| Objective: In this study,SGC-7901 cells,its resistant strain SGC-7901/VCR,si-XIAP/SGC-7901 and vector/SGC-7901 cells as the subjects to study whether DADS can enhance apoptosis and sensitivity of5-Fu in SGC-7901 cells,and further explore the possible relationship between DADS and SGC-7901 on the sensitivity of 5-FU chemotherapy to XIAP gene expression.Method: The XIAP plasmid was transfected into SGC-7901 cells by liposome,and the si-XIAP/SGC-7901 cells were established.MTT were used to detect the effect of proliferation in SGC-7901 cells and the sensitivity to 5-Fu under different concentration of 5-Fu.The apoptosis rate of each group was detected by flow cytometry.The expression of XIAP,caspase-3,Bcl-2 and P-gp were detected by immunofluorescence and Western blot.Results:1.Stable silencing of XIAP construction of SGC-7901 cellsThe purified XIAP vector and empty vector were transfected into SGC-7901 cells by pcDNA6.2-GW/EmGFP-miXIAP vector constructed by Yingwei Jieji Co.,Ltd..After transfection,the cells were observed by fluorescence microscopy.Fluorescent cells were added to the blast fungus for 14 days to form a fluorescent cell mass and then inoculated into a sixwell plate for continued culture2.Effects of DADS and Silencing XIAP on Proliferation in SGC-7901 Cells.MTT results confirmed that under different 5-FU concentrations.The inhibitory rate of proliferation of SGC-7901 was 2.092%,5.082%,13.602% and 20.179% respectively.The proliferation inhibition rate of SGC-7901 group was significantly increased by 4.291%,11.874%,22.745%,47.353%,59.942 %(P <0.05).The proliferation inhibition rates of SGC-7901 / VCR were 0.157%,0.470%,0.940%,2.031% and 4.073%,respectively(P <0.05).The proliferation inhibition rate of SGC-7901 /VCR was significantly increased by 1.229%,3.471%,7.809%,19.017%and 27.838%,respectively(P <0.05).Compared with SGC-7901 group,the proliferation inhibition rate of SGC-7901 / si-XIAP increased by2.882%,8.067%,18.022%,35.147% and 54.561%(P <0.05),It was confirmed that DADS and silencing XIAP could inhibit the proliferation of SGC-7901 cell line and increase the sensitivity to 5-Fu drug to a certain extent.3.Effects of DADS and Silencing XIAP on Apoptosis in SGC-7901 Cells.Flow cytometry test results confirmed:The apoptotic rates of SGC-7901 and SGC-7901 / VCR were 2.1% and 0.24%,respectively.The apoptotic rates of SGC-7901 and SGC-7901 / VCR groups were 27.8%and 18.7%,respectively,which were significantly higher than those in untreated group(P <0.05).The apoptotic rate of si-XIAP / SGC-7901 group Was 22.47%,which was significantly higher than that in SGC-7901 group(P <0.05).It was confirmed that DADS and silencing XIAP could increase the apoptosis rate of SGC-7901 cell line.4.Expression of XIAP,Caspase3,Bcl-2 and P-gp in each group of SGC7901 cells.Western blot analysis showed:The expression of XIAP,Bcl-2 and P-gp in SGC-7901 group and SGC-7901 / VCR group was downregulated(P <0.05),and Caspase3 was up-regulated(P <0.05).The expression of XIAP,BCL-2 and P-gp in SG-XIAP / SGC-7901 group was lower than that in SGC-7901 group(P <0.05),and Caspase3 was upregulated(P <0.05).Immunofluorescence showed:After treatment with DADS,SGC-7901 group and SGC-7901 / VCR group were weaker than those of untreated group,XIAP,Bcl-2 and P-gp fluorescence,and Caspase3 fluorescence enhanced in si-XIAP / SGC-7901 group compared with SGC-7901 group XIAP,BCL-2 and P-gp fluorescence weakened,Caspase3 fluorescence enhancement;consistent with the results of Western blot.Indicating that DADS and silencing XIAP can downregulate the expression of apoptotic proteins and drug-resistant proteins,up-regulate the expression of pro-apoptotic proteins.Conclusion:1.DADS can downregulateXIAP ? Bcl-2 and P-gp and upregulate Caspase3 to promote apoptosis and enhance the sensitivity of SGC7901 cells to 5-Fu.2.silencing XIAP through upregulating Caspase3 and downregulating Pgp and Bcl-2 can promote apoptosis and enhance the sensitivity of SGC7901 cells to 5-Fu. |
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