| Objective:To explore that whether overexpression PDX1 can effect the sensitility of gemcitabine to the PANC-1 cell.Methods:(1)The pdx1 gene was isolated from Hela cell,the expression vetor of PCMV6-Entry-pdx1 was constructed by inserting the CDS of pdx1 into p CMV6-Entry plasmid.(2)Cultured human pancreatic cancer PANC-1 cells,the cell growth in good condition in 24 well plate,PCMV6-Entry empty plasmid and PCMV6-Entry-PDX1 recombinant plasmid respectively transfected into the PANC-1 cells by Lipo3000.To form stable overexpression cells use G418 screened 2 weeks after culture 48 h.(3)When the overexpression cell culture expansion to be good condition,plated the cells in 24 well plates,the total protein was extracted to detect the expression of PDX1 and PI3K-AKT on whether there is any effect after cultured 72 h.(4)The overexpression cells were respectively planted into 96 –well plates,and treated with gemcitabine with 0,10,100,200,400 u M concentration cultured 48 h and 72 h.MTS method was used to measure the absorbance at 490 nm.Results:(1)The PDX1 gene was successfully acquired and constructed on the PCMV6-Entry empty plasmid to form PCMV6-Entry-PDX1 recombinant plasmid.Sequencing results showed that the recombinant plasmid sequence was correct.(2)Western blot resultes showed that PDX1 overexpressing PANC-1 cells were successfully constructed,and it could down-regulated the level of p-AKT,which could inhibit the PI3K-AKT signaling pathway.The MTS method to analyze the effect of gemcitabine on the expression of cell proliferation: compared with empty plasmid group,48 h and 72 h overexpression PDX1 inhibited cell proliferation in a time-dependent manner,but did not reflect the dose dependence.Conclusion: PDX1 can increase the sensitivity of PANC-1 to gemcitabine in pancreatic cancer cells,which is time dependent but not dose dependent.Objective:Study the influence of IL-15 on proliferation and apoptosis of pancreatic cancer PANC-1.Methods:(1)Detect that whether the PANC-1 cell express IL-15 receptors by PCR.Planted the PANC-1 cells into the cell plate and treated the cells with0,4,12,36,108,216 ng/ml concentrations of IL-15 after cultured 24 h,when the cell state is good.(2)Study the effect of different concentrations of IL-15 on proliferation of the PANC-1 cells by MTS assay.(3)Study the apoptosis effects of different concentrations of IL-15 treated the PANC-1 cell on 72 h by FCM.(4)Western blot analysis different concentrations of IL-15 on apoptosis protein(Bax,Bcl-2,Caspase-3)and cyclin Cyclin E.(5)Western blot analysis the effect on apoptosis protein(Bcl-2,Bax,Caspase 3)and cyclin Cyclin E with single concentration of IL-15 treated on 0,1,6,12,24,48,72 h.Results:(1)The PANC-1 cell expression the IL-15 RA but not the IL-15 RB and IL-15 RG.(2)The result of MTS showed that there was no significant difference between 48 h group and control group at different concentrations of PANC-1 cell,the lower dose in 72h group had not difference with the control group,but the high dose group was inhibited.(3)The results of flow cytometry showed that low dose of IL-15 did not promote the apoptosis of PANC-1 but the high dose can promote apoptosis.(4)Western blot analysis showed that IL-15 could enhance the expression of Bax and Caspase-3,and decrease the expression of Bcl-2 and p-AKT.(5)The results show that the expression of BAX and Caspase 3 increased and Bcl-2 decreased.The expression of Cyclin E increased at 6,12,24,48 h,while the expression decreased at 72 h and there was no statistical difference compared with the control group.The expression of p-AKT in 1,6,12,24,48,72 h decreased significantly compared with the control group,and the expression of p-STAT3 in 24,48 and 72 h increased significantly compared with the control group.Conclusion: IL-15 could inhibit the proliferation of PANC-1 cells in a dose and time dependent manner,and could induce the apoptosis of PANC-1 cells. |
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