脂联素在小鼠肝脏的高效稳定表达  

Efficient and stable expression of adiponectin in mouse liver

作　　者：王会中[1] 付秋霞[2] 陈竞新[2] 贾帅争[2] 詹林盛[2] 孙红琰[2] 王全立[3] 

机构地区：[1]解放军第305医院,北京100017 [2]军事医学科学院野战输血研究所,北京100850 [3]解放军总医院输血科,北京100853

出　　处：《军事医学科学院院刊》2005年第5期439-443,共5页Bulletin of the Academy of Military Medical Sciences

摘　　要：目的:获得小鼠脂联素(ad iponectin,ADPN)在肝脏的高效稳定表达,方法:构建含肝脏特异性启动子hAATp和增强子ApoEHCR的小鼠脂联素基因(mAd)的真核表达载体pAA-neo-mAd。以绿色荧光蛋白(green fluo-rescent prote in,GFP)为报告基因证明该载体在COS-7细胞中表达后,流体动力法(hydrodynam ics-based procedure)通过尾静脉注射将其导入小鼠肝脏,通过肝脏mAd mRNA定量(半定量RT-PCR法)、免疫组织化学检测及血清脂联素水平测定(定量ELISA法)鉴定脂联素在肝脏的表达及表达效率。结果:肝组织RT-PCR的结果显示,注射后12 h肝脏内即可检测到mAd mRNA的表达,且能持续表达6周以上。24 h后血清脂联素水平显著升高,48 h达到峰值,一直持续6周后仍明显高于正常水平。肝脏脂联素免疫组化染色呈阳性。结论:流体动力法能有效地将质粒载体pAA-neo-mAd导入小鼠肝脏,并能在小鼠肝脏高效稳定地表达重组小鼠脂联素。Objective： To obtain efficient and stable expression of recombinant adiponectin （ADPN） in mouse liver. Methods：Engineered adiponectin eDNA （mAd） was cloned into plasmid vector pAA-neo, which was constructed by replacing the CMV immediate-early enhancer/promoter region of vector pCI-neo with the liver-specific enhancer, human apolipoprotein E locus control region （ApoE-HCR） , and human α1-antitrypsin promoter （hAATp）. After verifying the expression of adiponectin in COS-7 cells with green fluorescent protein （GFP） as a reporter, the plasmid pAA-neo-mAd was transferred into mice by hydrodynamics-based procedure. Then, the plasma adiponectin level, immunohistology of the liver, and quantity of adiponectin mRNA in the liver were assayed to identify the expression of adiponectin in the livers. Resuits ： After plasmid administration, plasma adiponectin was significantly increased and the adiponectin-positive in livers was confirmed by immunostaining. Conclusion： Adiponectin gene cloned into the plasmid pAA-neo-mAd could be efficiently transferred into the livers of mice via tail vein rapid injection ,resulting in high level of adiponectin expression.

关 键 词：脂联素 流体动力法 肝脏 质粒载体 体内转基因表达 

分 类 号：R346[医药卫生—基础医学]