Construction of Dickkopf1-expressing recombinant adenovirus and its effect on ?-catenin expression in A375 cells
- VernacularTitle:人Dickkopf1基因腺病毒载体的构建及感染黑色素瘤后?-catenin表达的研究
- Author:
Hengjun CHEN
;
Hui LI
;
Jin CHEN
;
Xiaojuan LU
- Publication Type:Journal Article
- Keywords:
DKK1 genes;
adenovirus;
melanoma;
signal transduction;
Wnt proteins
- From:Journal of Third Military Medical University
2002;0(12):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a recombinant adenovirus vector expressing Dickkopf1 (DKK1) (Ad-DKK1) and study the expression of ?-catenin in melanoma cells A375 after Ad-DKK1 infection. Methods DKK1 gene was amplified by PCR and subcloned to shuttle plasmid Adeasy by molecular cloning and adenovirus recombination technique,then converted in BJAdeasy cells. The recombinant adenovirus plasmid was generated. After cut by PacⅠ enzyme,the recombinant adenovirus plasmid Ad-DKK1 was packaged and amplified in HEK293 cells. Ad-DKK1 effect group,Ad-SimDKK1 effect group,Ad-GFP effect group,Ad-RFP effect group and blank group were used to infect the A375 cells in multiplicity of infection (MOI) 80,and cell morphology was observed. The expression of DKK1 and ?-catenin in A375 cells was detected by RT-PCR. Cell vitality was detected by MTT assay. Results Ad-DKK1 was proved to be recombined successfully by identification with restriction enzyme and sequencing. The infection was efficient (80%) in A375 cells. The expression of DKK1 had significant difference between Ad-DKK1 effect group (1.638?0.067) and other 4 groups (0.718?0.086,1.424?0.125,1.414?0.089 and 1.423?0.088) (P
