骨髓间充质干细胞在去卵巢大鼠骨质疏松发病机理中潜在的作用

为探讨去卵巢骨质疏松大鼠骨髓间充质干细胞（Bone marrow mesenchymal stem cells，MSCs）成纤维细胞集落生成单位（CFU—Fs）、细胞周期、成骨分化能力和成脂分化能力的变化，选用3月龄和6月龄健康雌性SD大鼠行双侧卵巢切除术，建立骨质疏松动物模型，动物实验分为4组：（1）3月龄正常组（control-3）；（2）3月龄卵巢切除组（OVX-3）；（3）6月龄正常组（control-6）；（4）6月龄卵巢切除组（0VX-6）。采用密度梯度离心方法分别获取各组MSCs，体外培养，选用第3～4代MSCs用于实验。倒置相差显微镜观察CFU—Fs；流式细胞技术检测细胞周期、细胞增殖指数（proliferation index，PI）和细胞凋亡指数（apoptotic index，AD）。加入成骨诱导剂，采用碱性磷酸酶活性蛋白检测法（IFCC推荐法）检测MSCs碱性磷酸酶（alkalinephos phatase，ALP）分泌量；采用同位素标记方法检测骨钙索（Osteocalcin，OCN）分泌量；茜素红染色观察钙结节形成。加入成脂诱导剂，油红O染色观察MSCs内脂滴形成；RT-PCR方法检测MSCs脂蛋白脂酶（1ipoprotein lipase，LPL）mRNA的表达。结果表明：与相应对照组比较，OVX-3组和OVX-6组CFU—Fs、PI值明显降低（P〈0．05），AI值明显增加（P〈0．05）；OVX-6组CFU，Fs、PI值的降低和AI值的增加都明显大于OVX-3组（P〈0．05）。成骨诱导表明：与相应对照组比较，OVX-3组和OVX-6组ALP、BGP分泌量以及钙结节形成的数量均明显降低（P〈0．05），OVX-6组的降低最为明显。成脂诱导表明：与相应对照组比较，OVX-3组和OVX-6组，LPLmRNA的表达量明显增加（P〈0．05），OVX-6组LPL mRNA的表达量的增加明显高于其他各组（P〈0．05）。结论：去卵巢骨质疏松大鼠MSCs增殖能力明显下降，向成骨细胞分化能力下降，向脂肪细胞分化能力增强，其中60VX组MSCs的变化最明显。提示：去卵巢骨质疏松大鼠MSCs向成骨细胞分化能力的降低和向脂肪细胞分化能力的增强，可能与绝经后骨质疏松症的发生相关。

A Potential Role for the Bone Marrow Mesenchymal Stem Cell in the Pathogenesis of Osteoporosis by Ovariectomy in Rat

The purpose of this paper is to investigate the osteogenesis and adipogenesis in bone marrow mesenchymal stem cells(MSCs) isolated from normal rats and osteoporotic rats by ovariectomy.Osteoporotic animal model was established in 3 month-old and 6 month-old female Sprague-Dawley(SD) rats by ovariectomy. Animal experiments were divided into 4 groups: 1)control-3 group;2)ovx-3 group; 3)control-6 group and 4)ovx-6 group.MSCs were isolated by means of the density-gradient centrifugation method from each group,respectively.Colony-forming unit-fibroblast(CFU-Fs)number,CFU-Fs size distribution and cell density in CFU-Fs of primary passage MSCs were measured at the inverted phase contrast microscope.The cell cycle and proliferation index(PI) as well as apoptosis idex(AI)of MSCs were studied by(FCM).After osteogenic induction(OSI),calcium nodes of MSCs were marked by alizarin red staining(ARS);The expression level of alkaline phosphatase(ALP) was detected by dynamics method with substrate of phosphoric acid para-Nitro benzene and the content of osteocalcin(OCN) was detected with the isotope labelling method.After adipogenic induction(ADI),lipid droplet in MSCs were detected by oil red O staining and the mRNA level of lipoprotein lipase(LPL) was measured by RT-PCR.The results showed that CFU-Fs and PI are obviously decresed and AI are increased of MSCs in OVX-3 and OVX-6 groups(P<0.05).The secretory volume of ALP and BGP of MSCs and the content of calcium nods of MSCs are lower in OVX-3 and OVX-6 groups than that in control-3 and control-6 groups after osteogenic induction(P<0.05).The number of lipid droplet and the expression level of LPL mRNA are higher in OVX-3 and OVX-6 groups than that in control-3 and control-6(P<0.05).The result in our study suggested that depress of osteogenesis and the up-regulation of adipogenesis of MSCs in osteoporotic rats by ovariectomy may be relate close to the pathogenesis of osteoporosis.