Construction of expression vector containing rat rCB1 gene and its influence on the apoptosis in human cervical cancer CaSki cell line
10.3969/j.issn.1005-4847.2015.02.010
- VernacularTitle:大鼠CB1基因真核表达载体的构建及其对CaSki细胞凋亡的影响
- Author:
Lei YAN
;
Jing LI
;
Tingting ZHAO
;
Huijuan WANG
;
Lei WANG
;
Guoqi LAI
- Publication Type:Journal Article
- Keywords:
rCB1;
HEK293 cells;
CaSki cells;
Cell apoptosis
- From:
Acta Laboratorium Animalis Scientia Sinica
2015;(2):153-158
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct rCB1 gene eukaryotic expression vector, detect its expression in the cell, and explore its influence on apoptosis in human cervical cancer CaSki cells.Methods The total RNA was extracted from rat brains.The rCB1gene was amplified by RT-PCR.The pcDNA3.1(+)-rCB1 was constructed by enzyme digestion, purifi-cation, bind the PCR purification products and pcDNA3.1 (+) DNA.The pcDNA3.1 (+)-rCB1 plasmid was transfect-ed into HEK293 and CaSki cells by liposomes.The expression and localization of rCB1 were detected by Western blot and immunofluorescence combined with confocal laser scanning microscopy.The apoptosis rate of CaSki cells was detected by flow cytometry.The expression of rCB1, Bcl-2, Bax and Bad was detected by Western blot and real-time fluorescence quantitative RT-PCR (qRT-PCR).Results The 5300 bp pcDNA3.1(+) and 1500 bp rCB1 were obtained after diges-ting the pcDNA3.1 ( +)-rCB1.The result of sequencing was in agreement with the sequence of rCB1 gene ( NM_012784.4 ) .The rCB1 expressed in the membrane and cytoplasm when pcDNA3.1 (+)-rCB1 plasmid was transfected into HEK293 cells.The apoptosis rate of rCB1 group was increased compared with the blank group when pcDNA3.1 (+)-rCB1 plasmid was transfected into CaSki cells (P<0.05).Compared with the blank group, rCB1 gene upregulated the expres-sion of Bax and Bad, and suppressed the expression of Bcl-2.The statistical difference was significant ( P <0.05). Conclusions The pCDNA3.1(+)-rCB1 eukaryotic expression vector is constructed successfully.It is found that rCB1 is expressed in membrane and cytoplasm of HEK293 cells.rCB1 can significantly promote the apoptosis in cervical cancer CaSki cells by up-regulating the expression of Bax and Bad, and down-regulating the expression of Bcl-2 as well.
