| ObjectiveThe cytokines which exist in the cell surface involved in cell communication, proliferation,migration,differentiation and other physiological processes in the body.It has a complex role in biological control.It is discussed aminopeptidase-N inhibitor Ubenimex in vitro on human's myeloma cell growth,mitochondrial membrane potential and induction of apoptosis and its mechanism.MethodsThe experiments in 2007-12/2009-2 has been finished at the Central Laboratory of China Medical University.Experimental Materials:Human myeloma cell XG-1 is preserved by the Chinese Medical Hematologists.Ubenimex is donated by Zhejiang Pharmaceutical Co.,Ltd.(ky-ub-20050903).Experimental Methods:Get the logarithmic phase of myeloma cells to produce cell suspension by 1.5×103 / hole density of inoculation,then cultivate 24h cells films.Ubenimex Group Add 500,1000μg / ml of Ubenimex in Ubenimex Group,add 1640 culture medium in a blank control group.Experimental assessment:After the 24,48h effect of Ubenimex,using an optical microscope to observe the changes in the structure of cell morphology;Use the Rhodamine123 mark the living cells,use the flow cytometry detect the changes of mitochondrial membrane potential;use SABC assay cellular immune cells wither Detection of apoptosis-related proteins detect caspase-3 and bcl-2 activity,use DNA fragments in situ end labeling(TUNEL) detect apoptotic cells.ResultsThe morphology of apoptosis:It can be seen that cell volume reduced,the quality of nuclear pyknosis,nuclear membrane nucleolus fragmentation,dissolution, cytoplasmic condensation and typical apoptotic body by the 1000ug/ml Ubenimex effect in the cell after 48h.The myeloma cells which in endoscopic control group is transparent and clear cytoplasm,has a gathered growth status.Cells which is confirmed that myeloma have CD13 cell surface exist by immunohistochemistry.Apoptosis:it is (3.57±0.41)%,(58.35±8.45)%after 500,1000μg / ml Ubenimex's effect by 24 h;at 24～48 h,the apoptosis of cells progressive necrosis;Blank control group,the apoptosis rate of 24,48 h were 1%.Changes in mitochondrial transmembrane potential: 1000μg/ml Ubenimex's effect by 24,48 h,the mean fluorescence intensity of the mitochondrial membrane potential of Ubenimex cell was significantly lower than blank control group(P＜0.01).The expression of apoptosis-related proteins:500,1000μg/ml Ubenimex's effect in myeloma cells after 48 h,apoptosis-related protein(pro-caspase3, Bcl-2) reduced.ConclusionThe Ubenimex is able to inhibit myeloma XG-1 cell apoptosis.Maybe DNA damage is induced by cells through the mitochondrial pathway to intervene and lower protein content,such as apoptosis-related apoptosis implementation. |
