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- 2015
PPARγ及其辅激活子MED1重组腺病毒的制备与生物学功能分析
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Abstract:
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、2.59×1012VP/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。
ABSTRACT: 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 PPARγ and 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 A260. 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/PPARγ and Ad/MED1, whose concentrations were 2.51×1012 VP/mL, 1.57×1012 VP/mL and 2.59×1012 VP/mL, respectively. Ad/PPARγ injected 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 PPARγ mRNA 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
| [1] | ROSEN ED, WALKEY CJ, PUIGSERVER P, et al. Transcriptional regulation of adipogenesis[J]. Genes Develop, 2000, 14(11):1293-1307. |
| [2] | CRAWFORD SE, QI C, MISRA P, et al. Defects of the heart, eye, and megakaryocytes in peroxisome proliferator activator receptor-binding protein (PBP) null embryos implicate GATA family of transcription factors[J]. J Biol Chem, 2002, 277(5):3585-3592. |
| [3] | LUO J, DENG ZL, LUO X, et al. A protocol for rapid generation of recombinant adenoviruses using the AdEasy system[J]. Nat Protoc, 2007, 2(5):1236-1247. |
| [4] | MAJHEN D, CALDERON H, CHANDRA N, et al. Adenovirus-based vaccines for fighting infectious diseases and cancer: progress in the field[J]. Hum Gene Ther, 2014, 25(4):301-317. |
| [5] | WANG N, YIN R, LIU Y, et al. Role of peroxisome proliferator-activated receptor-gamma in atherosclerosis: an update[J]. Circ J, 2011, 75(3):528-535. |
| [6] | BLASCHKE F, TAKATA Y, CAGLAYAN E, et al. Obesity, peroxisome proliferator-activated receptor, and atherosclerosis in type 2 diabetes[J]. Arterioscler Throm Vasc Biol, 2006, 26(1):28-40. |
| [7] | GE K, GUERMAH M, YUAN CX, et al. Transcription coactivator TRAP220 is required for PPAR gamma 2-stimulated adipogenesis[J]. Nature, 2002, 417(6888):563-567. |
| [8] | HE TC, ZHOU S, DA COSTA LT, et al. A simplified system for generating recombinant adenoviruses[J]. Proc Natl Acad Sci USA, 1998, 95(5):2509-2514. |
| [9] | SILVA AC, FERNANDES P, SOUSA MF, et al. Scalable production of adenovirus vectors[J]. Methods Mol Biol, 2014, 1089:175-196. |
| [10] | MITTEREDER N, MARCH KL, TRAPNELL BC. Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy[J]. J Virol, 1996, 70(11):7498-7509. |
| [11] | DEAL C, PEKOSZ A, KETNER G. Prospects for oral replicating denovirus-vectored vaccines[J]. Vaccine, 2013, 31(32):3236-3243. |
| [12] | LI H, GADE P, NALLAR SC, et al. The Med1 subunit of transcriptional mediator plays a central role in regulating CCAAT/enhancer-binding protein-beta-driven transcription in response to interferon-gamma[J]. J Biol Chem, 2008, 283(19):13077-13086. |
| [13] | GRAHAM FL , PREVEC L. Adenovirus-based expression vectors and recombinant vaccines[J]. Biotechnology, 1992, 20:363-390. |
| [14] | WANG YX. PPARs: diverse regulators in energy metabolism and metabolic diseases[J]. Cell Res, 2010, 20(2):124-137. |
| [15] | REDDY JK. Peroxisome proliferators and peroxisome proliferator-activated receptor alpha: biotic and xenobiotic sensing[J]. Am J Pathol, 2004, 164(6):2305-2321. |
| [16] | TONTONOZ P, HU E, SPIEGELMAN BM. Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor[J]. Cell, 1994, 79(7):1147-1156. |
| [17] | ZHU Y, QI C, JAIN S, et al. Isolation and characterization of PBP, a protein that interacts with peroxisome proliferator-activated receptor[J]. J Biol Chem, 1997, 272(41):25500-25506. |
