| Research purposes:Through experiments,the effects of Fu-zheng-xiao-zheng Decoction on tumor growth and metastasis of gastric cancer and its molecular mechanism by regulating PI3Ky immune checkpoints and remodeling Tumor-associated macrophages(TAM)were observed.Research method:1.Establish TAM model.Cell morphology was observed under a microscope,cell marker antigens(M1:CD86;M2:CD206)were detected by flow cytometry,and TAM-related genes and protein levels were detected by real-time immunofluorescence quantitative PCR and Western blotting(M1:TNF-?;M2:IL-10)to identify the proportion of M1/M2 cells in the TAM model.The level of PI3Ky-related protein was detected by Western blot to compare the difference between MO and TAM.2.To observe the affection of PI3Ky inhibitors and agonists on HGC-27 through TAM.Western blotting was used to detect the expression of PI3Ky-related protein,flow cytometry was used to detect the cell marker antigen(M1:CD86;M2:CD206),and real-time immunofluorescence quantitative PCR was used to detect related genes(M1:TNF-?;M2:IL-10)Level.Cell invasion and scratch experiments were used to observe gastric cancer invasion and migration.Real-time immunofluorescence quantitative PCR and Western blotting were used to observe the expression of genes and proteins related to apoptosis and epithelial-mesenchymal transition(EMT).3.In vitro experiments to observe the effect and mechanism of Fu-zheng-xiao-zheng Decoction on gastric cancer.Western blotting was used to detect the protein expression level of PI3Ky pathway related molecules,flow cytometry was used to detect cell marker antigens,real-time immunofluorescence quantitative PCR and western blotting were used to detect TAM-related genes and proteins(M1:TNF-?,CCL22,IL-10;M2:IL-10,TGF-?)levels.Cell invasion and wound healing assays were used to observe gastric cancer invasion and migration,MTT cell viability experiment was used to observe proliferation,real-time immunofluorescence quantitative PCR and western blotting were used to observe apoptosis,epithelial-mesenchymal transition-related genes and protein expression.4.In vivo experiments to observe the effect and mechanism of Fu-zheng-xiao-zheng Decoction on gastric cancer.Gastric mouse axillary tumor model,observe the changes of the tumor body in each group after the effect of Fu-zheng-xiao-zheng Decoction,the changes in the weight of the mouse and the proportion of TAM phenotype in tissue samples by immunofluorescence and flow cytometry,Real-time immunofluorescence quantitative PCR and protein labeling detected cell apoptosis,expression of genes and proteins related to epithelial-mesenchymal transition,and re-verified its correlation with changes in tumor PI3Ky expression.Research result:TAM-related antigens and cytokines were significantly up-regulated in the TAM model.After PI3K? was inhibited,the expression of M2-related antigens and cytokines in the TAM model was significantly down-regulated,M1-related cytokines were up-regulated,and the levels of pro-apoptosis and EMT inhibitory factors in HGC-27 increased.The levels of anti-apoptosis and EMT-promoting factors are reduced,and the invasion of gastric cancer cells is reduced,and migration is slowed.The experimental results after addition and subtraction of Fu-zheng-xiao-zheng Decoction show the same trend as PI3K? inhibitor.Analysis conclusion:Tumor-associated macrophages express the M2 functional phenotype;macrophages PI3K?is an immune checkpoint that regulates TAM polarization in gastric cancer;addition and subtraction of Fu-zheng-xiao-zheng Decoction can inhibit PI3K? and promote TAM reprogramming,thereby promoting gastric cancer cell apoptosis,And inhibit gastric cancer cell EMT,and ultimately inhibit the growth and metastasis of gastric cancer. |
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