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本研究探讨蛋白酶体抑制剂硼替佐米在骨髓瘤骨病(myeloma bone disease,MBD)病理状态下对成骨细胞的影响。以小鼠骨髓瘤细胞RPMI8226与小鼠成骨细胞MC-3T3E1共培养和上清干预培养的方式,模拟体内骨髓瘤骨病状态,采用改良四甲基偶氮唑盐比色(MTT)法检测硼替佐米对MC-3T3E1细胞的增殖抑制,Annexin V/PI染色流式细胞术检测MC-3T3E1细胞的凋亡,RT-PCR及Western blot法比较加入硼替佐米后MC-3T3E1细胞成骨相关基因及蛋白Runx2/cbfa1、OSX(osterix)、骨钙蛋白(osteocalcin,OCN)的表达变化。结果表明:较高浓度的硼替佐米对成骨细胞MC-3T3E1的增殖抑制呈剂量依赖性,48小时IC50为38.1 nmol/L。低浓度(5 nmol/L)硼替佐米对单独培养的MC-3T3E1增殖无明显影响(p>0.1),但可使与骨髓瘤细胞RPMI8226共培养及上清干预培养的MC-3T3E1凋亡比例分别降低24.6%和32.5%,并且促使成骨细胞相关基因osx、ocn的mRNA及蛋白表达增加(p<0.05),而Runx2/cbfa1无明显变化(p>0.05)。结论:低剂量硼替佐米可通过活化成骨细胞内部机制减弱骨髓瘤细胞引起的成骨细胞凋亡,增强Runx2/cbfa1通路中成骨细胞相关基因osx、ocn mRNA及蛋白的表达,可能在骨髓瘤骨病中保护成骨细胞,维持成骨细胞生存。
This study was aimed to investigate the effect of bortezomib, a proteasome inhibitor, on osteoblasts in the pathological state of myeloma bone disease (MBD). The myeloma bone disease status was simulated by the co-culture of mouse myeloma cells RPMI8226 and mouse osteoblasts MC-3T3E1 and supernatant. The results of MTT assay The proliferation of MC-3T3E1 cells was inhibited by bortezomib. The apoptosis of MC-3T3E1 cells was detected by Annexin V / PI staining. The osteogenesis of MC-3T3E1 cells was observed by RT-PCR and Western blot Changes of expression of Runx2 / cbfa1, OSX (osterix) and osteocalcin (OCN). The results showed that the higher concentration of bortezomib inhibited the proliferation of osteoblast MC-3T3E1 in a dose-dependent manner, with IC50 of 38.1 nmol / L at 48 hours. Bortezomib at a low concentration (5 nmol / L) had no significant effect on the proliferation of MC-3T3E1 cultured alone (p> 0.1), but the percentage of apoptotic MC-3T3E1 cells co-cultured with myeloma RPMI8226 and the supernatant Decreased by 24.6% and 32.5% respectively, and promoted the mRNA and protein expression of osteoclast related genes osx and ocn (p <0.05), while Runx2 / cbfa1 showed no significant change (p> 0.05). CONCLUSION: Bortezomib can attenuate the apoptosis of osteoblasts induced by myeloma cells and increase the expression of osteoclast-related genes osx, ocn mRNA and protein in Runx2 / cbfa1 pathway by activating the internal mechanism of osteoblasts, Osteoblasts protect osteoblasts and maintain the survival of osteoblasts.