| Objective To investigate the biological changes such as osteogenetic ability, proliferative capacity, and transcription of DKK1, Collagenl,Decorin and Smad5 gene of BMMSCs co-cultured with U-266 myeloma cells.The results can help us to know whether the biological behavior of BMMSCs can be affected by myeloma cells when they cultured together, through which we can elucidate further the pathogenenic mechanism and provide the more abundant theoretical and experimental foundation to explore the new clinical treatment methods.Methods1.BMMSCs were obtained from the bone marrow of normal subjects.The BMMSCs on passage 3 were employed for further experiments.There were two groups:①BMMSCs were cultured in the absence of U-266 myeloam cell lines as controls;②MMSCs were co-cultured with U-266 cells for 5,8 or 12 days.2. The BMMSCs co-cultured with U226 cells were the experimental group and were compared with the controls.The morphology of The No.3 generation BMMSCs(have enough amount cells)which co-cultured with U226 cells were observed under the Inverted Microscopic when be continue cultured them for other 4 days.Meanwhile, we counted the number of the BMMSCs at 5,8,12 day and compared with control groups. 3. Affer five days., the L-DMEM which contained dexamethasone (10-8 mol/L),vitaminC(0.05g/L) and P-glycerophosphate(10-2mol/L) were added into BMMSCs co-cultured with U226 cells culture system and BMMSCs removed U266 cells to induce BMMSCs differentiation to OB.After added L-DMEM the BMMSCs were stained by alkaline phosphatase (ALP) and Acid red alizarine to identify the formation ability of BMMSCs differtiation to the OB at 14 day.4.BMMSCs were collected after 5,8 or 12 days'culture.The expression of Collagen1,Decorin,DKK1,Smad5 mRNA of the BMMSCs of two groups were measured by Real-time PCR and compared the expression levels of Collagen 1,Decorin,DKK1,Smad5 gene at different time.Results1.There were no evident morphological changes between the BMMSCs cocultured with myeloma cell lines and BMMSCs without MM cell lines.The proliferative capacity of BMMSCs in co-culture group have no evidence changes.2.The assays for osteogenic capacity of the BMMSCs indicated that the BMMSCs of two groups can be found to differentiate to osteoblast. But the BMMSCs co-cultured with U266 cells has the less mineralized matrix deposition than that found in control group and has the poor osteogenic differentiation potential.3.The expression levels of Collagenl,Decorin, DKK1,Smad5 genes were analyzed by Real-time PCR in both groups, the results showed that the expression levels of Collagenl,Decorin, Smad5 genes decreased in the BMMSCs co-cultured with U266 than in control group, but the expression levels of DKK1 increased. (1)Following the co-culture time, the levels of expression of Collagenl were respectively 19.8600±6.8429,20.8633±7.6858,15.8267±6.3454 at 5,8,12 day,50.2382±16.1061,42.2650±9.5823,57.9167±17.0791 in control group, which was on a declining trend compared with control group(P<0.05).The levels of Decorin expressions were decreased siginificantly (4.6657±0.9456, 5.9857±0.9894 and 5.5714±0.9292 for co-cultured group at 5,8 and 12 day respectively, while 14.8414±3.0398,5.6800±3.0845,12.9086±2.6157 for control group respectively, P<0.05).(2) The levels of Smad5 expressions at 5,8,12 day are respectively 2.7125±1.4307,1.7125±0.2288,2.8325±0.8263;5.2725±1.5321,11.5275±2.6250和7.2125±2.0749 in control group, the expression levels of it at 5,8,12 day were decreased (P<0.05)。The levels of DKK1 gene expression were 2.4757±0.6017,8.8821±2.0199,3.5250±2.7964 in control group,6.4537±1.3495,12.8947±7.9996,3.7819±1.3803 at 5,8,12 day respectively, the level of the expression of DKK1 gene increased in BMMSCs significancely at 5 day(P<0.05), but had no statistical significance at 8,12 day (P>0.05) compared with control group.Conclusion1.The proliferative and osteogenic ability of BMMSCs were impared when cocultured with myeloma cells,but have no evident morphological changes. 2.The osteogenic differentiated potential differentiate ability of the BMMSCs decreased when co-cultured with U266,the fact indicated that Myeloma cell lines had the ability to regulate BMMSCs biological activity.3.U226 cells significantly up-regulated osteoclastogenesis-associated gene expressions such as the DKK1 gene expressions, the results show that myeloma cells may took part in the dissolved bone pathological process by activating or inhibiting relative gene expression. U226 cells can reduced Collagen1,Decorin, Smad5 gene expression in BMMSCs.the results show that the myeloma cells in the bone marrow have profoud effects on marrow microenvironment componants BMMSCs.4.Those biological changes of BMMSCs influnced by myeloma cells may havecorrelations with the disorder of MM.
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