ABSTRACT
Objective: To compare the sensitivity of 8-color panels and next generation flow cytometry (NGF) for detecting minimal residual disease of multiple myeloma patients. Methods: 8-color-membrane antigens (8C-Mem) panel was built including CD45, CD38, CD138, CD19, CD56, CD81, CD27 and CD117 to identify the plasma cells, while 8-color-cytoplasmic antigens (8C-Cyto) panel was built including CD45, CD38, CD138, CD19, CD56, CD81, cKappa (cK) and cLambda (cλ) , and 8-color-two-tubes (8C-2tubes) panel were built including 8C-Mem and 8C-Cyto panels, the data of three groups was analyzed by Diva software. NGF uses Infinicyt software to fuse 8C-2tubes data to further analyze the expression of plasma antigens. Bone marrow aspiration obtained from 20 controls and 76 multiple myeloma patients who achieved complete remission were measured and analyzed. Results: Positive MRD samples were discriminated in 88.2% of the specimen evaluated through either abnormal plasma cells (aPCs) or clonal plasma cells (cPCs) by NGF antigens panel, Among of them, consistency was 94.7%. The median percentage of cPCs was 0.3530%, The lowest sensitivity of NGF was 0.0003%. In 8-color panels, the positive MRD rates of 8C-Mem, 8C-Cyto and 8C-2tubes panels were 84.2%, 85.5% and 86.8%, respectively, which lower than that of NGF (P<0.001) . The positive MRD rate of 8C-Mem and 8C-Cyto panels were lower than that of 8C-2tubes panel (P<0.001) , and the positive MRD rate of 8C-Mem panel was lower than that of 8C-Cyto panel (P<0.001) . Sensitivity and specificity of NGF was higher than that of 8-color panels. 8C-2tubes panel has the best sensitivity, accuracy, negative predicted value, positive predicted value and specificity than other 8-color panels. However, huge data and low efficiency for analysis is the disadvantage. 8C-Cyto panel was the second choice, and 8C-Mem panel was the last. Conclusions: Membrane and cytoplasmic light chain is a better method for multiple myeloma-MRD detection and NGF panel is an ideal approach. 8C-Cyto panel is recommended in 8-MFC groups.
Subject(s)
Humans , Bone Marrow , Flow Cytometry , Immunophenotyping , Multiple Myeloma/diagnosis , Neoplasm, Residual , Plasma CellsABSTRACT
<p><b>OBJECTIVE</b>To explore the regulatory effect of GRK6 on the proliferation of multiple myeloma cells and its mechanism.</p><p><b>METHODS</b>A lentivirus vector shRNA interfering in human GRK6 gene expression was constructed and trans-fected into multiple myeloma cells to obtain the cell line MM1R with stable down-regulation of GRK6 gene expression. The real-time quantitative PCR and Western blot were used to confirm the effectiveness of the GRK6 gene expression down-regulation mediated by lentivirus vector. The MM1R cells with most obvious down-regulation were selected to detect the effect of GRK6 gene on cell proliferation.</p><p><b>RESULTS</b>The lentivirus vector GRK6-shRNA interfering in human GRK6 gene was constructed succesufully and transfected into multiple myeloma cells, thereby the MM1R cell line with stable down-regulation of GRK6 gene was obtained. The CCK-8 assay showed that the proliferative viability of MM1R cells in experimental group was significantly lower than that in control group (P<0.05); the flow cytometry showed that cells in experimental group were arrested in G/Gphase(P<0.05); the Western blot detection showed that the Cyclin D1 and CDK4 levels in experiment group obviously decreased as compared with control group.</p><p><b>CONCLUSION</b>A lentivirus vector which can specifically interfere in GRK6 gene expression is constructed successfully, The MM1R cell line with stable down-regulation of GRK6 expression is obtained by transfection and screening. The down-regulation of GRK6 expression can arrest MM1R cells in G/Gphase, moreover inhibits the proliferation of MM1R cells by inhibition of Cyclin D1 and CDK4 levels.</p>
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of ADAM10 inhibitor GI254023X on the proliferation and apoptosis of multiple myeloma H929 cell line and its mechanisms.</p><p><b>METHODS</b>H929 cells were treated with different concentrations of GI254023X, the proliferation-inhibitive curve was assayed and plotted by CCK-8 method, the cell viability and apoptosis were detected by flow cytometry with Annexin V/7-AAD double staining. The cleavage of Notch1 protein (cleaved notch1) was determined by Western blot. The transcripts of Notch1 target gene Hes-1 were detected by real-time PCR.</p><p><b>RESULTS</b>The GI254023X inhibited the proliferation of H929 cells in the time- and dose- dependent manners. As compared with the control group, the apoptosis of cells increased along with enhancement of GI254023X concentration; The expression of cleaved Notch1 was down-regulated after the treatment with GI254023X. The levels of Hes-1 mRNA transcripts in H929 cells was reduced in GI254023X treated group.</p><p><b>CONCLUSION</b>GI254023X can remarkably inhibit the proliferation and induce the apoptosis of H929 cells. Its mechanism may be associated with inbihition of Notch1 activation.</p>