The Basic Research On The Proliferation And Apoptosis Of Post-radiation Residual Pancreatic Cancer Cells Induced By Exogenous High-mobility Group Box-1 Protein

Objective: To investigate the HMGB1 content in the Pancreatic cells supernatant after different irradiation,and to further explore its proliferation and apoptosis effect on post-radiation residual pancreatic cells in vitro,and the exact mechanisms.And then providing a certain experimental basis and theoretical basis for clinical treatment on pancreatic cancer.Methods:(1)The morphology of Pancreatic cancer cells(SW1990?Patu8988?PANC-1?Aspc-1)differently changed after different radiation dose by optical microscope.(2)The HMGB1 content in the Pancreatic cells(SW1990?Patu8988?PANC-1?Aspc-1)supernatant after different irradiation were determined using ELISA.(3)By CCK-8 assay to observe the proliferation effect of different concentrations of exogenous HMGB1 on post-radiation residual pancreatic cells in vitro and screening the best effect concentration.(4)By CCK-8 assay to observe the proliferation effect of post-radiation supernatant on residual pancreatic cells in vitro.(5)By Flow Cytometry to observe the apoptosis effect of post-radiation supernatant on post-radiation residual pancreatic cells in vitro.(6)By Western-Blot to analysis the expression of proliferation-related protein C-myc ?Ki67 and apoptosis-associated protein Bcl2/Bax?Caspase-3 ?Cleaved Caspase-3 in the different treatment groups.(7)Expression m RNA and protein levels of TLR2 were determined using q RT-PCR?Western-Blot and immunofluorescence in pancreatic cells(SW1990Patu8988?PANC-1?Aspc-1).(8)Transfection efficiency of sh-TLR2 in the pancreatic SW1990 cells were observed by inverted fluorescence microscope.Expression m RNA and protein levels of TLR2 were determined using q RT-PCR and Western-Blot in pancreatic SW1990 cells.(9)By Western-Blot to analysis the expression of related proteins in Wnt signaling pathway and proliferation-related protein ?apoptosis-associated protein in post-radiation residual sh-TLR2 SW1990 cells after the exogenous HMGB1 treatment.Results:(1)The morphology of Pancreatic cancer cells(SW1990?Patu8988?PANC-1?Aspc-1)differently changed after different radiation dose by optical microscope.(2)ELISA results showed the highest HMGB1 content in the Pancreatic SW1990 cells supernatant at 10 Gy irradiation.(3)CCK-8 assay showed that the exogenous HMGB1 could induce the proliferation of the post-radiation residual pancreatic SW1990 cells after 10 Gy with a significant difference at 100ng/m L.(4)CCK-8 assay showed that supernatant+EP(Ethyl pyruvate,inhibitor of HMGB1)group? supernatant group ?HMGB1(100ng/m L)group could induce the proliferation of the residual pancreatic SW1990 cells after 10Gy;and HMGB1(100ng/m L)group had a greater ability to induce the cells proliferation than supernatant group.(5)Flow Cytometry results showed that compared with PBS group,the percentage of early and later apoptotic cells had decreased in the supernatant+EP group? supernatant group ?HMGB1(100ng/m L)group;and compared with supernatant group,HMGB1(100ng/m L)group was more significant with the percentage of early and later apoptotic cells.(6)Western-Blot results showed that the expression of apoptosis-related protein Bcl2/Bax ?Caspase-3 and proliferation-related protein C-myc?Ki67 in the PBS group?supernatant+EP group?supernatant group?HMGB1(100ng/m L)group were successively decreased and increased,and apoptosis-related protein Cleaved Caspase-3 was increased.(7)Real-time PCR and Western-Blot results showed that SW1990 cells had the higher expression of TLR2 than Patu8988?PANC-1?Aspc-1 pancreatic cells.(8)Real-time PCR and Western-Blot results showed that of TLR2 stable down-regulation SW1990 cells.(9)Western-Blot results showed that compared with the control group,Wnt signaling pathway that GSK-3? expression was increased ? p-GSK-3? expression was decreased??-catenin expression was increased,in addition,the expression of apoptosis-related protein Caspase-3 was increased and Cleaved Caspase-3 was decreased,and proliferation-related protein Ki67 was decreased in the post-radiation residual sh-TLR2 group witch have a time-dependent significantly.Conclusion:(1)This study demonstrated that HMGB1 was in the supernatant of pancreatic cancer cell line SW1990 and was not significantly correlated with the dose of radiation.(2)This study demonstrated that the exogenous HMGB1 and supernatant could dramatically promote proliferation and inhibit apoptosis of post-radiation residual pancreatic cells in vitro.(3)This study demonstrated that the exogenous HMGB1 could adjust Wnt/?-catenin signaling pathway,further control proliferation and apoptosis resistance through TLR2 in post-radiation residual pancreatic cells in vitro.