Construction of recombinant plasmids with mutant C-kit cDNA in gastrointestinal stromal tumor
- VernacularTitle:胃肠道间质瘤中C-kit原癌基因突变体的构建及功能研究
- Author:
Qiang XIE
;
Xiaohong LIU
;
Chenguang BAI
;
Fei FENG
;
Dali MA
- Publication Type:Journal Article
- Keywords:
gastrointestinal stromal tumor;
C-kit protooncogene;
mutation
- From:
Academic Journal of Second Military Medical University
1985;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct the recombinant eukaryotic expression vector plasmids with mutant C-kit cDNA and to study the effect of the mutant C-kit gene on cell proliferation and cell cycle in gastrointestinal stromal tumor (GIST). Methods: Wild-type C-kit cDNA was cloned from human embryonic brain tissue by RT-PCR technique.Site-directed mutagenesis of the wild type C-kit cDNA was performed according to the C-kit mutations we cloned. Recombinant plasmids were stably transfected into human embryonic kidney cell line and the cells expressing mutant C-kit were selected by special cell culture medium containing G418.Expressions of C-kit protein of the transfectants were detected by Western blot.Cell proliferation and cell cycle of the transfectants were detected by MTT clolorimetic assay and flow cytometry,respectively.Whether HEK cell with mutated C-kit cDNA could grow autonomously in nude mice or not was also detected. pcDNA3 vector transfected and recombinant plasmids with wild-type C-kit cDNA transfected HEK cell were used as the control groups. Results: The mutant C-kit cDNA was obtained by site-directed mutagenesis of the wild type C-kit cDNA. Compared with the 2 control groups ,the growth rate and proliferative activity of the HEK cells with mutant C-kit cDNA were increased significantly.The analysis of cell cycle showed that more HEK cells with mutanted C-kit cDNA remained in proliferation phase (S+G 2-M)than the groups without mutated C-kit cDNA.HEK cells with the mutated C-kit also grew autonomously in nude mice.Conclusion: Mutation of C-kit gene can increase proliferation of human cells,causing malignant transformation of human normal cells,which may play an important role in the malignant transformation of GIST.
