Stimulation by 1,25-dihydroxyvitamin D3 of in vitro mineralization induced by osteoblast-like MC3T3-E1 cells

Although vitamin D is essential for mineralization of bone, it is as yet unclear whether vitamin D has a direct stimulatory effect on the bone mineralization process. In the present study, the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on in vitro mineralization mediated by osteoblast-like MC3T3-E1 cells was examined. MC3T3-E1 cells continued to grow after they reached confluency, and DNA content and alkaline phosphatase activity increased linearly until about 16 days of culture, whereas 45Ca accumulation into cell and matrix layer remained low. After this period, DNA content plateaued, and 45Ca accumulation increased sharply. Histological examination by von Kossa staining revealed that calcium was accumulated into extracellular matrix. In addition, needle-shaped mineral crystals similar to hydroxyapatite crystals could be demonstrated in between collagen fibrils by electron microscopy. Thus, MC3T3-E1 cells differentiate in vitro into cells with osteoblastic phenotype and exhibit mineralization. When MC3T3-E1 cells were treated with 1,25(OH)2D3 at this stage of culture, there was a dose-dependent stimulation of 45Ca accumulation by 1,25(OH)2D3, and a significant stimulation of 45Ca accumulation was observed with 3 x 10(-10) M 1,25(OH)2D3. Although 1,25(OH)2D3 enhanced alkaline phosphatase activity and collagen synthesis at the early phase of culture, it did not affect any of these parameters at the late phase when 1,25(OH)2D3 stimulated mineralization. Neither 24,25-dihydroxyvitamin D3 nor human PTH(1-34) affected mineralization in the presence or absence of 1,25(OH)2D3. These results demonstrate that 1,25(OH)2D3 stimulates matrix mineralization induced by osteoblastic MC3T3-E1 cells, and are consistent with the possibility that 1,25(OH)2D3 has a direct stimulatory effect on bone mineralization process.(ABSTRACT TRUNCATED AT 250 WORDS)     

黄瓜籽总皂苷对MC3T3-E1细胞分化及SPARC、OPG/RANKL表达的影响

目的观察黄瓜籽总皂苷提取物(cucumber seed saponins,CSS)对小鼠成骨细胞MC3T3-E1增殖、分化和矿化的影响,以及与骨质疏松相关的SPARC、OPG/RANKL/RANK信号通路的作用。方法通过MTT实验、碱性磷酸酶(alkaline phosphatase,ALP)活性检测、茜素红染色,考察不同浓度CSS对MC3T3-E1细胞增殖、分化及矿化的影响;采用RT-PCR方法检测SPARC、OPG/RANKL mRNA表达水平; Western blot检测SPARC、OPG/RANKL的蛋白表达量。结果与阳性对照组相比,CSS能明显促进MC3T3-E1细胞增殖(P0.05),CSS高、中剂量组能明显提高MC3T3-E1细胞ALP活性及钙化结节数量(P0.05);与空白组相比,CSS不同剂量组均明显上调SPARC、OPG/RANKL mRNA及蛋白表达水平(P0.05)。结论黄瓜籽总皂苷能够促进成骨细胞MC3T3-E1的增殖、分化及矿化能力,并通过上调SPARC、OPG/RANKL的表达水平提高MC3T3-E1细胞的成骨能力。     

健骨胶囊对成骨样细胞MC3T3-E1增殖及分化作用机制

目的 探讨国医大师刘柏龄“健骨胶囊”对MC3T3-E1成骨细胞分化及增殖的影响。方法 制备健骨胶囊水提物,采用CCK-8法和细胞迁移实验检测健骨胶囊提取物对MC3T3-E1细胞增殖和细胞迁移的影响;茜素红染色检测MC3T3-E1细胞的矿化能力;实时荧光定量PCR检测成骨分化基因Runx2、OCN、OPN、Col1a1、ALP、Bcl2、RASSF1A等mRNA表达水平;蛋白质印迹法Western blot检测Col1a1、Bcl2的蛋白表达水平。结果 通过实验结果比对得出,健骨胶囊提取物能促进MC3T3-E1细胞增殖、使细胞迁移率提高;同时健骨胶囊提取物组能明显提高MC3T3-E1细胞钙化能力(P＜0.01),促进Runx2、OCN、OPN、Col1a1、ALP、Bcl2的mRNA表达(P＜0.05),上调Col1a1、Bcl2蛋白量的表达。结论 健骨胶囊能促进成骨细胞MC3T3-E1的增殖及细胞迁移能力,并通过上调成骨基因的表达水平如Runx2、OCN、OPN、Col1a1、ALP、Bcl2等,提高MC3T3-E1细胞的成骨能力。     

Modulation of thrombospondin gene expression during osteoblast differentiation in MC3T3-E1 cells.

