西安交通大学学报（医学版）
西安交通大學學報（醫學版）
서안교통대학학보（의학판）
JOURNAL OF XI'AN JIAOTONG UNIVERSITY(MEDICAL SCIENCES)
2015年
3期
294-298
,共5页
白亮%李倩薇%赵四海%贾玉枝%REDDY Janardan K%刘恩岐
白亮%李倩薇%趙四海%賈玉枝%REDDY Janardan K%劉恩岐
백량%리천미%조사해%가옥지%REDDY Janardan K%류은기
PPARγ%MED1%重组腺病毒%CsCl 梯度离心
PPARγ%MED1%重組腺病毒%CsCl 梯度離心
PPARγ%MED1%중조선병독%CsCl 제도리심
PPARγ%MED1%recombinant adenovirus%CsCl gradient centrifuge
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 PPARγ mRNA 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。
目的：製備大量 PPARγ與 MED1腺病毒,探討其生物學功能。方法以實驗室現有的腺病毒原液為材料,用HEK293a 細胞包裝腺病毒,CsCl 梯度離心法純化病毒,A260法測定病毒顆粒,併通過細胞感染、小鼠活體尾靜脈註射病毒等實驗,用 HE 染色法、Real-time PCR 及免疫組化法進一步鑒定病毒的生物學功能。結果穫得 Ad/LacZ、Ad/PPARγ和 Ad/MED1,其濃度分彆為2.51×1012、1.57×1012、2.59×1012 VP/mL。C57BL/6J 小鼠經尾靜脈註射 Ad/PPARγ,小鼠齣現脂肪肝,肝細胞聚集大量脂滴,且 PPARγ mRNA 和蛋白錶達顯著增加。同樣,用 Ad/MED1感染3T3-L1細胞或給小鼠尾靜脈註射 Ad/MED1,MED1錶達水平都顯著升高。結論 Ad/PPARγ、Ad/MED1及對照Ad/LacZ 成功製備,為體外細胞病毒感染以及活體研究 PPARγ與 MED1的基因功能以及網絡調控奠定瞭實驗基礎。
목적：제비대량 PPARγ여 MED1선병독,탐토기생물학공능。방법이실험실현유적선병독원액위재료,용HEK293a 세포포장선병독,CsCl 제도리심법순화병독,A260법측정병독과립,병통과세포감염、소서활체미정맥주사병독등실험,용 HE 염색법、Real-time PCR 급면역조화법진일보감정병독적생물학공능。결과획득 Ad/LacZ、Ad/PPARγ화 Ad/MED1,기농도분별위2.51×1012、1.57×1012、2.59×1012 VP/mL。C57BL/6J 소서경미정맥주사 Ad/PPARγ,소서출현지방간,간세포취집대량지적,차 PPARγ mRNA 화단백표체현저증가。동양,용 Ad/MED1감염3T3-L1세포혹급소서미정맥주사 Ad/MED1,MED1표체수평도현저승고。결론 Ad/PPARγ、Ad/MED1급대조Ad/LacZ 성공제비,위체외세포병독감염이급활체연구 PPARγ여 MED1적기인공능이급망락조공전정료실험기출。
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 1 2 VP/mL,1.57×10 1 2 VP/mL and 2.59 × 10 1 2 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 .
