The Inhibitory Effect Of Acitretin On Proliferation And The Expression Of Livin And BFGF On Malignant Melanoma B16 Cells

Objective:Malignant melanoma(MM) is one of the skin cancer which threat to people's lives. Its cells come from black melanin cell. Malignant melanoma is in a high degree of malignancy and its biological behavior of high-grade, with invasion of organizations and continuedproliferation ability to evade immune surveillance. So far there is no effective prevention and treatment methods, and surgery remains the treatment of melanoma, but even surgical excision, the prognosis is still far from ideal. The average survival time of this advanced cancer patients is only 6 to 9 months. Therefore, the preliminary study the treatment of malignant melanoma of retinoids and its mechanism, to explore whether the mechanism of retinoids effects Livin, bFGF expression is related thereby inducing differentiation therapy for clinical melanoma provide an experimental basis.Retinoids includes vitamin A, natural and synthetic derivatives. It has a very important role in the normal differentiation of its epithelium. It also has a very important role in clinical practice for the prevention and treatment of various skin diseases. Recent scientific studies suggest, that retinoic induced differentiation and has a strong role in inhibiting proliferation of tumor cells.Methods:1 cell cultureContaining 10% fetal calf serum RPMI-1640 medium (including penicillin 100U/ml, streptomycin-containing 100U/ml, PH7.2)which cultured B16 cells were placed in saturated humidity, 37℃.5% CO2 Training Box routinely subcultured. The B16 cells were treated by acitretin in different concentration.2 Detection of the B16 cell proliferation in vivo by the MTT analysis method Detection of B16 cell proliferation in different acitretin concentration (0.1umol/L,1umol / L,10umol/L,100umol / L) for different time (24,48,72 hours).3 Observing B16 cell differentiation and morphological changing using microscope and HE staining after treatment with different concentrations of drugs.4 To detect different the expression of Livin and bFGF of B16 cell by immunohistochemical method with different concentrations of drug treatment.5 To detect cell cycle and apoptosis rate in progress by FCMThe cells were divided into five groups, of each group with different concentrations of drug intervention 48 hours, and collected cells by FCM to detect cell cycle distribution and cell apoptosis of each group .6 To detect different the expression of Livin and bFGF of B16 cell by FCM method method with different concentrations of drug treatment.7 To analysis statistic date use statistical software SPSS13.0Results:1 MTT assay showed that acitretin onthe proliferation of B16 cells significantly inhibited (relative inhibition rate). The differences between the various groups were significantly (p <0.05). The role of different concentrations of acitretin produced in the mouse B16 cells showed a significant inhibition of time-dependent effect relationship and dose-dependent effect relationship2 To observe the differentiation and morphological changing of B16 cells:Cell in the negative control group were spindle or polygonal, adherent growth of good, transparent, refractive index is good, strong proliferation; in the acitretin treatment group, the cells become round, poor adhesion, refraction weakened, wrinkle shrinkage, some cells appear at both ends of pseudopods, and the phenomena of cell bleaching, etc., and along with the time extend, and increasind the concentration these phenomena had became more apparent serious.3 To detect the expression of Livin and bFGF protein by cell immuno- chemistry:Livin, bFGF protein was mainly expressed in the cytoplasm, DAB stain brown. The different concentrations of drug treated cells with varying degrees of protein expression, negative control group expressed strong, with the increase of drug concentration levels varying degrees of staining decreased, the difference was significant (p <0.05).4 To detect the cell cycle distribution, apoptosis rate changes by flow cytometry(FCM):By flow cytometry showed: G1 phase delay, G1-S phase transformation of inhibition, G0/G1 phase of the rise in the ratio, S phase cells reduced proportionally.5 To detect the Livin,bFGF protein expression of B16 cells by flow cytometry(FCM) after different concentrations of drug treated:The expreesion of Livin,bFGF protein of B16 cells in the different concentrations of drugs, comparison with the negative control group , expr- esion Livin, bFGF positive cells gradually decreased, after 48 hours, Livin protein fluorescence index(FI) were respectively 1.6500, 1.4215, 1.2100, 1.1124, bFGF protein expression in fluorescence index(FI) were respectively 1.8421, 1.6446, 1.4300, 1.2218. There was significantly difference between the control and the experiment group(p<0.05).Conclusions:1 Acitretin can inhibit the proliferation of B16 cells, but also within a certain time and concentration on the inhibition of B16 cell proliferation in a time-dependent and dose-dependent.2 Four concentrations of acitretin can affect the B16 mouse melanoma cells from G0/G1 phase to S phase, however, apoptosis induced by B16 is not very clear.3 Acitretin on the inhibition of B16 cell proliferation may be related to reduced apoptosis Livin factor and basic fibroblast growth factor bFGF expression.