The levels of expression of two related extracellular matrix protein genes, thrombospondins 1 and 2 (TSP1 and TSP2), were analyzed in the mouse osteogenic cell line, MC3T3-E1. To monitor differentiation, we also measured two potential markers of the osteoblastic phenotype, alkaline phosphatase (ALP) activity, and alpha 1(I) collagen mRNA levels. TSP1 mRNA levels increased 10- to 15-fold during the first nine days of osteoblastic conversion, and then dropped to a level still significantly above baseline values. This increase in TSP1 mRNA closely paralleled that observed in ALP activity. In contrast, TSP2 mRNA levels were unchanged throughout the 21-day time course. These findings suggest that TSP1 is a marker for osteoblast differentiation and could play a role in the cellular changes that accompany acquisition of the osteoblastic phenotype in MC3T3-E1 cells.     

Osteopontin is a negative regulator of proliferation and differentiation in MC3T3-E1 pre-osteoblastic cells

Osteopontin (OPN) is an important mediator of bone remodeling. However, the role of OPN in the process of bone formation is not fully understood. In previous studies, we have shown that MC3T3-E1 pre-osteoblastic cells at higher passage number exhibited weakened osteogenic capacity and elevated OPN mRNA expression. In this work, we investigated the role of OPN on proliferation and differentiation of low-passage MC3T3-E1 cells by studying stable cell lines overexpressing either OPN mRNA or its antisense RNA. Overexpression was verified by both Northern and Western blot analyses. Overexpression of OPN markedly inhibited proliferation as determined by daily cell counts, while overexpression of antisense RNA stimulated cellular proliferation. We also examined the effect of OPN level on BMP-2-induced alkaline phosphatase activity. Overexpression of OPN inhibited BMP-2 responsiveness while overexpression of antisense RNA enhanced the effect of BMP-2 on alkaline phosphatase activity. Increased OPN expression also caused decreases in expression of osteocalcin and bone sialoproteins while a reduction of OPN level caused the opposite. Furthermore, endogenous OPN expression in response to BMP-2 exhibited a biphasic pattern, that is, it was initially inhibited and then enhanced by the treatment of BMP-2, indicating that OPN might function as a negative feedback regulator for osteoblastic differentiation. Finally, overexpression of OPN inhibited mineral deposition. In contrast, overexpression of antisense RNA enhanced mineral deposition. These results indicate that OPN is a negative regulator of proliferation and differentiation in MC3T3-E1 cells.     

铁调素对MC3T3-E1小鼠成骨细胞骨保护素和骨钙素基因表达的影响

目的研究铁调素对小鼠成骨细胞MC3T3-E1细胞骨保护素(OPG)和骨钙素(BGP)基因表达的影响。方法小鼠MC3T3-E1细胞体外培养后,以不同浓度(100 nmol/ml,200 nmol/ml,300 nmol/ml)的铁调素作用72 h,用RT-PCR方法检测OPG、BGP mRNA的表达水平。结果RT-PCR检测显示在不同浓度铁调素干预后,各组均有OPG mRNA和BGP mRNA表达;不同浓度组的OPG mRNA和BGP mRNA表达光密度比值不同,组间密度比值比较存在显著性差异(P0.05)。结论铁调素可上调MC3T3-E1细胞OPG及BGP mRNA表达,铁调素浓度增加转录水平逐渐增加,结果显示有浓度依赖性。     

Osteotropic agents induce the differential secretion of granulocyte-macrophage colony-stimulating factor by the osteoblast cell line MC3T3-E1

