Amplification of recombinant adenovirus vector carrying rat angiotensinⅡtype 2 receptor and transfection in INS-1 cells
10.7644/j.issn.1674-9960.2014.12.002
- VernacularTitle:血管紧张素Ⅱ2型受体重组腺病毒的扩增及其在INS－1细胞中的转染
- Author:
Xiaojing LI
;
Min LIU
;
Yan WANG
;
Yi HU
;
Shinan YIN
;
Yiming MU
- Publication Type:Journal Article
- Keywords:
receptor,angiotensin,type 2;
adenoviridae;
HEK 293A cell line;
rat;
insulinoma INS-1 cell line
- From:
Military Medical Sciences
2014;(12):927-931,935
- CountryChina
- Language:Chinese
-
Abstract:
Objective To amplify the recombinant adenovirus vector carrying rat angiotensin Ⅱ type 2 receptor (AT2R) gene using human embryonic kidney (HEK) 293A cell lines and to construct a pancreatic islet βcell model overexpressing AT2R by transfecting the adenovirus vector into rat insulinoma (INS-1) cell lines.Methods Recombinant adenovirus vector Ad-G-AT2R-EGFP and control vector Ad-CMV-EGFP were amplified with HEK 293A cells and the titer of the adenovirus was detected .After both adenovirus vectors were transfected into INS-1 cells,AT2R and angiotensin Ⅱtype 1 receptor(AT1R) gene expressions were tested using real-time PCR, Western blotting, immunofluorescence staining and confocal laser-scanning microscopy .Results The titer of amplified Ad-G-AT2R-EGFP and Ad-CMV-EGFP was re-spectively 9 ×109 pfu/ml and 8 ×109 pfu/ml.Transfection of Ad-G-AT2R-EGFP into INS-1 cells induced an increase in AT2R mRNA expression in a dose-dependent manner , and significantly increased AT2R mRNA and protein expression compared with Ad-CMV-EGFP-or mock-transfection.Conclusion The recombinant adenoviral vector carrying AT2R gene is successfully amplified and an INS-1 cell model overexpressing AT2R is constructed by transient transfection , which can contribute to further study of the role of AT2R in pancreatic islet βcells.
