The Study Between BH4/iNOS And Lymphangiogenesis In Gastric Adenocarcinoma

Objective:The main route of metastasis in patients with gastric cancer is the lymphatic system,and the degree of lymph node involvement affects the survival rate of patients.The growth of solid tumors depends on the proliferative activity of the tumor as well as the angiogenesis of the tumor.Many studies have shown that lymphangiogenesis is a more reliable parameter than vessel formation in predicting tumor progression.However,the biological role of gastric tumor cells in the progression of lymph node metastasis through potential lymphatic metastasis and tumor-associated lymphangiogenesis remains unclear.The relationship between BH4 and the expression of iNOS and D2-40 in gastric adenocarcinoma tissue was studied by pathological immunohistochemistry,Griess reagent,ELISA and Western blot technology.In addition,the relationship between BH4 and lymphatic vessel density(LVD)in gastric adenocarcinoma will be answered.It is significant to explore the role of BH4 in lymphangiogenesis of gastric adenocarcinoma and to elucidate its mechanism by animal experiments for understanding the lymphangiogenesis of gastric adenocarcinoma,which may provide a new target for the treatment of lymphangiogenesis of gastric adenocarcinoma,so as to improve the therapeutic effect of advanced gastric adenocarcinoma.Methods:1?Collect cancer tissues and adjacent normal tissues specimens from 40 patients with gastric adenocarcinoma.2?Detect the expression of iNOS and D2-40 in gastric adenocarcinoma tissues and adjacent normal tissues by immunohistochemistry.Detect lymphatic vessel density(LVD)in gastric adenocarcinoma tissues and adjacent normal tissues by D2-40 monoclonal antibody labeling.3?Analysis of positive expression rates of iNOS and D2-40 protein in gastric adenocarcinoma tissues and adjacent normal tissues by chi-square test statistical method.Correlation analysis of iNOS and D2-40 protein expression in gastric adenocarcinoma was performed by Spearman statistical method.4?Culture human gastric cancer cell line MKN-45 cells in DMEM/F12 medium,and establish a stable and reliable tumor-bearing model of human gastric cancer cell line MKN-45 by using BALB/c-nu mice.Divide the BALB/c-nu mice into 2 groups by random number table method,and the experimental group(BH4 group)was given BH4 intraperitoneal injection,and the control group(saline group)was given the same amount of normal saline.5?Detect the expression of iNOS and D2-40 in BH4 group and saline group by immunohistochemistry.Detect lymphatic vessel density(LVD)in BH4 group and saline group by D2-40 monoclonal antibody labeling.6?The level of BH4 was detected by ELSA method in the tumor tissues of the BH4 group and saline group.Analyze the BH4 level between the BH4 group and saline group by using two independent sample t-test statistical method.7?The level of NO was detected by Griess reagent in the tumor tissues of the BH4 group and saline group.Analyze the NO level between the BH4 group and saline group by using two independent sample t-test statistical method.8?The expressions of iNOS in the tumor tissues of the BH4 group and saline group were detected by Western blot.Results:1?Immunohistochemistry showed that the positive expression rate of iNOS and D2-40 protein in gastric adenocarcinoma tissues were significantly higher than that in adjacent normal tissues(P<0.05).2?The expression levels of iNOS and D2-40 protein in gastric adenocarcinoma were correlated with tumor invasion depth and lymph node metastasis/distance metastasis(P<0.05),and were not related to gender and age(P>0.05).3?There was a significant positive correlation between the expression of iNOS and D2-40 protein in gastric adenocarcinoma tissues(P<0.05).4?Immunohistochemistry showed that the positive expression rate of iNOS and D2-40 protein in BH4 group were significantly higher than that in saline group(P<0.05).5?The level of BH4 in BH4 group was significantly higher than that in saline group by ELISA,and the difference was statistically significant.(P<0.05).6?The Griess reagent assay showed that the level of NO in BH4 group was significantly higher than that in saline group,and the difference was statistically significant(P<0.05).7?Western blot analysis showed that the expression level of iNOS in BH4 group was significantly higher than that in saline group,and the difference was statistically significant(P<0.05).Conclusion:BH4/iNOS can promote lymphangiogenesis in gastric adenocarcinoma,and its mechanism is related to the coupling of BH4 and iNOS,which induces the production of high level of NO in the tumor.The result may provide a new possible basis for the inhibition of lymphangiogenesis in gastric adenocarcinoma.