外力诱导培养兔成骨细胞表达c—fos的实验研究

目的：探讨兔成骨细胞在机械力作用下将机械力信号转变为生物学效应的作用机理;方法：利用ＦＯＳ免疫组化技术观察在受到外力后培养的兔成骨细胞对立即早期基因ｃ－ｆｏｓ的蛋白产物（ＦＯＳ）的表达情况;结果：几乎所有的成骨细胞核均染成黄褐色,为ＦＯＳ免疫组化反应阳性;结论：ｃ－ｆｏｓ在兔成骨细胞的机械力信号向生物学效应转换的过程中可能发挥重要作用。     

Matricellular proteins: Extracellular modulators of bone development, remodeling, and regeneration

Matricellular proteins are components of the extracellular matrix which are highly expressed in the developing and mature skeleton. Members of this protein class serve as biological mediators of cell function by interacting directly with cells or by modulating the activity of growth factors, proteases, and other extracellular matrix proteins. Although skeletons of matricellular protein-null mice are grossly normal, they each display unique deficiencies that are often magnified under pathological conditions. In addition, bone cells from wild-type and matricellular protein-null mice behave differently in various in vitro models of bone matrix synthesis and turnover. In this review, osteopontin, bone sialoprotein, tenascin C, SPARC, and thrombospondins 1 and 2 will each be discussed in the context of bone cell biology. Because the biological effects of matricellular proteins are largely context dependent, in vivo and in vitro results must be considered together in order to fully appreciate the specific contributions that matricellular proteins make to bone physiology and pathophysiology. In particular, it is clear that although matricellular proteins are not required for bone development and function, the proteins act to modulate post-natal bone structure in response to aging, ovariectomy, mechanical loading, and bone regeneration.     

人成骨细胞与外周血单核细胞共同培养诱导破骨细胞样细胞

目的 探讨人外周血单核细胞与原代人成骨细胞共同培养体外转化为破骨细胞样细胞的条件,并观察其体外生长,功能情况。方法 分别分离培养人成骨细胞,外周血单核细胞,并在含1,25（OH）2D3（10^-7mol/L）,地塞米松（10^-8mol/L)及巨噬细胞集落刺激因子（M－CSF）（25ng/ml)的条件液中共同培养,用相差显微,抗酒石酸酸性磷酸酶染色（TRAP）观察破骨细胞样细胞生长情况,应用扫描电镜（SEM）观察骨片隐窝以反映其功能。结果 共同培养中的单核细胞逐渐融合;TRAP染色阳性多核细胞在第7天明显增多;骨片隐窝呈现多种形态。结论 人原代成骨细胞与人外周血单核细胞共同培养可以诱导出破骨细胞样细胞,但应该选择分化程度较低的细胞以及控制接种密度。     

脐血来源的间充质干细胞生物学特性与向成骨细胞分化能力的研究

目的研究人脐血来源的间充质干细胞（UCBMSC）的免疫表型、生物学特性及其向成骨细胞分化的能力。方法分离脐血单个核细胞，以干细胞培养液培养间充质干细胞（MSC），观察其体外生长特性；用流式细胞术进行免疫表型测定和细胞周期分析；以含地塞米松、β-磷酸甘油和维生素C的诱导液定向诱导向成骨细胞分化；以碱性磷酸酶染色、矿化结节染色、骨桥蛋白mRNA的表达以及I型胶原表达鉴定MSC向成骨细胞分化的潜能。结果从脐血中可以培养出MSC，为成纤维细胞样的贴壁细胞。免疫表型分析显示CD45、CD34、CD11a、CD14、HLA．DR阳性细胞占1％～3％；CD166、CD44、CD106、CD29、CD105、CD49e、CD90、CD73表达率达91％～98％。细胞周期分析显示95％以上的细胞处于G0／G1期。定向成骨细胞诱导后，可以检测到碱性磷酸酶、I型胶原的表达，矿化结节的形成和骨桥蛋白mRNA的表达。结论UCBMSC是基质细胞的祖细胞，在合适的条件下可被诱导向成骨细胞分化，因此有可能作为有潜力的骨组织工程的种子细胞来源。     

成人骨髓间充质干细胞分化为成骨细胞的研究

目的：通过成人骨髓间充质干细胞(mesenchymal stem cells，MSC)体外定向诱导为成骨细胞，探讨理想而有临床实用价值的成人骨髓MSC体外诱导培养体系。方法：体外分离、扩增成人骨髓MSC，流式细胞仪检测细胞表面抗原的表达。采用基础诱导培养液[地塞米松、β-甘油磷酸钠、L-抗坏血酸加不同浓度的重组人转化生长因子-β1(recombinant human transforming growth factor—betal，rhTGF-β1)]诱导成人骨髓MSC体外分化为成骨细胞。利用倒置光学显微镜、透射电镜、四甲基偶氮唑盐(MTT)比色、碱性磷酸酶(ALP)染色、ALP活性测定等方法研究成人骨髓MSC增殖和分化情况。结果：成人骨髓MSC的增殖和分化作用和rhTGF-β1有剂量依赖关系，低浓度时促进增殖，高浓度抑制增殖，5μg／L浓度达到高峰，其浓度升高促进MSC分化。结论：含有rhTGF-β1 5μg／L的基础诱导培养液是理想且有临床实用价值的成人骨髓间充质干细胞体外诱导为成骨细胞的培养体系。     

