siRNA沉默ERK5基因对MC 3 T3-E1成骨细胞增殖及 BMP2/BMP7的影响

目的观察ERK5信号通路对MC3T3-E1成骨细胞的增殖,及功能分化蛋白BMP2/BMP7的影响。方法应用小RNA分子干扰技术(siRNA)沉默ERK5基因,分别将浓度为20、40、60、80 nmol/L的ERK5 siRNA干预MC3T3-E1成骨细胞,筛选ERK5 siRNA转染的最佳浓度。噻唑蓝实验(MTT)检测转染后12 h、24 h、36 h和48 h细胞的增殖情况,实时荧光定量PCR检测ERK5、BMP2和BMP7 mRNA的表达。Westernblot检测不同干预组BMP2及BMP7蛋白的表达变化。结果当ERK5siRNA浓度为60 nmol/L时,MC3T3-E1成骨细胞的ERK5 mRNA的表达下降最为显著,沉默率为76.8±2.2%(P0.01)。ERK5 siRNA转染后12 h、24 h、36 h,成骨细胞的增殖受到明显抑制(P0.05),但48 h转染组未发现明显变化(P0.05)。ERK5 siRNA转染24 h后,实时荧光定量PCR结果显示:空白组与对照siRNA组成骨细胞的BMP2/BMP7 mRNA变化水平无显著性差异(P0.05),但ERK5 siRNA转染组的BMP2/BMP7 mRNA明显降低(P0.05);Westernblot结果显示,空白组与对照siRNA组成骨细胞的BMP2/BMP7蛋白未发现明显变化(P0.05),但转染组成骨细胞的BMP2/BMP7显著降低(P0.05)。结论 ERK5 siRNA对MC3T3-E1成骨细胞的增殖以及骨形态蛋白BMP2和BMP7的表达有明显抑制作用。     

Mitogenic Action of Adenosine on Osteoblast-Like Cells, MC3T3-E1

The purpose of this study was to investigate the mechanisms by which adenosine stimulates proliferation of osteoblast-like cells, MC3T3-E1. Adenosine by itself induces the stimulation of cell proliferation and accentuates the mitogenecity of PDGFs (AA and BB homodimers) for the cells. 8-Cyclopentyl-1,3-dimethylxanthine (CPX), a nonselective adenosine receptor antagonist, partially inhibited adenosine-induced DNA synthesis in a competitive manner, suggesting that the mitogenic action of adenosine is, at least in part, mediated by xanthine-sensitive receptors. In pertussis-toxin (PTX)-pretreated cells, adenosine- but not PDGF-BB-stimulated DNA synthesis was partially inhibited, and CPX did not exert a further inhibitory effect, suggesting an involvement of PTX-sensitive G-protein downstream of CPX-sensitive receptor. When adenosine uptake was prevented with dipyridamole, the stimulation of proliferation by adenosine was not decreased at all, indicating that the CPX-insensitive part of adenosine action is not associated with the uptake of adenosine and subsequent incorporation into the nucleotide pool. Adenosine did not influence the basal level or the PDGF-BB-induced increase in [Ca²⁺]i. Since it is known that the cAMP pathway acts in inhibiting osteoblast proliferation, the mitogenic action of adenosine would be dependent on neither the cAMP pathway nor the phospholipase C/Ca²⁺ pathway. It has been concluded that adenosine exerts a mitogenic effect via two pathways at least, one mediated by xanthine-sensitive receptor and PTX-sensitive G-protein and the other through an unknown xanthine- and PTX-insensitive process. Received: 21 May 1995 / Accepted: 28 June 1997     

Zinc Enhancement of 17β-Estradiol's Anabolic Effect in Osteoblastic MC3T3-E1 Cells

