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OBJECTIVE@#To investigate the expression of pyruvate kinase M2 (PKM2) in bone marrow mesenchymal stem cells (BMSCs) in myeloma bone disease (MBD) and its effect on osteogenic and adipogenic differentiation of BMSCs.@*METHODS@#BMSCs were isolated from bone marrow of five patients with multiple myeloma (MM) (MM group) and five with iron deficiency anemia (control group) for culture and identification. The expression of PKM2 protein were compared between the two groups. The differences between osteogenic and adipogenic differentiation of BMSCs were assessed by using alkaline phosphatase (ALP) and oil red O staining, and detecting marker genes of osteogenesis and adipogenesis. The effect of MM cell line (RPMI-8226) and BMSCs co-culture on the expression of PKM2 was explored. Functional analysis was performed to investigate the correlations of PKM2 expression of MM-derived BMSCs with osteogenic and adipogenic differentiation by employing PKM2 activator and inhibitor. The role of orlistat was explored in regulating PKM2 expression, osteogenic and adipogenic differentiation of MM-derived BMSCs.@*RESULTS@#Compared with control, MM-originated BMSCs possessed the ability of increased adipogenic and decreased osteogenic differentiation, and higher level of PKM2 protein. Co-culture of MM cells with BMSCs markedly up-regulated the expression of PKM2 of BMSCs. Up-regulation of PKM2 expression could promote adipogenic differentiation and inhibit osteogenic differentiation of MM-derived BMSCs, while down-regulation of PKM2 showed opposite effect. Orlistat significantly promoted osteogenic differentiation in MM-derived BMSCs via inhibiting the expression of PKM2.@*CONCLUSION@#The overexpression of PKM2 can induce the inhibition of osteogenic differentiation of BMSCs in MBD. Orlistat can promote the osteogenic differentiation of BMSCs via inhibiting the expression of PKM2, indicating a potential novel agent of anti-MBD therapy.
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Humans , Adipogenesis , Bone Diseases/metabolism , Bone Marrow Cells , Cell Differentiation , Cells, Cultured , Mesenchymal Stem Cells/physiology , Multiple Myeloma/metabolism , Orlistat/pharmacology , Osteogenesis/geneticsABSTRACT
Objective To understand the correlation of expression levels of serum stromal cell-derived factor 1α (SDF-1α), osteoprotegerin (OPG) and β2microglobulin (β2-MG) in patients with multiple myeloma (MM) with or without myeloma bone disease (MBD). Methods Eighty patients with MM who were admitted to the Affiliated Hospital of Inner Mongolia Medical University from January 2014 to June 2017 were selected; all of the patients met the international diagnostic criteria for MM. According to the symptoms such as bone pain, the patients were divided into group with MBD (45 cases) and group without MBD (35 cases). Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of SDF-1α and OPG, and radioimmunoassay was used to detect the expression of MM major prognostic indicator β2-MG. The MBD score was evaluated in 45 patients selected by random number table after sacroiliac joint X-ray and three-dimensional bone reconstruction. The χ 2test was used to compare the categorical variables; the two independent sample t-test was used to compare the continuous variables that conformed to the normal distribution between two groups, and the Pearson method was used for the correlation analysis. Results The expression level of serum SDF-1α in the group with MBD was significantly higher than that in the group without MBD [0.31±0.17) pg/ml vs. (0.18±0.06) pg/ml], and the difference was statistically significant (t ＝-4.21, P < 0.001). The expression level of serum OPG in the group with MBD was significantly lower than that in the group without MBD [(0.73±0.50) pg/ml vs. (1.08±0.31) pg/ml], and the difference was statistically significant (t＝ 3.62, P< 0.001). Pearson analysis showed that β2-MG level in the group was positively correlated with SDF-1α level (r＝ 0.84, P< 0.001), and negatively correlated with OPG level (r＝ -0.48, P<0.001). The β2-MG level in the group without MBD did not show a correlation with the SDF-1α and OPG levels. Conclusions In the serum of patients with MBD, the expression levels of β2-MG and SDF-1α are increased, and the expression level of OPG is decreased. SDF-1α and OPG may be new clinical biochemical indicators for diagnosis, treatment and prognosis assessment in MBD.
