去势大鼠骨量丢失过程中骨髓间充质干细胞的增殖分化功能

目的: 研究骨髓间充质干细胞(BMSCs)增殖分化功能在去势大鼠骨质疏松发病过程中对骨量丢失的作用。方法: 选用10周龄健康雌性SD大鼠行双侧卵巢切除术(OVX),建立骨质疏松症(OP)的动物模型;选用同一批次、周龄相同、体重相近的健康雌性SD大鼠行双侧卵巢附近脂肪组织部分切除术,建立假手术(sham)组,假手术大鼠组(sham group)。采用离心法、贴壁法和有限稀释法分离、培养、纯化大鼠BMSCs,体外培养传至3~4代后用于实验:流式细胞术进行BMSCs表型鉴定;克隆形成实验检测BMSCs增殖状况;MTT法测定BMSCs生长曲线;成脂诱导后脂滴油红O染色法检测比较2组大鼠BMSCs成脂能力;成骨诱导后钙化结节茜素红染色法检测比较2组大鼠BMSCs成骨能力;RT-PCR法检测大鼠BMSCs成骨相关蛋白Runx2、骨钙素(OCN)和骨桥蛋白(OPN)mRNA的表达。结果: 与sham组大鼠BMSCs相比,OVX组大鼠BMSCs克隆形成能力减弱,增殖能力降低,成脂向分化增强,成骨向分化减弱(P<0.05)。结论: 去卵巢骨质疏松大鼠BMSCs增殖及成骨分化减弱,成脂分化增强;这导致去势大鼠快速的骨量丢失,在去势大鼠OP发病过程中发挥着重要作用。

Proliferation and differentiation of bone marrow mesenchymal stem cells in process of bone loss in ovariectomized rats

AIM:To study the function of proliferation and differentiation of bone marrow mesenchymal stem cells(BMSCs) for bone loss in the pathogenesis of osteoporosis(OP) in ovariectomized rats.METHODS:Animal model of OP was established by ovariectomy(OVX,bilateral ovarian resection) in 10-week-old healthy female Sprague-Dawley (SD) rats.BMSCs were isolated,cultured and purified by the combination of density gradient centrifugation,adhesion separation and limited dilution method,and cultured in vitro to the 3rd～4th passage in all experiments.The BMSCs phenotype appraisal was studied by flow cytometry.Colony-forming assay was applied to detect the BMSCs proliferation ability. The MTT method was used to analyze the growth curves of BMSCs.After adipogenic induction(ADI),lipid drops were observed by oil red 0 staining to compare the adipogenic potential between the 2 kinds of BMSCs.After osteogenic induction (OSI),calcium nodules were observed by alizarin red staining(ARS).The mRNA expression levels of BMSCs osteogenesis-related proteins,for instance,Runx2,osteocalcin(OCN) and osteopontin(OPN ) were measured by RT-PCR. RESULTS:Compared with sham group,the colony-forming ability of BMSCs in OVX group became decreased,the proliferation capacity was declined,the osteogenic potential was decreased,and the adipogenic potential was increased (P <0.05).CONCLUSION:In ovariectomized OP rats,the proliferation and osteogenesis of BMSCs decrease,and the adipogenesis of BMSCs increases,which may cause rapid bone loss and play an important role in the pathogenesis of OP.