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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 mouse adiponectin (ADPN) in the liver.Methods: To construct an eukaryotic expression vector of mouse adiponectin gene (mAd) with liver-specific promoter hAATp and enhancer ApoEHCR pAA-neo-mAd. After the vector was expressed in COS-7 cells by using green fluorescent protein (GFP) as a reporter gene, hydrodynamic method was introduced into mouse liver by tail vein injection. The expression of adiponectin in liver and its expression efficiency were identified by mAd mRNA quantitative (semi-quantitative RT-PCR), immunohistochemistry and serum adiponectin levels (quantitative ELISA). Results: The results of RT-PCR in liver tissue showed that the expression of mAd mRNA could be detected in the liver within 12 h after injection, and could be continuously expressed for more than 6 weeks. Serum adiponectin levels increased significantly after 24 h, peaked at 48 h and remained significantly higher than normal after 6 weeks. Liver adiponectin immunohistochemical staining was positive. Conclusion: The hydrodynamic method can effectively introduce the plasmid vector pAA-neo-mAd into the liver of mice and efficiently and stably express recombinant mouse adiponectin in mouse liver.