The anabolic effect of 17β-estradiol in osteoblastic MC3T3-E1 cells was investigated. The cells were cultured for 3 days in the medium containing either vehicle or 17β-estradiol (10⁻¹¹–10⁻⁹ M). 17β-Estradiol significantly increased alkaline phosphatase activity and protein concentration in the cells. The steroid (10⁻⁹ M) also significantly elevated the cell numbers and the cellular DNA content. The anabolic effect by 17β-estradiol was blocked by the presence of dipicolinate (10⁻³ M), a chelator of zinc ion, suggesting a role of cellular zinc in osteoblastic cell function. The presence of zinc sulfate (10⁻⁵ M) or β-alanyl-L-histidinato zinc (AHZ) (10⁻⁵ M) significantly enhanced the 17β-estradiol (10⁻¹⁰ or 10⁻⁹ M)-induced increase of alkaline phosphatase activity and protein concentration in the cells; the effect of AHZ was greater than that of zinc sulfate. The enhancement by zinc compounds was not based on the augmentation of osteoblastic cell numbers. The co-addition of cycloheximide (10⁻⁶ M), an inhibitor of protein synthesis, completely blocked the zinc compound (10⁻⁵ M)-induced enhancement of 17β-estradiol's (10⁻⁹ M) effect to increase alkaline phosphatase activity and protein concentration in the cells. Moreover, the anabolic effect of 17β-estradiol together with or without zinc compounds was abolished by the presence of staurosporine (10⁻⁸ M), an inhibitor of protein kinase C, or of okadaic acid (10⁻⁷ M), an inhibitor of protein phosphatase. The present study demonstrates that the anabolic effect of 17β-estradiol is enhanced by zinc-chelating dipeptide in osteoblastic MC3T3-E1 cells, and that the enhancing effect may involve protein synthesis and protein kinase activity. Received: 25 September 1995 / Accepted: 21 August 1996     

Restoration of Mineral Depositions by Dexamethasone in the Matrix of Nonmineralizing Osteoblastic Cells Subcloned from MC3T3-E1 Cells

The original osteoblastic cell line, MC3T3-E1, was derived from normal mouse bone tissue and mineralized without any specific factors in vitro. This cell line may be slightly unstable because of high differentiation, and some of these cells sometimes lost the ability for mineral deposition. In this study, a new cell line was cloned which lost the ability for mineral deposition from MC3T3-E1 cells. This cell line, termed MC3T3-NM4, was not observed to undergo mineral deposition for up to at least 36 days even in media containing beta-glycerophosphate. The alkaline phosphatase (ALP) activity was also not increased. The lack of calcifying ability was found to be restored by the addition of dexamethasone in the media. This restoration was accompanied by an increase in ALP activity and osteocalcin level. It was suggested that this restoration was not due to artificial mineralization resulting from cell death. Received: 3 March 1999 / Accepted: 25 May 2000 / Online publication: 22 September 2000     

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     

小鼠骨髓间充质干细胞Osterix基因特异siRNA的设计和沉默作用

目的 探讨应用小干扰RNA(small interfering RNA,siRNA)特异性抑制骨髓间充质干细胞内Osterix基因表达,筛选高效特异性siRNA。方法 根据siRNA设计原则,针对Osterix基因序列特征设计Osterix特异siRNA( 1-3 ),转染骨髓间充质干细胞,用QPCR和Western blot方法检测siRNA对Oslerix基因的抑制效果。结果 Osterix siRNA-2可有效抑制骨髓间充质干细胞中Osterix基因的表达。随siRNA-2终浓度由50 nmol/L增加到100 nmol/L及200 nmol/L,抑制效率逐渐增强（P < 0. 05);siHNA-2以终浓度200 rnnol/L转染后48 h抑制效果最强,72 h逐渐减弱,但仍明显抑制（P< 0. 05 )。结论 应用RNA干扰技术可抑制骨髓间充质干细胞中Osterix基因的表达,其抑制作用具有明显的时间、浓度依赖性。     

Fibroblast Growth Factor 23 (FGF23) and Alpha-Klotho Stimulate Osteoblastic MC3T3.E1 Cell Proliferation and Inhibit Mineralization

