The Study Of Inhibitory Effect Of Brucea Javanica Oil Emulsion On SKMG-4 Glioma Cells

Objective:In this experiment, we use Brucea javanica oil emulsion to experiment to study the inhibition of SKMG-4 glioma cells and its mechanism, providing the basis for Chinese medicine in chemotherapy of glioma.Methods:Cultured SKMG-4 glioma cells, divided into experimental and control groups.According to the results of preliminary experiments, in the experimental group Brucea javanica oil emulsion injection into the concentration of 5g/L,2.5g/L,1.25g/L,0.625g/L.Control group without javanica oil emulsion injection, the others completely consistent with the experimental group. The experimental group and control group put in 5% CO2, 37℃incubator about 48h.1,Using Inverted microscope to observe the inhibition effect on proliferation of different concentrations of Brucea javanica oil emulsion for SKMG-4 golima;2,Using four methyl azo thiazole salt(MTT )colorimetric assay to measure the inhibitory effect on proliferation of Brucea javanica oil emulsion for SKMG-4 golima. Inhibition rate = (A control group-A experimental group)/A control group×100%;3,Using flow cytometry(FCM) analysis the different of the cycle of SKMG-4 glioma cells before and after using Brucea javanica oil emulsion injection; 4,Using the immunohistochemical to detect the expression of apoptosis gene Bcl-2 and matrix metalloproteinase MMP-2 bfore and after using drugs;5,Using Real-time quantitative RT-PCR to detect the change of Bcl-2 gene in experimental and control groups.Using spss13.0 software in this analysis, All results are used (x一±s).Using ANOVA analysis of the results, Test with P <0.05 was statistically significant.Results:1,Inverted microscope find that with the concentration of Brucea javanica oil emulsion injection increased the growth of glioma SKMG-4 cell density decreases, cell shrinkage, cells spacing increases, nuclear fragmentation, shrinkage of the round cells (apoptotic cells) increased.2,MTT showed that with the concentration of Brucea javanica oil emulsion injection increased, the inhibition of proliferation of SKMG-4 glioma cells gradually increased,and in a dose dependent. Compared with the control group was statistically significant(P<0.05).3,Flowcytometry analysis showed that, with the drug concentration increased, G1 phase gradually increased, S phase gradually decreased,Compared with the control group, when the concentration of 5g/L,2.5g/L,1.25g/L,the difference was statistically significant (P <0.05).When the drug concentration was 0.625g/L,the difference was not statistically significant (P>0.05).4,Immunohistochemistry showed that with the concentration of Brucea javanica oil emulsion injection increased, Bcl-2 and MMP-2 in SKMG-4 glioma cells expression decreased. (Brown particles in cytoplasm is positive).5,Real-time quantitative RT-PCR results show that with the concentration of Brucea javanica oil emulsion injection increased, the expression level of apoptosis gene Bcl-2mRNA decreased. Compared with the control group was statistically significant(P<0.01).Conclusion:1,Brucea javanica oil emulsion injection can significantly inhibit the glioma SKMG-4 cells proliferation, and can reduce the invasiveness of glioma cells, and this inhibition in a dose dependent.2,Brucea javanica oil emulsion injection can make SKMG-4 glioma cells produce significant changes in morphological and biochemical characteristics, thus promoting SKMG-4 glioma cells apoptosis.3,Brucea javanica oil emulsion injection restrain the proliferation of SKMG-4 glioma cell maybe by blocking cell cycle in the G1 phase and inhibiting the expression of Bcl-2 gene.4,Brucea javanica oil emulsion injection reduce SKMG-4 glioma cell invasiveness by inhibiting the expression of matrix metalloproteinases MMP-2 gene.