| Objective:Malignant melanoma is a common invasive tumor in the skin and mucous membranes.Over the past few decades,morbidity and mortality have increased year by year,how can better control the development of their condition and prolong the survival of patients,has become an main problem that the clinician facing.Itraconazole is a commonly used first-line triazole antifungal drugs,widely used in a variety of shallow and deep fungal infection,itraconazole injection is also used in systemic fungal infection.In recent years,W Shi and other scholars found that itraconazole has the role of inhibition of tumor endothelial cell proliferation [1].Inoue K,through the study found that itraconazole significantly inhibited the proliferation and migration of endometrial cancer cells [2].Lockhart NR found that when treated with itraconazole in the treatment of a fungal infection in a patient with advanced pancreatic cancer,the patient who had lost the chance of surgery had undergone 9 months of treatment and the condition was controlled,the tumor was reduced and regained surgical resection of the tumor.No evidence of recurrence and metastasis was found in the follow-up of several days after the whipple operation [3].Itraconazole as a new antitumor drug has been recognized by a growing number of medical workers.Coix seed oil is a kind of active substance extracted from the plant nuts,and its main active ingredient is fatty acid and lipid,and its anti-tumor component is triglycerides.P Pei and so found that Coix Seed oil(Kang Lai Te)in lewis lung cancer treatment process showed a strong anti-tumor and immune stimulating activity [4].Lu X and other scholars reported that through the basic experiments found that coix seed extract on lung cancer A549 cell proliferation was significantly inhibited,and can induce A549 cell apoptosis [5].In this paper,the effects and mechanism of itraconazole and coix seed oil on the mouse melanoma B16 cell line were observed by experiment.Methods:1 Cell culture2 MTT colorimetric method was used to construct the growth curve of mouse B16 cells,.and the growth characteristics were observed.3 MTT assay was used to detect the effects of two drugs on mouse B16 cells at different concentrations and time.4 The morphological changes of B16 cells were observed by inverted microscope and HE staining.5 The cell cycle and apoptosis of B16 cells were detected by flow cytometry(FCM).6 The expression of vascular endothelial growth factor(VEGF)was detected by immunohistochemistry.7 SPSS21.0 was used for statistical analysis.Results:1 MTT method to draw the B16 cell growth curveCells were cultured in 96-well plates for different times and the OD values??were plotted using a microplate reader to plot the growth curve near the "S" type.2 MTT assay was used to detect the effect of drug interventionMethods:1 Cell culture2 MTT colorimetric method was used to construct the growth curve of mouse B16 cells,.and the growth characteristics were observed.3 MTT assay was used to detect the effects of two drugs on mouse B16 cells at different concentrations and time.4 The morphological changes of B16 cells were observed by inverted microscope and HE staining.5 The cell cycle and apoptosis of B16 cells were detected by flow cytometry(FCM).6 The expression of vascular endothelial growth factor(VEGF)was detected by immunohistochemistry.7 SPSS21.0 was used for statistical analysis.Results:1 MTT method to draw the B16 cell growth curveCells were cultured in 96-well plates for different times and the OD values ??were plotted using a microplate reader to plot the growth curve near the "S" type.2 MTT assay was used to detect the effect of drug interventionAccording to the MTT results we found that,the inhibitory effect of itraconazole on the B16 cells was higher(P <0.05)with the increase of the drug concentration and the prolongation of the time.The inhibition of coix seed oil on B16 cells was stronger(P <0.05)with the increase of drug concentration and the prolongation of the time.Between the two experimental groups,the effect of the two drugs after 24 hours,1.41ug/ml itraconazole compared with 20?l/ml coix seed oil,2.82?g/ml itraconazole compared with40?l/ml coix seed oil,5.65?g/ml of itraconazole compared with 80?l/ml coix seed oil,11.30?g/ml itraconazole compared with 160?l /ml coix Seed oil were significantly different(P <0.05).After 48 hours of two drugs treatment,1.41?g/ml of itraconazole compared with 20?l/ml of coix seed oil inhibition rate showed no statistical significance(P> 0.05).The difference between the other groups was statistically significant(P <0.05).There is a significant differences between the two groups after 72 hours durg action.The group of11.30 ?g/ml concentration of itraconazole do maximal suppression on B16cells3 Morphological characteristics of cells under the inverted microscope and the color change of the mediumThe color change of the medium:The color change from pale yellow to dark red,and the longer concentration,the longer time of action,the more obvious of the color change.The color change of coix Seed oil was less obvious.Morphological changes observed by the inverted microscope: B16 cells in the control group grew well,the cells were polygonal or rhombic,the cells were tightly connected,adherent uniform growth,cell membrane integrity,cytoplasm translucent,visible nuclei.After the drug effect,the number of cells can be seen reducing under the microscope,the cells become round,the volume becomes smaller,the pseudo-foot reduced or even disappeared,the cells become shrinking and floating,showing cell debris,part of the group,the cytoplasm is not uniform.Compared with coix seed oil group,itraconazole group changes more obvious.4 HE staining to observe the morphological characteristics of B16 cellsThe cells were stained by HE,the nuclei were blue,the cytoplasm was stained evenly pink,and some cells were seen as multinucleated and mitotic images.The membrane was complete and the morphology was regular and the cells were closely connected.After the drug intervention,the number of adherent cells decreased,the shape became round,the volume shrinks,partially shedding,and the cytoplasm concentration was marginalized.Compared with the inverted microscope,the morphological changes of the cells were more intuitive and clear.Compared with the two experimental group,the effect of the itraconazole group on the cell morphology was more significant than that of the coix seed oil group.5 Effects of drugs on cell cycle and apoptosis of B16 cellsEffects of Drugs on cell cycle: Two drug groups compared with the control group,with the increase of drug concentration,G0 / G1 phase of the cell gradually increased,S phase cells gradually decreased,and which were statistically significant(P < 0.05).Effects of drugs on cell apoptosis: 24 hours after drug action,DNA histograms did not show significant apoptotic peaks.6 On the expression of VEGF protein in B16 cellsCompared with the control group,the expression of VEGF protein decreased(P<0.05).The itraconazole group:The expression of VEGF decreased with the increase of drug concentration.(P <0.05).The coix seedoil group: With the increase of drug concentration,the VEGF expression decreased,which was statistically significant(P<0.05).Comparison between the two experimental groups,the expression of VEGF protein in itraconazole group was more obvious.Conclusion:1 Both of the itraconazole and coix seed oil can inhibit the proliferation of mouse melanoma B16 cells.In a certain range,the inhibitory effect increased with the drug concentration up and the action time prolong.2 Itraconazole and coix seed oil both can inhibited the express-ion of VEGF protein in mouse melanoma B16 cells in a concentration-dependent manner,and the inhibitory effect of itraconazole was stronger than that of coix seed oil.Thus,inhibition of VEGF protein expression may be one of the two drugs anti-B16 cell proliferation.3 Both itraconazole and coix seed oil can block the cell cycle of mouse melanoma B16 cells in G0 / G1 phase.In this study,no significant apoptotic peak was detected,the possible cause of the occurrence of apoptosis in this experiment is at an early stage,the apoptotic peak is not obvious,the two drugs through the mechanism of apoptosis to inhibit the proliferation of the role remains to be further experimental confirmed.4 The inhibitory effect of itraconazole on the proliferation of melanoma cells in mice was stronger than that of coix seed oil. |
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