| Background and Objective:Multiple Myeloma(MM) is a clonal plasma cell neoplasm that leads to the accumulation of malignant plasma cells.It's a high-incidence malignant blood tumor which often occurs in the middle-aged and elder.Due to the tumor cells' long proliferation time and their resistance to multidrug chemotherapy,most MM patients respond badly to traditional treatment and relapse easily.Therefore,a new and effective treatment target needs to be discovered urgently.Recently, with the deepening research on the etiological factors and biological mechanism of MM,there is a new understanding of the MM cell growth control and the survival factors.The main reason for disordered amplification of malignant cells,thus inhibiting the proliferation and increasing the apoptosis of tumor cells become an important research direction in tumor therapy.PDCD5(programmed cell death 5) is one of the apoptosis-related genes cloned from the human leukemia cell line TF-1 cells.Studies showed that its mRNA is widely expressed in human tissue.Its expression is reduced in a variety of malignant tissues.And some studies have shown that PDCD5 alone can not influence cells after entering a variety of cells through the eukaryotic expression vector or E.coli expression of recombinant protein,but could significantly promote cell apoptosis by adding an apoptosis-inducing factor.In the process of cell apoptosis,PDCD5 appear to the nucleus from cytoplasm and the inversion of nuclear transfer phenomena exists extensively.Our previous research showed that MM patients' bone marrow mononuclear cells have significantly down-regulated expression of PDCD5,but it is unclear whether human PDCD5 gene product rhPDCD5 can promote apoptosis of Multiple Myeloma cells.This present experiment will adopt PDCD5 recombinant protein alone or combine it with apoptosis-inducing agent dexamethasone,to observe the effect of PDCD5 protein on the apoptosis of KM3 Multiple Myeloma induced by dexamethasone,and its mechanism.Methods:Add rhPDCD5 protein alone(different concentrations of 5mg/L,10mg/L,15mg/L,20mg/L) or conjunction with dexamethasone (8mg/L) to Multiple Myeloma KM3 cells in phase of logarithmic growth, then collect the cells after co-culture for following experiments.1.Cell morphology was observed by inverted microscope directly and by fluorescence microscope after staining with DAPI.2.Flow cytometric analysis of the effects of rhPDCD5 protein and dexamethasone on the apoptotic rate of Multiple Myeloma cells through Annexin V-FITC & PI double staining.3.Western-blotting to detect Caspase-3 activity of KM3 cells4.Immunocytochemistry to detect the Survivin protein expression of KM3 cells.Results:In groups of different concentrations of rhPDCD5 protein with dexamethasonum for 16h,we observed the cell shrinkage, membrane protrusions and irregular bubble-shaped depression,nuclear pyknosis,nuclear fission and other morphological characteristic of apoptosis,while in single-treatment groups there were some but relatively little morphological changes of apoptosis.PBS control group had no significant changes in cell morphology.Using FCM to analyze the apoptosis rate,we found that apoptosis rate increased obviously in the combination groups than in the single-treatment groups.Western blotting showed that Caspase-3 protein expression and cleavage activation were raised significantly in the combinaton groups than in the single-treatment groups.ICC indicated that the expression of survivin protein reduced significantly in GC group than single-treatment group,not obviously in the control group.Conclusion:1.Human recombinant PDCD5 protein can enter KM3 multple myeloma cells and induce apoptosis of KM3 cells.Human recombinant PDCD5 protein accelerates apoptosis of KM3 cells induced by dexamethasone.2.Human recombinant PDCD5 protein may reduce expression of Survivin protein and increase activation of Caspase-3 cleavage to play its role in promoting apoptosis.
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