Osteoblasts play a central role in the regulation of bone remodeling. Not only are they responsible for the formation of new bone, but they also regulate bone resorption. These cells also exert regulatory influences outside the bone in that they are able to regulate hematopoiesis. However, obtaining pure populations of osteoblasts devoid of contaminating cell types remains problematic. One approach to this problem is the use of cloned osteoblastic cell lines. To this end we have used MC3T3-E1, a cloned murine osteoblast cell line of C57BL/6 origin. We report that MC3T3-E1 cells respond to lipopolysaccharide (LPS) and, to a lesser extent, parathyroid hormone (PTH) by the secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF). However, 1,25-(OH)2D3, a potent activator of osteoblasts, fails to induce these cells to secrete GM-CSF. These results suggest that MC3T3-E1 cells respond to osteotropic agents in a hierarchical fashion. Secretion of GM-CSF is not constitutive but rather requires active induction of the cells. MC3T3 cells fail to secrete detectable levels of interleukin-2 (IL-2), IL-3, or IL-4, regardless of whether or not the cells are activated. The data indicate that MC3T3-E1 cells secrete cytokines in response to osteotropic agents in a way similar to that of normal primary osteoblasts. Therefore, MC3T3-E1 cells may serve as a good in vitro model for primary osteoblasts.     

流体剪切力对MC3T3-E1细胞OPG、RANKL蛋白表达的实验研究

目的探讨流体剪切力(fluid shear stress,FSS)作用下,两种调节骨骼重建的重要分子骨保护素(osteoprotegerin,OPG)和细胞核因子κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)的蛋白表达情况。方法采用体外模型对MC3T3-E1细胞加载流体剪切力,细胞经不同时间加力后(0,30,60,90,120min),对细胞分别进行染色和裂解,运用免疫荧光和蛋白印迹法对OPG和RANKL的蛋白表达水平进行定量分析。结果 FSS作用30,60,90,120min后能够显著增加OPG的蛋白表达(P0.05),减少RANKL的蛋白表达(P0.05)。两者共同作用使得OPG/RANKL值显著增高(P0.05)。结论流体剪切力刺激提示OPG/RANKL的比值可能在成骨细胞和破骨细胞联合调节骨骼形成和吸收的过程中起着重要的调节作用。     

Bone Morphogenetic Protein-6-loaded Chitosan Scaffolds Enhance the Osteoblastic Characteristics of MC3T3-E1 Cells

The purpose of this study is to investigate the convenience of bone morphogenetic protein-6 (BMP-6)-loaded chitosan scaffolds with preosteoblastic cells for bone tissue engineering. MC3T3-E1 cells were seeded into three different groups: chitosan scaffolds, BMP-6-loaded chitosan scaffolds, and chitosan scaffolds with free BMP-6 in culture medium. Tissue-engineered constructs were characterized by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay, scanning electron microscopy (SEM), mineralization assay (von Kossa), alkaline phosphatase (ALP) activity, and osteocalcin (OCN) assays. BMP-6-loaded chitosan scaffolds supported proliferation of the MC3T3-E1 mouse osteogenic cells in a similar pattern as the unloaded chitosan scaffolds group and as the chitosan scaffolds with free BMP-6 group. SEM images of the cell-seeded scaffolds revealed significant acceleration of extracellular matrix synthesis in BMP-6-loaded chitosan scaffolds. Both levels of ALP and OCN were higher in BMP-6-loaded chitosan scaffold group compared with the other two groups. In addition, BMP-6-loaded scaffolds showed strong staining in mineralization assays. These findings suggest that BMP-6-loaded chitosan scaffold supports cellular functions of the osteoblastic cells; therefore, this scaffold is considered as a new promising vehicle for bone tissue engineering applications.     

THSD4基因在小鼠间充质干细胞及MC3T3-E1细胞成骨分化中的作用

目的 探讨THSD4基因对小鼠间充质干细胞和MC3T3-E1细胞成骨分化的影响。方法 提取绝经后骨质疏松症患者的骨髓间充质干细胞进行基因测序分析,与骨关节炎患者的骨髓间充质干细胞进行比较,分析基因表达差异。通过提取不同分化阶段的小鼠骨髓间充质干细胞（M-BMSC）及MC3T3-E1细胞的mRNA来检测THSD4 基因以及成骨分化的标志性基因（ALP、Runx2、Osx）的表达水平。通过构建慢病毒表达载体来实现对M-BMSC及MC3T3-E1细胞中THSD4的敲减及过表达,并观察其对M-BMSC及MC3T3-E1细胞成骨分化能力的影响。结果 THSD4基因在绝经后骨质疏松症患者骨髓间充质干细胞中明显下调,且通过KEGG以及GO富集分析发现THSD4基因可能与PI3K-AKT信号通路及Wnt信号通路相关。随着成骨诱导分化时间的延长,THSD4 mRNA和成骨分化标志性基因（ALP、Runx2、Osx）mRNA在MC3T3-E1以及M-BMSC中表达量均逐渐增加。过表达THSD4可以增强MC3T3-E1细胞和M-BMSC的成骨分化能力,而敲减THSD4则减弱了MC3T3-E1细胞和M-BMSC的成骨分化能力。结论 THSD4基因在绝经后骨质疏松症患者骨髓间充质干细胞中明显下调,且THSD4基因可以增强MC3T3-E1细胞以及M-BMSC的成骨分化能力。     

