| Objective:The efficacy and clinical safety of the treatment of intratumoral injection of recombinant human type 5 adenovirus(oncorine)under the guidance of endoscopic ultrasound combined with capecitabine formoderateand advancedpancreaticcancerwereinitially researched.Then,by establishing an animal model of pancreatic cancer xenografts in nude mice,we further validated the therapeutic efficacy of the treatment of Oncorine combined with capecitabine on pancreatic cancer and explored its potential anti-tumor mechanism.Method:1.Ten patients with moderate and advanced pancreatic cancer which had lost the chance of surgery without any antitumor treatment were selected.And oncorine was injected into the pancreatic tumor under the guidance of endoscopic ultrasound,then oral capecitabine chemotherapy(1250mg/m~2,2 times/day,14 consecutive days,stop 7 days,1 cycle per 21 days)was performed in the third day after treatment.The volume of the tumor,the elasticity coefficient of the tumor,the serum tumor markers,karnofsky score,pain score,patient’s survival time,adverse reaction and complication were observed and recorded before and after the treatment.2.Western Blot was used to test the expression type of p53 protein in pancreatic cancer cells Bxpc3.The subcutaneous xenograft model of nude mice was constructed by subcutaneously embedding the tumor block.Observe the general condition of each group of nude mice,the growth of the tumor,depict the tumor growth curve,and calculate the tumor inhibition rate.HE staining was performed to observe the morphological changes of tumor tissues in each group.Immunohistochemical method was used to detect the expression of wild-type p53 protein and ki67protein in tumor tissue of each group.TUNEL was used to detect apoptosis in each group.Elisa was used to detect the levels of IL-1β,IL-18 and IL-10 in the supernatant of the tumor tissue.Electron microscopy was used to observe the ultrastructure of cells in each group.Result:1.According to the RECIST 1.1 criteria,in 10 patients with pancreatic cancer after 3 weeks of treatment,1 patient had PR,8 patients had SD,1 patient had PD,and none had CR.Among 8 patients who received SD,5 patients appeared liquefaction and necrosis in the tumor after 6 weeks of treatment.The elastic coefficient of the primary pancreatic tumor after 6 weeks of treatment was statistically different from that before treatment(P<0.05).The pain scores of patients were reduced from 5.50(4.00~7.00)before treatment to 0.00(0.00~2.00)at 3weeks after treatment,and the Karnofsky score was increased from(64.00±12.65)before treatment to(90.00±14.14)at 3 weeks after treatment.There was a significant difference between the two indicators before and after the treatment(P<0.01).The volume of pancreatic tumors,the concentrations of serum CEA and CA199 in patients with pancreatic cancer at 3 weeks,6 weeks,and 9 weeks after treatment were not significantly different from those before the treatment(P>0.05).10patients with pancreatic cancer followed up for(8.5±6.1)months.At the end of follow-up,1 patient was survived,the shortest survival period was2 months,and the longest survival period was 22 months.The median survival period was 6 months and the median progression-free survival was 4 months.After treatment,1 patient had mild symptom like influenza,3 patients had low fever,1 patient had high fever.Except 1patient’s had mild hyperamylasemia,the rest had no adverse reaction and operation-related complication.2.Western Blot results showed that human pancreatic cancer cells Bxpc3 express mutant p53 protein.The tumor growth rate of the nude mice after combined treatment was significantly slower than that of the chemotherapy and control groups(P<0.05).The tumor volume at 1 and 2weeks after treatment in the combination group and the tumor volume at1 week after treatment in the virus group were slightly smaller than those before treatment(P>0.05).The tumor inhibition rate was 47.7%in the combined group,40.7%in the virus group,and 21.0%in the chemotherapy group.The difference between the combined group and the virus group compared with the control group was statistically significant(P<0.01).HE staining showed a large area of necrotic lesions in the combined group,and lymphocytes accumulated at the edge of the necrotic area.The area of necrosis in the virus group was surrounded by tumor tissue and there was a great deal of inflammatory cell infiltration.A small amount of focal necrosis was seen in the chemotherapy group.The control group was mainly solid tumor tissue.The distribution of the wild-type p53 protein positive cells in the combined group and ki67protein positive cells in the control group was relatively wide under microscope,and the difference between the four groups was statistically significant(P<0.05).The proportion of apoptotic cells in the combined group was higher than the other three groups(P<0.01).The levels of IL-18 and IL-1βin the combined group were higher than those in the other three groups,and the IL-10 level was decreased(P<0.05).Electron microscopy showed that pancreatic cancer cell membrane of the two groups which injected virus was ruptured,organelle pyknosis,structure disappearance,nuclear condensation,chromatin condensation,edge set,intracellular membranous vacuolar structure and autophagy.Conclusion:1.The intratumoral injection of oncorine under the guidance of endoscopic ultrasound combined with capecitabine,which can relieve the pain in patients with advanced pancreatic cancer,improve the patient’s life quality and extend patient’s survival time.The treatment is safe and effective.2.The results of animal experiments show that the combination therapy of oncorine and capecitabine is more advantageous than monotherapy,which can reduce the tumor volume,inhibit tumor growth,increase the expression of wild-type p53 protein in tumor tissue,and induce apoptosis and pyroptosis. |
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