Elevated serum levels of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23) are found in patients with phosphate wasting diseases and chronic kidney disease-mineral and bone disorder (CKD-MBD). These diseases are associated with rickets and renal osteodystrophy, respectively. FGF23 is secreted from osteoblastic cells and signals through FGFRs, membrane coreceptor alpha-Klotho (Klotho), and, possibly, a circulating form of Klotho. Despite the absence of detectable Klotho on osteoblastic cells, studies have suggested that forced FGF23 expression in osteoblasts inhibited mineralization. Thus, we examined the effects of exogenously applied FGF23 on osteoblastic MC3T3.E1 cell proliferation and differentiation, with and without soluble Klotho. MC3T3.E1 cells were cultured in osteoblast differentiation medium, supplemented with FGF23 (0.1–1,000 ng/mL), Klotho (50 ng/mL), the combination FGF23 + Klotho, and FGF2 (100 ng/mL) as a control. Neither FGF23 nor Klotho exposure affected proliferation of day 4 growth phase cells or mineralization of day 14 cultures. In contrast, FGF23 + Klotho resulted in inhibition of mineralization and osteoblast activity markers at day 14, and a slight, reproducible induction of proliferation. Inhibition of FGFR1, but not FGFR2 or FGFR3, completely restored FGF23 + Klotho-induced inhibition of alkaline phosphatase (ALP) activity at day 7. ALP activity was partially restored by the MAPK inhibitor U0126 but not inhibitors p38 and P13K. Thus, soluble Klotho enables FGF23 signaling in MC3T3.E1 cells, likely through FGFR 1(IIIc). Elevated FGF23 actions, in part, appear to parallel FGF2 with lower potency. In addition to affecting bone via indirect phosphate wasting pathways, supraphysiological FGF23 and soluble Klotho may directly impact bone in diseases with elevated FGF23 levels.     

Prostaglandin E2 (PGE2) Autoamplifies its Production Through EP1 Subtype of PGE Receptor in Mouse Osteoblastic MC3T3-E1 Cells

Prostaglandin E₂ (PGE₂) is known to autoamplify its production in the osteoblasts through the induction of prostaglandin G/H synthase-2 (PGHS-2), which is the inducible form of the rate-limiting enzyme in PG synthesis, PGHS. To elucidate the cellular mechanism mediating this process, we have employed the PGE₂ analogs, which are specific agonists for four subtypes of PGE receptor, and studied the potency of these analogs to induce PGHS-2 mRNA in mouse osteoblastic MC3T3-E1 cells. The induction was mainly observed by 17-phenyl-ω-trinor PGE₂ (EP₁ agonist) and sulprostone (EP₃/EP₁ agonist), but not by butaprost (EP₂ agonist) or 11-deoxy PGE₁ (EP₄/EP₂ agonist). Since EP₃ subtype was undetectable in MC3T3-E1 cells, these data indicate that PGHS-2 mRNA induction is mediated through EP₁ subtype of PGE receptor in MC3T3-E1 cells. PGE₂ production determined by radioimmunoassay was also increased by 17-phenyl-ω-trinor PGE₂ and sulprostone. The autoamplification of PGE₂ production is considered to be important in elongating the otherwise short-lived PGE₂ action in certain physiological conditions such as mechanical stress and fracture healing, as well as the pathological inflammatory bone loss. The observations in the present study provide us with the better understanding of these processes. Received: 29 April 1997 / Accepted: 22 August 1997     

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。     

牵拉应力下成骨细胞(MC3T3-E1)的增殖与matrilin-2 mRNA的表达

目的观察三维培养的克隆鼠颅骨成骨细胞(MC3T3-E1)在特定的周期性牵拉应力刺激下,其增殖能力、碱性磷酸酶(ALP)活性及matrilin-2mRNA表达的变化。方法以明胶海绵(2cm×2cm×0.25cm)作为MC3T3-E1的三维培养支架,每块明胶海绵种植细胞悬液100μl,细胞数目为1.25×105个。明胶海绵拉伸度为5%、频率60r/min、15min/h,牵拉后2、4、6、8、10d分别从牵拉组和对照组中各取3个样本,行细胞计数、细胞及培养液的ALP活性测定、细胞的matrilin-2mRNA测定。结果第2天牵拉组的细胞数目即多于对照组(P<0.05),细胞倍增时间从71h提前至55h。除第2天外,牵拉组细胞的ALP活性低于对照组(P<0.05),两组培养液中ALP活性均处于低水平,无显著差别。第4天牵拉组matrilin-2mRNA的表达水平高于对照组(P<0.01),之后保持高水平表达。结论周期性牵拉应力促进三维培养的MC3T3-E1的增殖,抑制细胞的ALP活性,促进matrilin-2mRNA的表达,细胞的分化能力增强。     