Expression profiles of phosphodiesterase 4D splicing variants in osteoblastic cells

The promotion of osteoblastic differentiation by bone morphogenetic proteins (BMPs) is accelerated by chemical compounds that increase the intracellular concentration of cyclic 3′,5′-adenosine monophosphate (cAMP). cAMP is synthesized from adenosine triphosphate (ATP) by adenyl cyclase and degraded by phosphodiesterase (PDE) family enzymes. Inhibition of PDEs leads to prolonged accumulation of cAMP within cells and Camp-mediated reactions. Rolipram, a specific inhibitor of PDE4, is a compound effective in inducing osteoblastic differentiation. Four PDE4 family members are transcribed from four distinct genes (4A, 4B, 4C, and 4D). Expression of PDE4A and PDE4D has been observed in osteoblastic cells. We identified PDE4D splicing variants that expressed in ST2 or primary calvarial osteoblasts by rapid amplification of the 5′-ends of cDNA when they were cultured with BMP. PDE4D9 mRNA was identified from ST2, and PDE4D1 and −4D2 mRNAs were identified from primary calvarial osteoblasts. Expression of these three variants of PDE4D mRNA was found in ST2, MC3T3-E1, C3H10T1/2, C2C12, and primary calvarial osteoblasts by RT-PCR, but not PDE4D1 or −4D2 in ST2 or PDE4D2 in MC3T3-E1. Expression of these three variants was detectable in brain, heart, lung, liver, kidney, placenta, and femur, and was thus ubiquitous. Purified recombinant PDE4D9 protein exhibited phosphodiesterase activity, which degraded cAMP to AMP, and this activity was inhibited by rolipram. These findings suggest that PDE4D1, −2, and −9 play some roles in bone formation.     

Inhibitory Effects of 1,25(OH)2D3 on Membrane-Associated and Cytosolic Casein Kinase Activity in MC3T3-E1 Osteoblast-like Cells

The secretion of phosphorylated matrix proteins is high in osteoblasts. Phosphorylation of these proteins may be catalyzed by casein kinases (CK), and CK may play an important role in the site of bone mineralization. In this study, we examined the effects of 1,25(OH)₂D₃ on CK activities in MC3T3-E1 osteoblast-like cells. Different concentrations (ranging from 10⁻⁷ to 10⁻¹¹M) of 1,25(OH)₂D₃ were included in a culture medium. After incubation for various lengths of time, MC3T3-E1 cells were homogenized and segregated into cytosolic (c) and microsomal (m) fractions. To measure CK activity, each fraction was used as an enzyme source to phosphorylate casein. MC3T3-E1 cells showed the highest cCK activity after incubation for 21 days, and showed the highest mCK activity after incubation for 14 days. 1,25(OH)₂D₃ inhibited mCK activity at the early stage of culture, but inhibited cCK activity at the late stage of culture. In contrast, 1,25(OH)₂D₃ had a slight stimulatory effect on CK activity in the culture medium of MC3T3-E1 cells. Our data suggest that cCK and mCK may play different roles in the function of osteoblasts, and 1,25(OH)₂D₃ regulates intracellular and extracellular casein kinase activities related to the function of osteoblasts. Received: 26 June 1997 / Accepted: 23 March 1998     

不同大小机械牵张力对MC3T3-E1成骨样细胞MMP-13/TIMP-1 mRNA表达的影响

目的:研究不同大小的机械牵张力对成骨细胞MMP-13/TIMP-1mRNA表达的影响。方法:通过自制的多通道细胞牵张应力加载系统对小鼠成骨样细胞MC3T3-E1同时施加6%、12%和18%的机械牵张力,作用24h后,用RT-PCR方法检测细胞受力后MMP-13/TIMP-1mRNA表达的变化。结果:细胞受力后,其MMP-13/TIMP-1mRNA表达随牵张力值的增大明显增加。结论:不同大小的机械牵张力可以影响成骨细胞的MMP-13/TIMP-1mRNA表达,进而影响骨改建。