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Multiple myeloma (MM) originates from malignant plasma cells, leading to multiple destructive lytic bone lesions that occur in more than 80%of MM patients. MicroRNAs have been reported to be involved in the development of bone lesions in MM. However, it is still unclear that microRNAs can be considered as diagnostic and prognostic biomarkers for bone lesions. MiR-214 and miR-135b may participate in the pathogenesis of bone marrow, which has shown a certain guiding significance in its prognosis. This paper will introduce the relationship between miR-214, miR-135b and myeloma bone disease.
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Multiple myeloma is a more common malignant disease in blood medicine,which is characterized by hyper-proliferation and accumulation of malignant plasma cells in the bone marrow,resulting in a large number of monoclonal immunoglobulins and their fragments,leading to damage to the terminal organs.Of which about 80％ of patients have multiple myeloma bone diseases (MBD),which seriously affected the quality of life and prognosis of patients.We have found that the main cause of MBD are contributed to inhibition of osteoblasts,activation of osteoclast and affecting the occurrence by a variety of cytokines and pathways.This article will review and introduce the occurrence and development of MBD related to the latest factors and treatment.
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Objective To explore the functions of neuron‐specific enolase(NSE) and human multiple myeloma U266 cells on osteoclast‐like cells(OLC) function .Methods Normal human peripheral blood mononuclear cells were induced and cultured by adding RANKL and M‐CSF to get OLC ;the experiment was divided into 3 groups ,the NSE group:OLC were cultured in the 6‐well culture plate for 14 d and added with 100 ng/mL recombinant human NSE to culture for 24 ,48 ,72 h;the co‐culture group:OLC were cultured in the lower well of 6‐well Transwell chamber for 14 d ,then added with 1 × 105/well U266 cells in each upper well and conducted the co‐culture for 24 ,48 ,72 h;the control group :OLC were cultured alone .The influences of NSE and U266 cell line on RANKL ,OPG ,IL‐6 and TRAP mRNA transcriptional level of OLS were compared by using real‐time fluorescent quantitative PCR .Results RANKL ,OPG ,IL‐6 mRNA had no expression on OLC in the co‐culture group ,NSE group and control group ;com‐pared with control group ,the TRAP mRNA expression level in the co‐culture group and the NSE group was increased ,the differ‐ence was statistically significant(P<0 .01);the increase of TRAP mRNA expression level was obvious especially at 48 ,72 h .Con‐clusion OLC expressing TRAP and NSE may be one of the factors for promoting OLC differentiation and maturation in myeloma bone disease ,prompting that NSE could increase the OLC viability .
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Objective:To explore the role of γδ T cells in the transdifferentiation of immature dendritic cells(imDC) into osteoclasts(OC). Methods:(1) Peripheral blood mononuclear cells(PBMNC) were cultured with zoledronate(Zol) and recombinant human interleukin-2(IL-2),and PBMNC from healthy volunteers were cultured with granulocyte macrophage colony-stimulating factor (GM-CSF) and recombinant human interleukin-4(IL-4) to differentiate into imDC,which were then cultured with receptor activator nuclear factor к B ligand(RANKL) and macrophage colony-stimulating factor(M-CSF) to differentiate into OC. The purity of γδ T cells,and phenotype changing of OC transdifferentiated from imDC were investigated by flow cytometry. (2) Co-culture system was es-tablished using millicell inserts.γδT cells isolated with immune magnetic bead were placed in the upper compartment and imDC in the lower compartment in the ratio of 10∶1. To explore the role of γδ T cells during differentiation of imDC into OC,tartrate resistant acid phosphatase( TRAP) staining and bone resorption observation staining were used. Tumor necrosis factor-alpha( TNF-α) of supernatant liquid from different cultures was measured using ELISA(Enzyme linked immunosorbent assay) kit. Results:(1) γδT cells can be ex-panded from PBMNC of MM patients, and the production capacity was similar to that of healthy volunteers ( 68. 87%± 20. 94% vs 69. 33%±16. 84%,P>0. 05 ) . ( 2 ) OC could be transdifferentiated from imDC when cultured with RANKL and M-CSF. ( 3 ) The number of TRAP+ multinuclear cell and the absorption area of dentine were significantly lower in the group of imDC indirectly co-cultured with γδ T cells than in the group of control imDC(5.67±0.58 vs 28.33±2.08,4.97%±4.3% vs 28.47%±12.8%, respectively). (4) Under the circumstance of γδ T cell-imDC indirect coculture,TNF-α got significantly higher. Conclusion: γδ T cells might inhibit the transdifferentiation of imDC into OC.γδ T cells-based immunotherapy is expected to be a new treatment for myeloma bone disease.
