| Objective:In recent years,the incidence of melanoma increased obviously around the world,and it has become the leading cause of skin cancer deaths.It was mostly found in adults over 30 years of age,and occured in the skin,mucous membranes and internal organs.It is a tumor caused by the combinational effects of genetic factors and environmental factors,with more malignancy and early metastasis via bloodstream and lymphatic system,and the metastasis is the main cause of death.With the development of medicine,people pay more attention to the prevention and treatment of the disease and prognosis,and in modern medicine,a simple surgical resection,radiotherapy,chemotherapy and other therapy are used,but unsatisfactory,and still with a high recurrence rate and mortality.Vascular endothelial growth factor(VEGF)is one of the most important regulators of angiogenesis,especially for neovascularization of tumor.Neonatal vasculature is an important structural basis of solid tumor growth,metastasis and invasion,which mainly through the specific binding of vascular endothelial cell corresponding receptor,vascular endothelial growth factor receptor(VEGFR),to activate downstream pathway,thereby promote the growth of tumor neovascularization.In a variety of human tumors,the VEGF receptors are not only expressed in tumor vascular endothelial cells,but also expressed in tumor cells,which account for the mechanism of the tumor cell autologous secretion of VEGF.VEGFR2 is the receptor of VEGF and VEGF binding to VEGFR2 can lead to a series of changes in endothelial cells,promoting tumor angiogenesis,invasion and metastasis.More and morestudies have shown that the expression of VEGF and its receptors in a variety of tumors increased.There are data shown that VEGF and VEGFR2 are highly expressed in lung cancer tissues and are closely related to the presence of lymph node metastasis,clinical stage and tumor differentiation,suggesting that it plays an important role in the progression of lung cancer.Some studies have also found that besides vascular endothelial cells,VEGF and its receptor VEGFR2 highly expressed in ovarian cancer cells.In the colorectal cancer study it is found that VEGFR2 can promote the invasion,growth and metastasis of colorectal adenocarcinoma,with a high expression of VEGFR2 in patients with poor prognosis.Previous studies have shown that melanoma cells overexpressing VEGF that are closely related to the pathogenesis and prognosis of melanoma.Tas et al.demonstrated that plasma levels of VEGF in melanoma patients were significantly elevated and VEGF expression was associated with disease progression.People have increasingly payed more attention to the role of tumor angiogenesis in tumor occurrence,development,infiltration,metastasis,and the inhibition of VEGF,reduction of angiogenesis,prevention from tumor blood supply,have become hot spots of research for the cancer therapy.Ganoderma lucidum of Polyporaceae fungal fruiting bodies is a very precious edible fungus,which has high nutritional value and medical.That in medicinal history China for thousands of years,its drugs associated with edible methods.There are a lot of tonic effect on the body after taking it,example the treatment of neurasthenia,arrhythmia hypertension,invigorating qi and promoting blood circulation,soothe the nerves,also can prevent and cure diseases of chronic bronchitis.The Ganoderma lucidum polysaccharide(Ganoderma lucidum polysaeeharides,GL-PS)is one of the main active ingredient which extracted from Ganoderma lucidum,which can enhance immunity,resistance Oxidative and hypoglycemic and anti diabetic complications.Numerous Clinical studies have shown that Ganoderma lucidum polysaccharides can indirectly inhibit tumor cell proliferation by regulating immune function,and even directly kill tumor cells.In this study,different concentrations of GL-PS were used in B16F10 melanoma cells to observe the effect of GL-PS on the expression of VEGF and its receptor VEGFR2 in B16F10 cells,thus providing a new direction for the treatment of melanoma.Methods:1.Cells: The melanoma B16F10 cells used in the experiment were derived from Central Laboratory of Chengde Affiliated Hospital2.Experimental group and the intervention: Five groups have been set up.According to the concentrations of GL-PS in RPMI-1640 complete medium to culture melanoma cells,0?g/ml group,0.2?g/ml group,0.8?g/ml group,3.2?g/ml group,12.8?g/ml group were set up.RPMI-1640 complete medium group without GL-PS was set up as a control group.3.qRT-PCR: the transcription expression of VEGF and VEGFR2 in each group were detected by q RT-PCR.4.Westeren-blot: the protein expression of VEGF and VEGFR2 in each group were detected by Westeren-blot.5.Immunohistochemistry: the expression of VEGF and VEGFR2 in each group were detected by Immunohistochemical method.6.The statistical analysis was performed with SPSS21.0 using one-way analysis of variance(ANOVA).P<0.05 was considered statistically significant.Result:1.The effects of Gl-PS on VEGF and VEGFR2 expression in melanoma cells was detected at the gene level and protein level: under the action of different concentrations of Gl-PS(0?g/ml,0.2?g/ml,0.8?g/ml,3.2?g/ml,12.8?g/ml),detection using qRT-PCR technology showed the results of the mRNA expression level of VEGF: 1.28±0.16,1.13±0.09,0.97±0.08,0.88±0.06,0.75±0.14,repectively,and the mRNA expression level of VEGFR2: 1.01±0.06,1.11±0.09,0.8±0.059,0.71±0.03,0.43±0.02,repectively.The mRNA expression of VEGF and VEGFR2 in B16F10 cells decreased gradually with the increasing concentration of Ganoderma lucidumpolysaccharide,seemingly in a dose-dependent manner.The analysis of variance indicated the difference was significant(VEGF: F=7.006,P<0.05;VEGFR2:F=48.033,P<0.005).2.Inhibitory effects of Ganoderma lucidum polysaccharides on the secretion of vascular endothelial growth factor and its receptor in mouse melanoma cells: under the action of different concentrations of Gl-PS(0?g/ml,0.2?g/ml,0.8?g/ml,3.2?g/ml,12.8?g/ml),the detection using Western-blot technology showed the results of protein expression level of VEGF: 1.63±0.47,1.77±0.16,0.74±0.07,0.82±0.07,0.41±0.10,repectively,and the protein expression level of VEGFR2: 2.16±0.11,1.97±0.07,1.51±0.06,1.35±0.09,1.04±0.16,repectively.With the concentration of GL-PS increasing,the protein expression level of VEGF and VEGFR2 was getting lower and lower,seemingly in a dose-dependent manner.The analysis of variance indicated the difference was significant(VEGF:F=19.931,P<0.005;VEGFR2:F=57.155,P<0.005).3.The detection of expression of VEGF and VEGFR2 in melanoma cells at the protein level under the action of different concentrations of Gl-PS using immunohistochemical method was performed.The positive expression of VEGF and VEGFR2 in B16F10 in the control group was brownish yellow,while the positive expression of VEGF and VEGFR2 in the experimental group treated with GL-PS was significantly less than that in the control group,and the results showed a decreasing trend with the increase of the concentration.4.Conclusion:1.Ganoderma lucidum polysaccharide can inhibit the secretion of vascular endothelial growth factor and its receptor in mouse melanoma cells... |
PDF Full Text Request