ABSTRACT
Objective: To establish a HEK-293T cell model with sodium channel function similar to that in PRKAG2 syndrome. Methods: Vectors ofPRKAG2 gene and SCN5A gene were constructed and were used to co-transfect HEK-293T cells. Immunofluorescence, real-time PCR and Western blotting analysis were used to examine the transfection results and the expression of interested mRNA and protein. Results: Imunofluorescence findings showed red fluorescence and green fluorescence in HEK-293T cells 48 hours after co-transfection. Real-time PCR and Western blotting analysis showed SCN5A expression in pure SCN5A group, SCN5A + wild-type group, SCN5A + R302Q mutant group, and SCN5A + G100S mutant group, but not in the blank control group, showing significant significance (P<0.05). Expression of PRKAG2 gene and protein in SCN5A+PRKAG2 group was significantly higher than those in the blank control group, pure SCN5A group, SCN5A+R302Q, and SCN5A+G100S groups (P<0.05). Conclusion: We have successfully established a HEK-293T cell model with sodium channel function similar to that in PRKAG2 syndrome, paving a way for future study.
ABSTRACT
Objective: To establish a human lung cancer cell line A549 highly expressing human 8-oxoguanine-DNA glycosylase (hOGG1) by co-transfecting pcDNA 3.1 (+)/Myc-HisA-hOGG1 and PGL3 promoter, and to observe the biological behavior of the transfected cells. Methods: PcDNA3.1 (+)/Myc-His A-hOGG1 and PGL3 promoter were steadily co-transfected into A549 cells via mediation of Fu GENE 6 (transfected group); untransfected cells served as blank control and cells transfected with PGL3 + pcDNA3.1 (+)/Myc-HisA served as negative control. The hOGG1 mRNA expression in A549 cells was detected by Bio-luciferase activity and the hOGG1 protein expression by Western blotting analysis. Cells in the three groups were exposed to hyperoxia condition, and the morphological changes were observed by phase-contrast microscope. Comet cell rate and olive tail moment of cells were tested after different exposure periods. After exposure, the cells were incubated for 0, 60, 120, and 180 min, and the same indices were determined by modified comet assay; changes of DNA oxidative damage markers 8-hydroxy-desoxoguanosine (8-OHdG) was tested at the same time. Results: The bio-luciferase activity was stable in the co-transfected cells. Western blotting analysis showed that the expression of hOGG1 protein in co-transfected A549-T cells was significantly higher than those in the other two groups, indicating the successful establishment of hOGG1 highly expressing cells. Under the same hyperoxia condition, the morphological changes of transfected cells were greatly alleviated, and the Comet cell rate and olive tail moment of cells were obviously lower than those of the other two groups(P< 0.05). Transfected A549-T cells had significantly increased ability of DNA repair and shorter repair time compared with cells in the other two groups (P<0.05). Furthermore, the level of 8-OHdG in transfected A549-T cells was significantly lower than those of the other two groups under the same hyperoxia condition, indicating a significantly higher ability of DNA damage resisting and repair (P<0.05). Conclusion: High hOGG1 expression can decrease the cell sensitivity to DNA damage and improve the repair ability of cells.
ABSTRACT
Objective To observe the inhibitory effects of local co-transfection of tissue-type plasminogen activator(tPA) gene and proliferating cell nuclear antigen antisense oligodeoxynucleotides(PCNA-ASODN) on the intima proliferation and restenosis of autograft artery in rabbits. Methods One hundred and twenty male Zelanian rabbits were randomly divided into four groups(n=30, in each group): control group, PCNA-ASODN group, tPA group and tPA+PCNA-ASODN group. The left and right external iliac arteries (length 1.0 cm) were transplanted reciprocally. The transplanted arteries were respectively soaked in lipofection, PCNA-ASODN, pBudCE4.1/tPA and pBudCE4.1/tPA+PCNA-ASODN solution about 15 minutes. The transplanted arteries were sutured with 9-0 sutures soaked in PCNA-ASODN and pBudCE4.1/tPA solution. Each group were divided into five subgroups(n=6, in each subgroup) according to the sacrifice time (3 d, 7 d, 14 d, 28 d and 56 d after operation). On every sacrifice time point, the vascular specimens were harvested. The thrombocyte assembling and thrombus forming lining vessel wall were observed by scanning electron microscope. The pathological morphology of transplanted arteries were observed under microscope(HE). The intimal areas and stenosis ratio(%) of transplanted arteries were calculate and analyzed statistically among groups by computer system. The mRNA expression of tPA gene in transplanted ressel wall was detected with vevere transcription-PCR(RT-PCR). The number of PCNA positive cells in transplanted vessel wall was counted by SP immunochemisty. Results The mRNA expression of tPA gene in the transplan-ted vessel wall in tPA and tPA+PCNA-ASODN groups was higher than that of the other two groups (P