RNA干扰抑制胸膜间皮细胞水通道蛋白1表达的实验研究  

Inhibition of the aquaporin-1 gene expression by RNA interference:experiment with cultured rat pleural mesothelial cells

作　　者：张伟[1] 谢灿茂[1] 李志平[1] 朱智文[1] 严英硕[1] 杜宏春[1] 

机构地区：[1]中山大学附属第一医院呼吸科,广州510080

出　　处：《中华医学杂志》2007年第25期1773-1777,共5页National Medical Journal of China

基　　金：教育部博士点基金(20050558060);中国博 士后科学基金(2005038182);广东省自然科学基金(5300762)

摘　　要：目的研究 RNA 干扰技术对大鼠胸膜间皮细胞水通道蛋白1(AQP1)基因表达的影响,探讨应用该技术治疗胸腔积液的可行性。方法体外培养大鼠胸膜间皮细胞,细胞鉴定后,采用免疫细胞化学、逆转录-聚合酶链反应(RT-PCR)方法检测 AQP1表达;设计并构建2个靶向大鼠AQP1基因的短发夹 RNA(shRNA)重组质粒,脂质体转染大鼠胸膜间皮细胞48 h 后,采用 RT-PCR 及Western 印迹方法分别在 mRNA 和蛋白质水平检测 AQP1基因表达的抑制效果。结果胸膜间皮细胞存在 AQP1表达,所设计的2个 AQP1-siRNA 重组质粒可不同程度地抑制 AQP1在胸膜间皮细胞中的表达,在 mRNA 水平抑制率分别为83.5%和90.9%;在蛋白质水平抑制率分别为41.2%和67.6%,与空白对照组和阴性对照组比较,其差异有统计学意义(P<0.01)。结论质粒介导的 RNA干扰可明显抑制大鼠胸膜间皮细胞 AQP1基因的表达,且抑制率具有序列相关性,是潜在的胸腔积液基因治疗新方法。Objective To investigate the influence of RNA interference （RNAi） on the expression of aquaporin-1 （ AQP1 ） gene and to investigate the feasibility of gene therapy for pleural effusion. Methods Two recombinant plasmids with shRNAs targeting the AQP1 gene, AQPl-l-pGenesil and AQP1-2-pGenesil-1 were constructed. Pleural mesothelial cells were obtained from rats, cultured, and randomly divided into 5 groups： blank control group, Lipofectamine 2000 control group, HK negative control group, AQPI-1- pGenesil-1 transfected group, and AQP1-2-pGenesil-1 transfected group. RT-PCR and Western blotting were used to detect the mRNA and protein expression of AQP1. Results The mRNA expression levels of aquaporin-1 of the AQPl-l-pGenesil-1 and AQP1-2-pGenesil-1 transfected groups were inhibited by 83.5% and 90.9% respectively, and the protein expression levels of the AQPl-l-pGenesil-1 and AQP1-2-pGenesil- 1 transfected groups were inhibited by 41. 2% and 67. 6% respectively. Conclusion RNAi can successfully inhibit the expression of AQP1 and has the feature of sequence correlation of shRNA in the cultured rat pleural mesothelial cells. It may be used as a potential new approach for gene therapy of pleural effusion.

关 键 词：胸膜 上皮细胞 基因表达 RNA干扰 水通道蛋白1 

分 类 号：R346[医药卫生—基础医学]