| Objective:1.Gastric cancer is the most common malignant tumor of the digestive system.The incidence rate of gastric cancer is increasing year by year.and the mortality rate is extremely high.At present,immunotherapy has made a breakthrough in the treatment of solid tumors.Among them,PD-1/PD-L1 inhibitors are the most representative monoclonal antibody drugs.It is reported that a PD-L1 monoclonal antibody drug named Atezolizumab has been approved by the American FDA for the treatment of urinary system tumors.But so far,there is no effective immunotherapy to alleviate the development of gastric cancer.2.Based on clinical samples,PD-1/PD-L1 expression in gastric cancer tissues and paracancerous tissues was detected,and differential intestinal probiotics in gastric cancer patients and normal controls were screened.The therapeutic effect of PD-L1 monoclonal antibody and probiotics on gastric cancer was observed by cell experiment and in vivo experiment.It provides a new way to increase the immunotherapeutic effect of gastric cancer,prolong the survival of gastric cancer patients and delay the progress of disease.Methods:1.Part 1: To explore the expression of PD-1/PD-L1 in gastric cancer and analyze the difference of intestinal bacteria.From November 2017 to November 2019,we collected and screened 80 patients who had undergone radical gastrectomy in The First Affiliated Hospital of Jinzhou Medical University and proved to be gastric cancer by pathology,and randomly selected the normal gastric tissue of 20 patients as the blank control.The expression of PD-1,PD-L1 and CD8 were detected by IHC,and the expression of PD-1,PD-L1 were detected by Western Blot and q RT-PCR.At the same time,the survival of patients with gastric cancer was analyzed.Bacterial culture was used to identify the differential flora in the feces of patients with gastric cancer and normal physical examinees.Probiotics were used to intervene SGC7901 gastric cancer cells,which were divided into the cell control group and the probiotics+ control group.Flow cytometry and Transwell were used to detect the apoptosis,cycle change and invasion ability of the two groups of cells.The cell line was used for subsequent experiments?2.Part2: To observe the efficacy of pd-l1 monoclonal antibody combined with probiotics in the treatment of gastric cancer,we conducted experiments on tumor-bearing mice.The experimental groups were as follows: SGC7901 gastric cancer model group,probiotics + model group,pd-l1 monoclonal antibody + model group and pd-l1 monoclonal antibody + probiotics + model group.the cervical vertebrae were removed and the mice were sacrificed at 21 days.Tumor tissue was taken,weighed and the tumor volume was calculated.The expression of pd-1 / pd-l1 in tumor tissues of mice in each group was detected by Western-Blot and q RT-PCR?3.Part3: In order to explore the mechanism of the combined effect of pd-l1 monoclonal antibody and probiotics on the treatment of gastric cancer,the second part was divided into groups of in vivo experiments.Serum Th1 cytokines il-2,IFNequity,TNF-equity and Th2 cytokines il-4,il-6 and il-10 were determined by ELISA.Levels of chemokines CXCL9 and CXCL4.Flow cytometry was used to detect the levels of CD4+T cells and CD8+T cells in spleen.The expressions of CD4,CD8,pd-l1 and pd-1 in tumor tissues were detected by western-blot and q RT-PCR?Results:1.Part 1: IHC results showed that,compared with the para-carcinoma tissues,pd-1and pd-l1 were highly expressed in the cell membrane of gastric cancer(P<0.05),and CD8 was highly expressed in the cell nucleus of gastric cancer(P<0.05).Western Blot and qrt-pcr showed that the expression levels of pd-1 and pd-l1 in gastric cancer tissues were significantly higher than that in adjacent tissues(P<0.05).Survival analysis showed that tumor stage and infiltration of CD8+T cells could be used as independent prognostic factors for postoperative recurrence and overall survival of gastric cancer.The expression of pd-l1 was positively correlated with the degree of infiltration of CD8+T cells(P<0.05).The application of probiotics can promote the apoptosis of gastric cancer cells(P<0.05),promote the gastric cancer cells to stay in the G2/M phase(P<0.05),and reduce the proliferation of gastric cancer cells(P<0.05).At the same time,the content of intestinal bifidobacteria in gastric cancer patients was significantly lower than that in healthy control group(P<0.05),which was used for subsequent experiments.2.Part2: In vivo results: the combination of pd-l1 monoclonal antibody and bifidobacterium could inhibit the growth of gastric cancer(P<0.05).Western Blot and q RT-PCR results showed that pd-l1 monoclonal antibody combined withbifidobacteria could reduce the expression of pd-1 and pd-l1 in tumor tissues of mice in each group(P<0.05).3.Part3: The combination of pd-l1 monoclonal antibody and bifidobacterium could increase serum il-2,IFN-equity,TNF-equity and other Th1 cytokines in tumor-bearing mice(P<0.05).The levels of Th2 cytokines(il-4,il-6,il-10,etc.)and CXCL9 and CXCL4 were decreased(P<0.05).The results of flow cytometry showed that the combination of pd-l1 monoclonal antibody and bifidobacteria could increase the contents of CD4+T and CD8+T cells in the spleen of mice(P<0.05).Western Blot and q RT-PCR results showed that the combination of pd-l1 monoclonal antibody and bifidobacterium could reduce the expression of pd-1 and pd-l1 in tumor tissues(P<0.05)and promote the expression of CD4 and CD8(P<0.05).Conclusion:In this study,it was found that the intestinal flora of patients with gastric cancer was significantly different,and the number of probiotics was significantly less than that of the normal control group,proving that probiotics play an important role in the occurrence and development of gastric cancer.This paper mainly through the PD-L1 single joint resistance of probiotic method,through clinical gastric cancer samples,cell and in vivo experiment and so many Angle,discovery and application of PD-L1 alone,compared to single resistance or probiotics combined use of PD-L1 single resistance and probiotics can reduce gastric cancer group and PD-PD-1 the expression of L1,inhibit the development of gastric cancer,provides new thinking for clinical treatment of gastric cancer,to provide basic data for the related scientific research support?... |
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