The Study Of The Mechanisms Of B7-H3 �Reverse Signals� Effect On Pancreatic Cancer Resistant To Gemcitabine Induced Apoptosis

Part I Effect of B7-H3 on Clinical Outcomes of Patients with Pancreatic CancerObjective:This study further explore the expression of B7-H3 in pancreatic cancer and the effect of B7-H3 on clinical outcomes of patients with pancreatic cancer.Methods:Immunohistochemical staining and real-time polymerase chain reaction?PCR?were used to determine the expression of B7-H3 in 31 clinical specimens.Clinical data were applied to survival analysis.Results:The expression of B7-H3 mRNA was found to be higher in tumor tissues?2.04�0.78?than in tumor-adjacent tissues?0.64�0.90?of pancreatic carcinoma.Higher expressions of B7-H3 were detected in pancreatic cancer than in tumor-adjacent pancreatic tissue??²=11.33,P=0.0008?detected by immunohistochemical staining.Survival analysis revealed that patients in the low-B7-H3 expression group were likely to have a longer overall survival compared with those in the high-expression group?P<0.05?.Conclusions:The expression of B7-H3 was found to be higher in pancreatic cancer,closely related with the prognosis of patients with pancreatic cancer,and is expected to become one of the important marker for monitoring the progress of pancreatic cancer.Part II Lentivirus Transfection Technique Construct a HumanPancreatic Cancer Cell Line Panc-1 of Stably Over-expressing B7-H3Objective:To construct the stable over-expressing gene B7-H3 of human pancreatic cancer cell line Panc-1,and provide tools for further investigate the function and biological significance of B7-H3 in pancreatic cancer.Methods:PCR technology was used to obtain the aim gene fragment and to construct recombinant B7-H3-GV287 plasmid vector.Then,prepared competent cells and transformed the cells with B7-H3-GV287 plasmid vector.Through positive clone sequencing,293T cells were transfected with the vector and Western Blot detected the expression of B7-H3 protein.Lentiviral vectors were packaged and the titer was determined.Then the recombinant lentivirus was used to infect Panc-1 cell.Real-time PCR and Western Blot was used to verify the expression of B7-H3,and FCM detected the the expression of B7-H3.Results:Positive cloning sequence comparison results show that the test was passed.The expression of B7-H3 protein was significantly enhanced by Western Blot test.The titer of lentiviral vectors was 2�10⁸ TU/mL.Real-time PCR and Western Blot verified the expressions of B7-H3 higher in B7-H3⁺of Panc-1 cell than in the wild type ofPanc-1cell?P<0.05?.FCM also confirmed that the expressions of B7-H3 higher in B7-H3⁺of Panc-1 cell than in the wild type of Panc-1 cell?P<0.05?.A human pancreatic cancer cell line Panc-1 of stable over-expressing B7-H3 was established.Conclusions:The present study successfully constructed a B7-H3 lentiviral vector and a human pancreatic cancer cell line Panc-1 of stable over-expressing B7-H3,and can be used in further study.Part III The Mechanisms of B7-H3�Reverse Signals�Impact on Human Pancreatic Cancer Cells in the Signal TransductionObjective:To further investigate the mechanisms of B7-H3�reverse signals�impact on human pancreatic cancer cells in the signal transduction.Methods:Phosphoprotein was purified from cultured Patu8988 cells using the Phosphoprotein Purification Kit.Cell apoptosis was detected using propidium iodide�Annexin V staining to investigate the relation between the expression of B7-H3 and Patu8988 cells treated with gemcitabine.Western blot was used to determine the effect of B7-H3 on the expression of proteins including extracellular signal�regulated kinase?ERK?1/2,epidermal growth factor receptor?EGFR?,and Inhibitor of NF-?B?IkB?in Patu8988 cells;B7-H3 was activated by 4H7.Results:B7-H3 activated by 4H7 could reduce gemcitabine-induced apoptosis in Patu8988 cells.Activation of B7-H3 by 4H7 induced variations in ERK1/2,EGFR,and IkB protein levels.When B7-H3 was upregulated,the expression levels of EGFR and ERK1/2 proteins significantly increased?P<0.05?,but the expression level of IkB significantly decreased?P<0.05?,especially in the gemcitabine-treated group.Conclusions:1.B7-H3 was phosphorylated after stimulation,indicating signal conduction in the cells.2.B7-H3 can affect human pancreatic cancer cells by raising the expression of EGFR in signal transduction.3.B7-H3 could deliver�reverse signals�to pancreatic cancer cells to combat apoptosis induced by gemcitabine.