骨髓瘤细胞抑制成骨细胞活性和Runx2表达

have a Inhibitory effect of myeloma cells on osteoblast activity and Runx2 expression

目的建立MM细胞系(U266细胞株)和MSCs共培养体系,研究骨髓瘤细胞对MSCs增殖能力、OB形成能力和Runx2表达的影响。方法研究对象为2例缺铁性贫血和1例特发性血小板减少性紫癜。采用密度梯度离心结合贴壁培养法分离纯化骨髓MSCs,培养3～5代后,诱导MSCs向OB分化,待细胞融合至60%～70%时,分两组,一组加U266细胞株共同培养(共培养组),另一组不加U266细胞株(对照组)。采用CCK-8试剂盒检测MSCs向OB分化过程中的增殖能力,以平均光密度值(AOD)表示;碱性磷酸酶(ALP)染色和钙结节(Von-Kossa)染色检测MSCs的OB形成能力,分别以IOD sum/Area sum值和平均钙结节个数表示;RT-PCR技术检测MSCs向OB分化过程中Runx2的表达,以Runx2/GAPDH平均光密度值表示。结果在共培养体系中,U266细胞株和MSCs共培养的前3d,MSCs增殖能力无明显变化,两组AOD值分别为0.731±0.020和0.805±0.152(P>0.05),至第6天,共培养组的MSCs增殖能力明显低于对照组,AOD分别为0.560±0.034和2.283±0.145(P<0.05)。与对照组相比,共培养组MSCs向OB分化过程中ALP的表达显著降低,IOD sum/Area sum值分别为0.138±0.038和0.376±0.044(P<0.01),钙结节的形成减少,平均钙结节个数分别为(6.7±2.75)个和(15.3±8.99)个(P<0.01)和Runx2的表达显著降低,Runx2/GAPDH平均光密度值分别为0.238±0.168和0.653±0.343(P<0.01)。结论骨髓瘤细胞可抑制骨髓MSCs向OB分化的增殖能力和OB形成能力,也抑制其Runx2表达。

Objective To investigate the effect of myeloma cells line（U266） on proliferation,osteoblast formation and Runx2 expression of MSCs when cocultured with U266 cell lines.Methods Bone marrow was collected from 2 patients with iron deficiency anemia and 1 with idiopathic thrombocytopenic purpura.At the passage 3 to 5,MSCs were induced to differentiate into OB in L-DMEM conditioned medium.MSCs were divided into two groups when they are fusion 60% to 70%,one group was cocultured with U266 cell line（coculture group）,the other without U266 cells line as a control.CCK-8 kit was employed to analyze the proliferation of MSCs,defined as the average optical density（AOD）.Alkaline phosphatase staining and Von-Kossa staining were performed to assess osteoblast formation,defined as IOD sum/Area sum and the average of calcium nodules respectively.RT-PCR assays were applied to detect Runx2 expression of MSCs,defined as Runx2/GAPDH average optical density.Results CCK-8 assay showed that no significant difference was found in proliferation of MSCs between coculture and control group when cultured up to day 3.AOD were 0.731±0.020 and 0.805±0.152,respectively（P0.05）.However,significant decrease of AOD was found in coculture group up to day 6.AOD were 0.506±0.034 vs 2.283±0.145,respectively（P0.05）.Compared with the control group,the ALP expression of coculture group decreased significantly.IOD sum/Area sum were 0.138±0.038 and 0.376±0.044,respectively（P0.01）.Besides,the calcium nodules formation was also significantly less in coculture than that of control group.The average of calcium nodules were 6.7±2.75 and 15.3±8.99,respectively（P 0.01）.Runx2 expression of MSCs was significantly lower in coculture than control group.Runx2/GAPDH average optical density were 0.238±0.168 and 0.653±0.343,respectively（P0.01）.Conclusions Myeloma cells have a inhibitory effect on proliferation and osteoblast formation of MSCs as well as their Runx2 expression.