 

分 类 号：R686[医药卫生—骨科学]