重组人成骨生长肽治疗去卵巢大鼠骨质疏松的实验观察

目的探讨重组人成骨生长肽(recombinanthumanosteogenicgrowthpeptide,rhOGP)对去卵巢大鼠骨质疏松的治疗作用。方法通过切除大鼠卵巢建立骨质疏松模型,经rhOGP治疗12周后,测定骨密度(BMD)、血清中的酸性磷酸酶(TRAP)、碱性磷酸酶(ALP)和骨钙素(BGP)。结果rhOGP治疗组碱性磷酸酶(233.77±42.07)U/L、骨钙素(1.95±0.15)μg/L和骨密度明显高于骨质疏松模型组(156.23±23.38)U/L、(1.33±0.24)μg/L(P<0.01),而酸性磷酸酶(19.27±4.24)U/L,明显低于骨质疏松模型组(29.98±3.08)U/L(P<0.05)。结论rhOGP能刺激成骨细胞的活性,增加骨密度,提高骨量,对去卵巢大鼠所引起的骨质疏松有效,有可能成为治疗骨质疏松症的有效药物。     

Wnt signaling and osteoblastogenesis

Wnts are a large family of growth factors that mediate fundamental biological processes like embryogenesis, organogenesis and tumorigenesis. These proteins bind to a membrane receptor complex comprised of a frizzled (FZD) G-protein-coupled receptor (GPCRs) and a low-density lipoprotein (LDL) receptor-related protein (LRP). The formation of this ligand-receptor complex initiates a number of intracellular signaling cascades that includes the canonical/β-catenin pathway, as well as several GPCR-mediated noncanonical pathways. In recent years, canonical Wnt signaling has been shown to play a substantial role in the control of bone formation. Clinical investigations have found that mutations in LRP-5 are associated with bone mineral density and fractures. For example, loss-of-function mutations in LRP-5 cause osteoporosis pseudoglioma syndrome, while gain-of-function mutations lead to high bone mass phenotypes. Studies of knockout and transgenic mouse models for Wnt pathway components like Wnt-10b, LRP-5/6, secreted frizzled-related protein-1, dickkopf-2, Axin-2 and β-catenin have demonstrated that canonical signaling modulates most aspects of osteoblast physiology including proliferation, differentiation, bone matrix formation/mineralization and apoptosis as well as coupling to osteoclastogenesis and bone resorption. Future studies in this rapidly growing area of research should focus on elucidating Wnt/FZD specificity in the control of bone cell function, the role of noncanonical pathways in skeletal remodeling, and direct effects of Wnts on cells of the osteoclast lineage.     

组织金属蛋白酶抑制因子3重组蛋白诱导小鼠成骨细胞MC3T3-E1凋亡

目的 研究组织金属蛋白酶抑制因子3（TIMP-3）重组蛋白对MC3T3-E1成骨细胞凋亡的影响。方法 细胞存活率和凋亡分别用MTT及ELISA检测。Fas，FasL，Bcl-2，Bax，caspase-3，caspase-8，细胞色素c的表达以及JNK，p38，细胞外信号调节激酶（ERK）1／2的磷酸化水平用Western印迹检测。结果 MTT及ELISA检测提示TIMP-3降低MC3T3-E1细胞存活率，诱导MC3T3-E1细胞凋亡。TIMP-1增加Fas、FasL蛋白表达，对Bax、Bcl-2蛋白表达无影响，但可诱导细胞色素c的释放及caspase-8、caspase-3的活化。TIMP-3促进p38和ERK磷酸化，而PD098059（ERK阻断剂）和SB203580（p38阻断剂）消除p38和ERK的促凋亡作用。结论 TIMP-3诱导MC3T3-E1细胞凋亡的信号途径为凋亡受体Fas介导，并有p38、ERK信号转导途径参与。     

不同龄小鼠成骨细胞OPG和RANKL表达的变化

目的观察来源于不同龄小鼠成骨细胞经雌激素作用后,分泌骨保护素(osteoprotegerin,OPG)、核因子κB受体(receptor activator of nuclear factor-κB ligand,RANKL)的变化.方法经雌激素作用前后,反转录-聚合酶链反应(RT-PCR)以及酶联免疫吸附试验(ELISA)测定OPG、RANKL表达的变化.结果经雌激素作用后,幼年组和成年组OPG分泌显著增多,而老年组RANKL表达明显下调.结论雌激素可以刺激成骨细胞分泌OPG,减少RANKL表达,很可能是骨质疏松的发生机制之一,是绝经后骨质疏松发生的重要原因.     

Epidermal growth factor and calcitriol synergistically induce osteoblast maturation

Calcitriol (1,25(OH)₂D₃) plays a key role in the differentiation of osteoblasts, the cells responsible for the formation and maintenance of healthy bone matrix. Recently it has emerged that calcitriol influences the trafficking or stability of epidermal growth factor (EGF) receptors. However, how these agents might work together in regulating growth and differentiation has not been examined. Using the human osteoblast cell line, MG63, we were able to induce a profound differentiation response by treating these cells with a combination of calcitriol (100 nM) and EGF (10 ng/ml). Co-stimulation of MG63 osteoblasts with calcitriol and EGF led to synergistic increases in osteocalcin and alkaline phosphatase (ALP), proteins expressed by differentiating cells. Inhibition of differentiation was accomplished by MEK and protein kinase C (PKC) inhibitors. Other ligands known to signal via receptor tyrosine kinases could not substitute for EGF in the maturation response. These novel findings may help identify new processes that drive osteoblast differentiation.