硼替佐米减弱小鼠骨髓瘤细胞所致成骨细胞凋亡的作用

Bortezomib Depresses Osteoblast Apoptosis Induced by Mouse Myeloma Cells

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。

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 superantant.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 cocentration inhibited proliferation of MC-3T3E1 cells in a dose-dependent manner,with the IC50 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 （p0.10）,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 （p0.05）,while no obvious change of Runx2/cbfa1 expression was observed（p0.05）.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.