Study On The Protease Inhibitor To Inhibit The Cell Of Glioma U251

Glioma is one of frequent malignant tumor in central nervous system,accounting for 30%-60% of intracranial tumour.Its morbility reason is not very understand, and there has been no obviously progress on treatment at the present,the main treatment is the surgical operation to remove the tumor with radiationg and chemoeherapy.But the glioma growing with infiltration, the tumor and the brain tissue's boundary is not clear, all of these reasons reduce the effect to a certain extent.So we need the effective drug in the clinical work in treating the glima,and it also become one of research direction for tumor treating.Ubiquitin-proteasome pathway is one of the main pathway of intracellular protein degradation, which participating many biochemistry responses in the biology, involving the cell's growth,proliferation,differentiation and apoptosis, etc,and the abnormal of pathways is relating with the mechanism of tumor,neurodegeneratie diseases,genetic diseases,inflammation and haematology, etc. Proteasome inhibitor can inhibit the growth of tumor cell and promote tumor cell apoptosis by the way of UPP, it can causes cell cycle arrest by effecting on the normal cell body,and induces tumor cell apoptosis when it effects tumor cells,but the mechanism is unclear.Cell apoptosis including Mitochondria pathway,Endoplasmic reticulum pathway and Death receptor pathway, Caspase-3 is the main enzyme in the apoptosis process.The change of Cytc causes cell membrane potential changes is the key steps.In our experiments,we detect the expression of Bcl-2/Bax,Caspase-3 and cell membrane potential changes to determine the type of apoptosis and the effects of proteasome inhibitor on glioma cells by MTT,RT-PCR,Rh123 and Western Blotting,etc.Purposes:We investigate the effects of proteasome inhibitor Lactacystin on rat U251 glioma cells to explore its relationship and mechanism on cell apoptosis,so we can provide a certain direction of reaserch and theoretical basis for treating glioma in clinical.Methods:Cultivating the U251 glioma cells in vitro,and select the cells in the logarithmic growth.The cells were divided into the control group, LAC 5μmol.L-1 Group, LAC 10μmol.L-1 Group and LAC 15μmol.L-1 Group, detecting the change of each group's cells viability by MTT assay; detecting the level of the cells Bcl-2/Bax and the expression of Caspase-3 by the method of RT-PCR; detecting the mitochondrial membrane potential and the change of the cells nuclei by confocal microscope.Results:1,Compared with the Con group, in LAC 5μmol.L-1 Group, LAC 10μmol.L-1 Group and LAC 15μmol.L-1 Group, the rate of the U251 cells viability were decreased(P<0.05) by the method of MTT assay.2,Compared with the Con group, in LAC 5μmol.L-1 Group, LAC 10μmol.L-1 Group and LAC 15μmol.L-1 Group, the level of the U251 cells Bcl-2/Bax were decreased(P<0.05) and the expression of Caspase-3 were increased(P<0.05) by the method of RT-PCR,which showed that the mitochondrial pathway participates the cell apoptosis.3,Compared with the Con group, in LAC 5μmol.L-1 Group, LAC 10μmol.L-1 Group and LAC 15μmol.L-1 Group, the U251 intracellular red fluorescence intensity decreased significantly by the method of Rhodamine 123,which showed that the mitochondrial membrane potential was decreased, suggestting the apoptosis.4,Compared with the Con group, in LAC 5μmol.L-1 Group, LAC 10μmol.L-1 Group and LAC 15μmol.L-1 Group,the U251 cells form obvious changed,cells nuclei obvious corrugation and fission by the method of Hoechst 33342,which suggested the apoptosis. 5,Compared with the Con group, in LAC 5μmol.L-1 Group, LAC 10μmol.L-1 Group and LAC 15μmol.L-1 Group, the level of the U251 cells Bcl-2/Bax were decreased(P<0.05) obviously by the method of Western blotting,which suggested that the mitochondrial pathway participates the cell apoptosis.Conclusions:1,Proteasome inhibitor Lactacystin can inhibit the proliferation of the U251 glioma cell.2,Proteasome inhibitor Lactacystin can induction the cell apoptosis of the U251 glioma cell.3,Proteasome inhibitor Lactacystin can induces the apoptosis of U251 glioma cell, which may be through the mitochondrial pathway.