Zhao Hui, Bai Qing-Xian, Huang Gao-Sheng, Yang Li-Jie, Yue Peng, Zhang Xiao-Yan, Wang Lu, Fan Lin-Ni, Zhu Jin
Department of Hematology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2010 Oct;18(5):1186-91.
The purpose of this study was to explore the effect of proteasome inhibitor, bortezomib (Bzb), on osteoblast in pathologic status of myeloma bone disease. The myeloma bone disease was modeled by co-culture of mouse myeloma cell RPMI8226 with osteoblast line MC-3T3E1 from mouse calvaria, and intervenient culture of supernatant. The inhibitory effect of Bzb on proliferation of MC-3T3E1 assayed by modified MTT method, the apoptosis of MC-3T3E1 cells was determined by flow cytometry with Annexin V/PI staining, the expressions of osteoblast markers, Runx2/cbfa1, osteocalcin (OCN) and osterix (OSX) in MC-3T3E1 treated with Bzb were detected by RT-PCR and Western blot respectively. Experiments were divided into 3 group: single cultured, co-cultured and supernatant-interveniently cultured groups. The results showed the Bzb in higher concentration inhibited proliferation of MC-3T3E1 cells in a dose-dependent manner, with the IC(50) of 38.1 nmol/L for 48 hours, the Bzb in low concentration (5 nmol/L) did not show the inhibitory effect on proliferation of MC-3T3E1 in single cultured group (p>0.10), but could decrease apoptotic rate of MC-3T3E1 by 32.5% and 24.6% respectively in cocultured and supernatant-interveniently cultured groups, moreover increased the expression of osteoblast-related gene OSX, OCN mRNA and protein (p<0.05), while no obvious change of Runx2/cbfa1 expression was observed (p>0.05). It is concluded that the proteasome inhibitor, Bzb, in low concentration promotes the activity of osteoblast internal mechanisms, and prevents the apoptosis of osteoblasts induced by myeloma cells. In addition, it can up-regulate transcription and expression of osteoblast markers related to Runx2/cbfa1 path way, thus may protect osteoblasts in myeloma bone disease.
本研究旨在探讨蛋白酶体抑制剂硼替佐米（Bzb）对骨髓瘤骨病病理状态下成骨细胞的影响。通过将小鼠骨髓瘤细胞RPMI8226与来自小鼠颅骨的成骨细胞系MC - 3T3E1共培养，并对培养上清液进行干预培养来建立骨髓瘤骨病模型。采用改良MTT法检测Bzb对MC - 3T3E1增殖的抑制作用，通过Annexin V/PI染色利用流式细胞术测定MC - 3T3E1细胞的凋亡情况，分别采用RT - PCR和Western blot检测经Bzb处理的MC - 3T3E1中成骨细胞标志物Runx2/cbfa1、骨钙素（OCN）和osterix（OSX）的表达。实验分为3组：单培养组、共培养组和上清液干预培养组。结果显示，高浓度的Bzb以剂量依赖方式抑制MC - 3T 3E1细胞的增殖，48小时的IC(50)为38.1 nmol/L，低浓度（5 nmol/L）的Bzb在单培养组中对MC - 3T3E1的增殖未显示抑制作用（p>0.10），但在共培养组和上清液干预培养组中可分别使MC - 3T3E1的凋亡率降低32.5%和24.6%，此外还增加了成骨细胞相关基因OSX、OCN的mRNA和蛋白表达（p<0.05），而Runx2/cbfa1的表达未观察到明显变化（p>0.05）。结论是，低浓度的蛋白酶体抑制剂Bzb可促进成骨细胞内在机制的活性，防止骨髓瘤细胞诱导的成骨细胞凋亡。此外，它可上调与Runx2/cbfa1途径相关的成骨细胞标志物的转录和表达，从而可能对骨髓瘤骨病中的成骨细胞起到保护作用。
