| Multiple myeloma (MM) is a common malignant tumor, which is about 1% of all mankind malignancies or 10% to 15% of malignant hematological diseases. It is characteriszed by abnormal plasma cells infinite expansion in marrow. Multiple myeloma bone disease (MBD) is one of its clinical characteristics, which mainly shows osteoporosis, osteolytic injury, pathologic fracture, osteodynia hypercalcemia and nerve compression syndromes. MBD is due to osteolytic injury and bone formation obstacles. In marrow microenvironment, myeloma cells,marrow stromal cells,osteoblast(OB) and osteoclast(OC) express and produce some cytokines such as IL-6,1L-1β,vascular endothelial growth factor (VEGF),macrophage inflammatory protein-la (MIP-la),nuclear factor (NF)-κB, as well as osteoprotegerin(OPG), etc. These factors interact and strengthen activity of OC, leading to bone damage and new bone formation obstacles. This is the main mechanism of MBD. Myeloma cells themselves are also the important direct or indirect factors during MM bone damage development. With the action of some cell factors, the strengthened OC further promotes the MBD.Mesenchymal Stem Cells (MSCs) are a group of non-hematopoietic cells that stem from bone marrow, which support bone marrow in space as well as adjust hematopoiesis subtly. MSCs self-renewal and have multiplex differentiation potential, they are ideal seed cells for adult cell and gene therapy based on stem cells. On certain conditions, MSCs can be differentiated to cartilage cells, osteoblasts, muscle cells, tendon cells, fat cells and fibroblasts, etc. The surface of MM MSCs express lower VCAM-1 and adhesion while higher IL-1βand TNF-a comparing with normal MSCs. Through producing cytokines and contacting among cells, MSCs provides important paracrine factors for myeloma cells in the marrow. They also produce cytokines including IL-6, G-CSF and GM-CSF, which are relative to normal steady hemopoiesis. Simply to say, MSCs involve in the occurrence and development of MM through those cytokines.In MM patients, the activity of Runx2/Cbfal is obviously inhibited during pre-osteogenesis cells. As a co-stimulating factor of Runx2/Cbfal, TAZ can combine with Runx2/Cbfal, which can promote MSCs to differentiate. Wnt/p-catenin pathway plays an important role during transformation to OB from MSCs.Objectives To investigate the effects of TAZ and Wnt/β-catenin genes on the osteogenesis potential of MSCs, to analyse TAZ and Wnt/β-catenin mRNA expressions in post-osteogenesis cells of MM patients, to discuss the value of TAZ and Wnt/β-catenin as they may be MBD potential therapeutic targets.Methods We collected 10 marrow specimens of newly diagnosed MM patients, contrasted with 10 cases of normal ones. Bone marrow mononuclear cells (BMNC) were separated, purified and cultivated. The cell morphology was observed with inverted phase contrast microscope. The cell immunophenotype and purity were identified with flow cytometric analysis. During culture process, the MSCs on passage 3 were cultivated in DMEM with 1 x 10-8mol/L dexamethasone (Dex),10 mmol/L beta-glycerophosphate (beta-GP),50 ug/ml ascorbic acid (Asc) and 10% inactivated fetal bovine serum (FBS) up to 21 days, inducing osteogenesis differentiation. Alizarin red staining method was used to observe mineralization. Fluorescence quantitative RT-PCR methods were adopted to measure the mRNA of TAZ and Wnt/β-catenin and other osteoblast related factors such as OPN, OC, ALP and Cbfal. We analysed the the differences of TAZ and Wnt/β-catenin mRNA expression in the two groups. Statistical methods were adopted to analyse the data. When the data met normal distribution and homoscedasticity, we adopted Independent-Samples T Test. When the data did not meet normal distribution and homoscedasticity, we adopted Two Independent Sample Non-parameter Test, the test standard a=0.05, P< 0.05 was considered as statistically significant differences.The experimental results were combined with clinical analysis.Results Alizarin red staining obviously presented red color and the mRNAs of OPN,OC,ALP,Cbfal increased in post induction MSCs of MM patients; the mRNAs of TAZ and p-catenin were 2.2315±1.0723,0.5801±0.2159 respectively in post induction MSCs of MM patients, vs (4.414±0.8325.0.9516±0.292) of control groups, the differences were statistically significant (P<0.05)Conclusions The mRNAs of OPN, OC, ALP, Cbfal, the osteogenesis related genes, decrease in post induction MSCs of MM patients, which showes the MSCs have been successfully induced to OB. Comparing with control groups, we have proofed the osteogenesis potential of experimental groups is lower. The mRNAs of TAZ and Wnt/p-catenin also are lower than the normal. TAZ is a kind of transcription factor, which can adjust osteogenetic differentiation and restrain fat differentiation. The classic Wnt/β-catenin pathway is an important signaling pathway in the process of osteogenesis, the non-phosphorylatedβ-catenin enters into nucleus and stimulates the expression of osteogenesis gene, Wnt/β-catenin signaling pathway activates the expression of Runx2/Cbfal gene directly, promoting osteogenesis formation. Based on the above research, TAZ and Wnt/β-catenin might act as the possible target treatment for MBD. Though inducing and modifying MSCs to "whole function" OB, we could establish the foundation for autologous MSCs treatment for MBD in the future. |
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