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Objective To discuss function of the fusion cells of human bone marrow stromal cells (BMSCs) and human myeloma cell RPMI 8226 in the pathogenesis of multiple myeloma bone disease.Methods The cells,labeled by cell tracer green fluorescent probe (CMFDA) and red fluorescent probe (CMTMR),respectively,were induced into fusion by chemical polyethylene glycol (polyethyleneglycol,PEG-1000),and cell fusion model was set up.Whether fusion cells had nucleus fusion was determined by Karyotype analysis.The expressions of stemness-related genes,SIRP α gene and DC-STAMP gene in fusion cells were identified.Results Polyethylene glycol (PEG-1000) could mediate the integration of BMSCs and RPMI 8226 cells.The number of chromosomes in more than 80 ％ the hybrid cells was about 80.Fusion cells not only showed that BMSCs,stemness-related genes of c-myc,Klf-4 and OCT-4 genes expressed positively,but also the fusion-related genes SIRPα and DC-STAMP expressed positively.Conclusion BMSCs and RPMI 8226 cells can form fusion cells,and the cells have the potential for further integration,which is one of the important reasons for the promotion of muhiple myeloma bone destruction.
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Objective To investigate the relationship between macrophage inflammatory protein 1-α( MIP-1α) ,sclerostin and multiple myeloma bone disease( MBD) .Methods 52 patients with multiple myeloma were selected as the observation group,among them,9 cases without MBD,43 cases with MBD;MBD grade:9 cases with grade 0,8 cases with grade 1,21 cases with grade 2,14 cases with grade 3.25 healthy examinee were selected as the control group.MIP-1αand sclerostin were observed.Results MIP-1α,sclerostin in the observation group[(158.43 ± 78.75)pg/mL,(0.62 ±0.24)pg/mL] were higher than those in the control group[(52.46 ±21.35)pg/mL,(0.31 ± 0.14)pg/mL](t =9.635,P <0.05;t =8.844,P <0.05);MIP-1α,sclerostin of MBD patients[(175.55 ± 62.75)pg/mL,(0.84 ±0.54)pg/mL]were higher than those of non MBD patients[(89.83 ±41.57)pg/mL,(0.42 ± 0.25)pg/mL](t=7.665,P<0.05;t=6.834,P<0.05).There were positive relation between MIP-1α,sclerostin and the grade of MBD(r=0.572,P<0.05;r=0.683,P<0.05).There was positive relation between the expressions of MIP-1αand sclerostin(r =0.522,P <0.05).Conclusion MIP-1αand sclerostin of patients with MM have increased.There is a significant relation between MIP-1α, sclerostin and MBD.United detection of MIP-1αand sclerostin may be an index used as evaluation of MBD.
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ObjectiveTo detect IL-17 level of bone marrow in patients with multiple myeloma bone disease,and to investigate its clinical significance.MethodsThe bone marrow IL-17 levels were quantified in 33 cases of multiple myeloma patients and 20 normal control by ELISA assay. RANKL mRNA expression were detected by using RT-PCR.ResultsIn bone marrow supernatant,IL-17 was detected in both groups,and RANKL mRNA were detected in both groups too. IL-17 and RANKL mRNA levels in bone marrow of patients with MM were significantly higher than those in the control group(P＜0.05). The bone marrow concentrations of IL-17 and bone marrow mononuclear cells' RANKL mRNA expression in active stage were significantly higher than those in stable stage (P＜0.05).The bone marrow IL-17 and RANKL were significantly correlated (r =690,P＜0.05).ConclusionIL-17 may play an important role in the pathogenesis of MM.
