蛋白酶体抑制剂硼替佐米对MC3T3-E1细胞成骨分化和增殖的影响

Effects of proteasome inhibitor bortezomib on the osteogenic differentiation and proliferation of MC3T3-E1 cells

目的观察蛋白酶体抑制剂硼替佐米(bortezomib)对于小鼠MC3T3-E1细胞系成骨分化、增殖和凋亡的影响。方法用不同浓度梯度的硼替佐米作用于培养的MC3T3-E1细胞,利用茜素红染色检测成骨分化,CCK-8法检测细胞增殖,流式细胞术分析细胞周期和凋亡,Westernblot分析细胞周期相关蛋白变化。结果①硼替佐米剂量依赖性地抑制MC3T3-E1细胞的增殖活力[IC50=(7.37±0.34)nmol/L];②低浓度硼替佐米能够诱导MC3T3-E1细胞发生成骨分化;③高浓度硼替佐米对于MC3T3-E1细胞表现出明显的毒性,诱导细胞凋亡发生;④低浓度硼替佐米所诱导的MC3T3-E1成骨分化进程中,伴随有明显的G0/G1期细胞周期阻滞。Westernblot检测发现,G0/G1期细胞周期阻滞与细胞周期素依赖性激酶CDK2和CDK4表达水平降低,以及细胞周期蛋白内质网应激活化引起的细胞周期抑制蛋白p21Cip1和p27Kip1的表达上调有关。结论低剂量蛋白酶体抑制剂硼替佐米能够诱导成骨前体细胞MC3T3-E1发生成骨分化,并引起G0/G1期细胞周期阻滞介导的增殖抑制。

Objective To investigate the effects of bortezomib on the osteogenic differentiation,proliferation,and apoptosis of MC3T3-E1 cells so as to explore the possible mechanisms involved. Methods Mouse MC3T3-E1 cells were cultured in the presence of different doses of bortezomib at different time points. Then the effects on osteogenic differentiation were determined by alizarin red staining,cell viability was measured by cell counting Kit-8 assay,cell cycle and apoptosis were examined by flow cytometry,and the protein level changes of cell cycle-associated proteins were analyzed by Western blot. Results ① Bortezomib inhibited MC3T3-E1 cell viability in a dose-dependent manner [IC 50 =(7.37±0.34)nmol/L].② Low-dose bortezomib induced osteogenic differentiation of MC3T3-E1 cells.③ However,high-dose bortezomib showed significant toxicity to MC3T3-E1 cells and induced high level of apoptosis.④ Cell cycle analysis showed that the number of cells at G 0/G 1 phase was significantly increased by low-dose bortezomib. Western blotting analysis further demonstrated that the decreased CDK2 and CDK4,and the increased p21 Cip1 and p27 Kip1 expressions were tightly related to the bortezomib-induced G 0/G 1 phase cell cycle arrest. Conclusion Low-dose bortezomib can induce the osteogenic differentiation of MC3T3-E1 cells and lead to G 0/G 1 phase cell cycle arrest. Our findings may improve the understanding of the mechanism of bortezomib in the treatment of myeloma bone disease.