| Objective: To explore the relevant mechanism of SDF-1/CXCR4 axis in gastric cancer metastasis,and to provide an important theoretical basis for the prevention and treatment of gastric cancer metastasis.Methods:(1)Expression analysis of SDF-1/CXCR4 in gastric cancer.290 cases of gastric cancer patients from March 2016 to March 2018 were selected as subjects,and lesion tissues,adjacent tissues and normal gastric tissues were collected.CXCR4 protein expression was determined by immunohistochemical method;CXCR4 mRNA level was determined by real-time fluorescent quantitative PCR technology;medical records were consulted,CXCR4 and CXCR4,VEGF-C,MMP-2 mRNA levels under pathology were analyzed and correlation analysis was completed.(2)Expression analysis of SDF-1/CXCR4 in gastric cancer.(1)Construct the HS-764 T cell line of gastric cancer cells stably expressing CXCR4 in vitro,add chemokines to complete in vitro chemotaxis experiments,and detect the chemotactic and invasive response of gastric cancer cells to SDF-1/CXCR4 at different expression levels of CXCR4;(2)Up-regulating CXCR4 and in vitro invasion experiments to determine the relationship between CXCR4 expression and gastric cancer invasion ability in vitro;(3)Establishing nude mice human gastric cancer metastasis animal models to form nude mice in situ tumors and metastases,determining the up-regulated expression of CXCR4,gastric cancer invasion and in vitro Changes in transfer capacity.(3)The mechanism of SDF-1/CXCR4 in the invasion and metastasis of gastric cancer.Take CXCR4-HS-746T and HS-746T cells to construct a CXCR4 overexpression cell line as a basis to increase CXCR4 expression;flow cytometry to detect cell apoptosis,cycle and intracellular calcium ion concentration level;use real-time fluorescent quantitative PCR method to determine The expression levels of VEGF-C and MMP-2 mRNA in different cells;Western Blot was used to determine the expression levels of VEGF-C and MMP-2 related proteins and the ERK phosphorylation determination of the APK signaling pathway protein was completed.Results: Part 1:(1)The positive rates of CXCR4,VEGF-C and MMP-2 in gastric cancer tissues were all higher than those in adjacent tissues and normal tissues(P<0.05).The positive rates of CXCR4,vegf-c and mmp-2 in para-carcinoma tissues and normal tissues were not statistically significant(P>0.05).(2)The positive rates of CXCR4,VEGF-C,MMP-2 and CXCR4 in gastric cancer tissues are statistically significant in TNM tumor staging and differentiation types(P<0.05);(3)The level of CXCR4 mRNA in gastric cancer tissue was higher than that in adjacent tissues and normal tissues(P<0.05);(4)The level of CXCR4 mRNA in gastric cancer tissue was statistically significant with TNM tumor stage and differentiation type(P<0.05);(5)SPSS The results of Pearson correlation analysis showed that the expression level of CXCR4 protein in gastric cancer tissue was positively correlated with the expression levels of VEGF-C and MMP-2(P<0.05).The second part:(1)Select PEGFP-C1-CXCR4 and p EGFP-C1 recombinants and digest them.The selected enzymes are Eco RI and HindⅢ respectively.After digestion,a CXCR4 c DNA fragment with a size of 1.2kb,p EGFP-C1 c DNA fragment,the size is 4.7kb,indicating that CXCR4 is contained in PEGFP-C1-CXCR4;the T7 site on p SUPER was used for sequencing and identification,and the results proved that the gene fragment carried by the plasmid was the target CXCR4 c DNA;(2)Fluorescence microscope It can be seen that the cells are round and have green fluorescence,and the interference rate is >60.0%,indicating that the transfection is successful;(3)The Western Blot assay results show that: Group C p EGFP-C1-CXCR4 plasmid transfected human gastric cancer cell line HS-The level of CXCR4 protein in 746T cells was higher than that in groups A and B(P<0.05);(4)The number of cell invasion and migration after cell intervention in group A and B was not statistically significant(P>0.05);The number of migration and invasion of gastric cancer cells after transfection in group C was higher than that in group A and B(P<0.05);Under the chemotaxis of SDF-1α,cells in group C had stronger migration ability(P<0.05);After CXCR4 antibody,the cell migration ability of the three groups was affected(P<0.05);(5)The average tumor mass after HS-746T cell inoculation was(2.98±0.43)g lower than CXCR4-HS-746T cell(4.64±0.51)g(P<0.05);The tumors and lungs of nude mice were isolated in situ,and HE staining was performed.The results showed that metastases were found in the liver and lung tissues of CXCR4-HS-746T cells,while metastases occurred in the median of HS-746T cells.Regulating the expression of CXCR4 can inhibit the invasion and metastasis of tumors in vivo.The third part:(1)The ratio of CXCR4-HS-746T is 98.3% compared with 90.1% of HS-746T cells,which is not statistically significant(P>0.05);the cell cycle changes after the intervention of CXCR4-HS-746T and HS-746T;(2)The average fluorescence intensity of CXCR4-HS-746T cells at 10 min,15min,and 20 min after intervention was higher than HS-746T cells(P<0.05);(3)VEGF-C and MMP-2 mRNA levels in CXCR4-HS-746T cells Higher than HS-746T cells(P<0.05);Western Blot results showed: VEGF-C and MMP-2 protein levels in CXCR4-HS-746T cells were higher than HS-746T cells(P<0.05);in SDF-1α Under chemotaxis,the cells of group C have stronger migration ability(P<0.05);after adding CXCR4 antibody,the migration ability of the three groups of cells is affected(P<0.05);(4)After SDF-1α is used for 15 min and 30 min,Both ERK and AKT are phosphorylated,and the phosphorylation level of CXCR4-HS-746T cells is higher.Conclusion:(1)CXCR4 is highly expressed in gastric cancer tissues,and is related to the degree of differentiation and TNM staging.It plays an important role in the occurrence,invasion and metastasis of gastric cancer;(2))By up-regulating CXCR4,a stable HS-T746T cell model of gastric cancer can be constructed,and SDF-1α can improve the migration,metastasis and tumor formation ability of gastric cancer cells,indicating that the SDF-1/CXCR4 axis can directly participate in gastric cancer metastasis;(3)CXCR4 Overexpression can activate cellular calcium influx and accelerate the invasion and metastasis of gastric cancer,which may be related to the regulation of MAPK/ERK and PI3K/AKT signaling,and is expected to become a new therapeutic target for gastric cancer. |
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