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ObjectiveTo investigate the gene expressions of TAZ and Wnt/β-catenin on the postosteogenic cells of mesenchymal stem cells(MSC)in multiple myeloma(MM)patients and to explore the potential therapeutic target of multiple myeloma bone disease (MBD).MethodsBone marrow mononuclear cells MNC from MM and controls were isolated,cultured,expanded and then induced to osteogenic differentiation.Realtime quantitative RT-PCR was employed to detect the osteogenic markers (TAZ,Wnt/β-catenin,OPN,OC,ALP and Cbf α1); and alizarin red staining for mineral deposition.The mRNA expressions of TAZ and Wnt/β-catenin in the two groups were analysed.ResultsAlizarin red staining was positive and the red calcium nodules were appeared on the post-osteogenic cells of MSC.The mRNA expressions of OC,ALP and Cbf α1 were 2.0958±0.5665,2.6670±0.3847,0.8463±0.3473,respectively,on the post-osteogenic cells of MSC in the experimental groups,which were significantly higher than those of pre-osteogenic cells(1.3487±0.9291,1.1452±0.6054,0.4439±0.2945) (t =2.171,6.709,2.795; all P ＜ 0.05).The mRNA expressions of OPN,OC,ALP and Cbf α1 were 2.1096±0.8267,2.8991±0.3531,4.3045±0.2844,1.3273±0.4075,respectively,on the post-osteogenic cells of MSC in the controls,which were significantly higher than those of pre-osteogenic cells (1.2200±0.9091,0.8780±0.3927,1.9161±0.2684,0.6736±0.2513) (t =2.289,12.103,25.134,4.411; all P ＜ 0.05).The mRNA expressions of OPN,OC,ALP,Cbf α1 were 1.2710±0.5636,2.0958±0.5665,2.6670± 0.3847,0.8463+0.3473,respectively,on the post-osteogenic cells of MSC in the experimental groups,which were significantly lower than those of control groups(2.1096 ±0.8267,2.8991 ±0.3531,4.3045±0.2844,1.3273±0.4075) (t =-2.650,-3.805,-10.822,-2.841; all P ＜ 0.05).The mRNA expression of TAZ and β-catenin were 2.2315±1.0723 and 0.5801±0.2159 on the post-osteogenic cells of MSC in MM patients,which were significantly lower than those of control groups (4.4140±0.8325,0.9516±0.2920) (t =±5.085,-3.235;both P ＜ 0.05).ConclusionThe gene expressions of OPN,OC,ALP and Cbf α1,the osteogenesis related genes,are increased in post-osteogenic cells of MSC,which showed the MSC have been successfully induced to osteoblasts.Comparing with control groups,the osteogenic potential of MSC in MM patients is lower.Based on the above research,TAZ and Wnt/β-catenin may present a novel target for the future therapy of MBD.
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Myeloma bone disease (MBD) with the characteristic of progressing osteolysis, is one of the main clinical manifestations of multiple myeloma (MM). In recent years, it was revealed that a kind of glycoprotein - Dickkopf (DKK1) associated with embryonic development and tumor progression can inhibit the Wnt signal pathway. The DKK1 efficacy in inhibiting the steoblasts and activating the osteoclasts appears to play an important role in MBD. The ongoing MBD-targeting therapies involving DKK1 neutralizing antibody, proteasome inhibitor, vaccine and regulatory factor on the basis of this mechanism have shown some benefit for MBD. Copyright© 2011 by the Editorial Board of Tumor.
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Objective To deplore the immunoregulatory function changes of mesenchymal stem cells(MSCs)from multiple myeloma(MM)patients and its effects on the pathogenesis of myeloma bone disease.Methods MSCs from MM patients and normal controls were isolated and the immunophenotype was detected.Real-time PCR was performed to detect the expressions of TGF-β1,TGF-β2,TGF-β3,IL-6,IL3,TNF-α,FasL and RANKL of MSCs.Transwell coculture systems were performed between MSCs and T cells.Lymphocyte proliferative assay was employed to detect the effect of MSCs on T cell proliferation.The effect of MSCs on T cell cycle and T cell activation markers CD25 and CD69 expression were analyzed by flow cytometry.Cleaved caspase 3 protein by western blot and hoechst 33258 staining were employed to detect the apotosis of T cells.Influence of T cells on the osteogenesis potential of MSCs were detected by Von kossa stain,real-time PCR and Western blot.Results MSCs from both MM patients and normal controls possessed similar morphology and immunophenotypes.MM derived MSCs exhibited increased expressions of TGF-β1,IL-6,IL-3,TNF-α and RANKL and decreased expression of TGF-β2,TGF-β3 and FasL.The inhibitory effect of MM derived MSCs on T cell proliferative ability was attenuated compared to control MSCs.MSCs from normal controls silence more T cells in Go/G1 phase than those from MM patients.The daupening effect of MM derived MSCs on activation-induced T apoptosis seemed to be enhanced.Expression of T cell activation markers were significantly inhibited by MSCs from normal controls.Both T cells cocultured with MM deprived MSCs and T cells directly from MM patients inhibited osteogenesis potential of MSCs from normal controls.Conclusion MSCs from MM patients showed impaired immunoregulatory capability on T cells.The activated T cells,in turn,inhibited the osteogenesis potential of MSCs.This may participate in the pathogenesis of myeloma bone disease.