The Role Of Vitamin C Playing In Human Glioma SHG44Cells And Subcutaneously Transplanted Tumor Of Nude Mice

Part One:The effect of vitamin C on human glioma SHG44cellsPurposes:Through treating human glioma SHG44cells with cisplatin as positive control group, and dealing with other human glioma SHG44cells with different concentrations of vitamin C,our purpose is to observe the influence of vitamin C on SHG44cell proliferation,apoptosis and the expression level of HIF-1alpha and VEGF mRNA in cell.Also we explore the possible mechanism of action of vitamin C on SHG44cells.Methods:On the one hand,we treated human glioma SHG44cells in logarithmic phase with different concentration of vitamin C (0,0.5,1.0,2.0,4.0,8.0,10.0mmol/L),and then detected cell IC50alue by MTT colorimetric methods. On the other hand, different concentration of vitamin C (0,0.5,0.75,1.0,1.25,1.5,1.75,2.0mmol/L) and cisplatin (20mg/L) were applied to SHG44cells in logarithmic phase, and the cell growth inhibition rate was assayed with MTT colorimetric methods. Moreover,human glioma SHG44cells in logarithmic phase were respectively treated by the vitamin C (0,1.5mmol/L) and cisplatin (20mg/L) for24hours, and the cell cycle and apoptosis were detected by flow cytometry instrument methods.The expression of HIF-1a and VEGF mRNA in cells was assayed with RT-PCR methods.Results:Vitamin C inhibited the growth of SHG44cells in a concentration dependent manner,and in the570-nm wavelength, after24hours, the IC50alue was1.695mmol/L. In line with cisplatin group,when the concentration of vitamin C was greater than1.25tendency/L, SHG44cells in each group were all dead after72hours. In both cisplatin and vitamin C treatment groups, the incident of early stage apoptosis cells was more than that of blank control group, and the number of SHG44 cells in S stage was less than that of it, the differences were both statistically significant (P<0.05). VS blank control group, under conventional oxygen concentration, both vitamin C and cisplatin could increase the expression of HIF-1alpha mRNA in SHG44cells, and reduce the expression of VEGF mRNA (P<0.05). The differences of the expression of VEGF mRNA in Vitamin C group and cisplatin group were not significant statistically (P>0.05).Conclusions:vitamin C can inhibit the proliferation of human glioma SHG44cells and promote their apoptosis. The mechanism of action of vitamin C may be through reducion of the expression of VEGF mRNA in cells and inhibition of angiogenesis of tissue cells. Part Two:The effect of vitamin C on subcutaneous transplanted tumor in the nude micePurposes:By establishing subcutaneous transplanted tumor model of human glioma SHG44cells of BALB/c in the nude mice, with cisplatin treatment group as the positive control group, we observed the inhibition effect of different doses of vitamin c on the growth of subcutaneous transplanted tumor in the nude mice, and we also observed the effect of vitamin C on the expression of HIF-1alpha and VEGF mRNA of the transplanted tumor tissues. Through analyzing the possible mechanism of vitamin C in the treatment of human glioma tumor, we try to provide laboratory basis for the treatment of human glioma tumor.Methods:1. Firstly,we cultivated and passaged human brain glioma SHG44cells. Secondly,we took some logarithmic growth phase cells, and made them into single cell suspension with density of2x107/ml,then we took0.2ml to inject into the lateral subcutaneous of right forelimbs of nude mice back. Two weeks later after inoculation,If lump grows in the subcutaneous of vaccination sites,and the volume of subcutaneous transplanted tumors is measured about100mm3, we regard it as a successful glioma model.2. A total of28,4-5weeks of female nude mice were randomly divided into four groups, each group of seven.Blank control group were given normal saline for0.4ml/each, intraperitoneal injection, once every two days, a total of three weeks; Low and high dose group of Vitamin C were respectively given2g/kg、4g/kg each, intraperitoneal injection, once every two days, a total of three weeks; Cisplatin group was given cisplatin2g/kg, intraperitoneal injection, once every two days, a total of three weeks. On the next day after different treatment with drugs,we put each group of mice to death,and calculated tumor volume and tumor inhibition rate. RT-PCR methods were used to detect the expression of HIF-1alpha and VEGF mRNA in each group of tumor tissues.Results:1.Subcutaneous transplanted tumor models of nude mice were successfully established, and the success rates were as high as100%.2. Vitamin C could inhibit the growth of subcutaneous transplanted tumor in the nude mice, the tumor inhibition rate of high dose group was higher than that of low dose group. Tumor volume in both blank control group and experimental group was statistically different (P<0.05).3. Compared with blank control group,low and high dose group of vitamin C can both reduce the expression of HIF-1alpha and VEGF mRNA, and the expression of high dose group was less than that of low dose group; the differences were statistically significant(P<0.05), low and high dose group of vitamin C and cisplatin group were statistically different (P<0.05).Conclusions:It suggested that human brain glioma SHG44cells establish subcutaneous transplanted tumor model in the nude mice is reliable, which is suitable for glioma experimental research of prevention and treatment.High doses of vitamin C in certain dose ranges can inhibit the growth of subcutaneous transplanted tumors in the nude mice, and show some anti-tumor effect.The mechanism may be related to that vitamin c can inhibit the expression of HIF-1alpha and VEGFmRNA in tissue cells and reduce the flow of blood to the tumor tissue.14charts,1table,60references.