PPARγ及其辅激活子MED1重组腺病毒的制备与生物学功能分析  

Preparation of recombinant adenoviruses of PPARγand its transcriptional coactivator MED1 and their biological function analysis

作　　者：白亮[1,2,3] 李倩薇[1,4] 赵四海[1,3] 贾玉枝 REDDY Janardan K 刘恩岐[1,3] 

机构地区：[1]西安交通大学医学部心血管中心动脉粥样硬化与脂代谢研究室,陕西西安710061 [2]Department of Pathology,Feinberg School of Medicine,Northwestern University [3]西安交通大学医学部医学实验动物中心,陕西西安710061 [4]陕西中医学院,陕西咸阳712046

出　　处：《西安交通大学学报（医学版）》2015年第3期294-298,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金(No.81200207);中央高校基本科研业务费专项资金资助~~

摘　　要：目的制备大量PPARγ与MED1腺病毒,探讨其生物学功能。方法以实验室现有的腺病毒原液为材料,用HEK293a细胞包装腺病毒,CsCl梯度离心法纯化病毒,A260法测定病毒颗粒,并通过细胞感染、小鼠活体尾静脉注射病毒等实验,用HE染色法、Real-time PCR及免疫组化法进一步鉴定病毒的生物学功能。结果获得Ad/LacZ、Ad/PPARγ和Ad/MED1,其浓度分别为2.51×1012、1.57×1012、2.59×1012 VP/mL。C57BL/6J小鼠经尾静脉注射Ad/PPARγ,小鼠出现脂肪肝,肝细胞聚集大量脂滴,且PPARγmRNA和蛋白表达显著增加。同样,用Ad/MED1感染3T3-L1细胞或给小鼠尾静脉注射Ad/MED1,MED1表达水平都显著升高。结论Ad/PPARγ、Ad/MED1及对照Ad/LacZ成功制备,为体外细胞病毒感染以及活体研究PPARγ与MED1的基因功能以及网络调控奠定了实验基础。Objective To obtain a large number of recombinant adenoviruses of PPARγ （Ad/PPARγ）and MED1 （Ad/MED1）and investigate the biological function of nuclear receptor PPARγand its coactivator mediator 1 （MED1）.Methods HEK293a cell line was amplified and used to package the adenovirus stocks （Ad/PPARγ or Ad/MED1）.CsCl gradient centrifuge was used to purify the adenovirus,and virus particles were determined by A2 60 .After cell infection or mouse injection with adenovirus,the biological function of Ad/PPARγ and Ad/MED1 was analyzed using HE staining,Real-time PCR or immunohistochemistry.Results We obtained control Ad/LacZ,Ad/PPARγand Ad/MED1,whose concentrations were 2.5 1×10^12 VP/mL,1.57×10^12 VP/mL and 2.59 × 10 ^12 VP/mL,respectively.Ad/PPARγinjected into the tail vein led to hepatic steatosis in C57BL/6J mice.HE and oil red O staining results showed that many lipid droplets accumulated in hepatocytes. Real time PCR and immunohistochemistry showed that the expressions of PPARγmRNA and protein were remarkably increased.Also, MED1 mRNA expression was significantly increased in 3T3-L1 cell line or C57BL/6J mice after Ad/MED1 treatment compared with that in control group. Conclusion Ad/PPARγ and Ad/MED1 were prepared successfully,which provides materials for cell infection,especially for gene function and network regulation in vivo .

关 键 词：PPARΓ MED1 重组腺病毒 CsCl梯度离心 

分 类 号：Q812[生物学—生物工程]