Microcarriers facilitate mineralization in MC3T3-E1 cells

Summary MC3T3-E1 cells showed mineral deposits after about 1 week of culture when incubated in the presence of microcarrier beads. These deposits appeared as white spots on the dish surface, and under light microscopy the cells showed multiple cell layers and mineralization around the microcarriers. The deposits stained positive with calcium-specific Von Kossa's method. Using conventional assay, alkaline phosphatase activity (ALP) and parathyroid hormone-stimulated intracellular cAMP production were lower in the microcarrier cultures than in the control, but using cytochemical methods, high alkaline phosphatase activity was found around the microcarriers. These results indicate that microcarriers facilitated the formation of multiple cell layers and provided a culture environment for mineralization.     

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.     

Lysyl hydroxylase-2b directs collagen cross-linking pathways in MC3T3-E1 cells.

To elucidate the roles of LH2b in collagen cross-linking, MC3T3-E1 cell clones expressing higher (S) or lower (AS) levels of LH2b were established. Compared with controls, the collagen cross-linking pattern was shifted toward hydroxylysine-aldehyde (S clones)- or lysine-aldehyde (AS clones)-derived pathways. The data indicate that LH2b directs collagen cross-linking pathways through its action on telopeptidyl lysine residues. INTRODUCTION: Lysine (Lys) hydroxylation is a post-translational modification of collagen critical for cross-linking and glycosylation. Currently, three isoforms of lysyl hydroxylase (LH) have been identified, but their specific functions are still not well defined. Recently, we proposed that LH2 might modulate collagen cross-linking pattern through its action on Lys residues located in the telopeptide domains of collagen. MATERIALS AND METHODS: To directly test this hypothesis, several MC3T3-E1 cell-derived clones expressing higher (sense [S]) or lower (antisense [AS]) levels of LH2b, the predominant form of LH2 in this cell line, were established and cultured for 2 weeks, and collagen cross-links and precursor aldehydes in the matrices were analyzed. RESULTS: In S clones tested, the ratio of dihydroxylysinonorleucine (DHLNL) to hydroxylysinonorleucine (HLNL) was significantly higher than the average of controls (76% and 140% increase, respectively), and the level of pyridinoline (Pyr) was elevated (100% and 150% increase, respectively). In contrast, when MC3T3-E1 cells were transfected with a LH2b antisense construct (AS clones), the DHLNL/HLNL ratios were significantly lower than that of controls (56% and 73% decrease, respectively), and Pyr was not detected. Furthermore, significant amounts of an aldol-derived cross-link, dehydrohistidinohydroxymerodesmosine, were produced ( approximately 0.3 mol/mol of collagen) in AS clones. CONCLUSIONS: The data clearly show a critical role of LH2b in determining collagen cross-linking pathways, most likely through its action on telopeptidyl Lys residues.     

MC3T3‐E1 Cells Behavior on Surfaces Bombarded by Argon Ions in Planar Cathode Discharge

To evaluate the effect of topography in nanoscale, titanium surfaces were bombarded by argon ions (a chemically inert gas), in an atmosphere of plasma. The effects of surface parameters on morphology, adhesion, proliferation, and MC3T3‐E1 preosteoblasts differentiation were analyzed. Nontreated (smooth) surfaces were used as a control. The levels of average roughness (Ra) observed in bombarded and smooth titanium surfaces were of 95 and 14 nm, respectively. The wettability increased on treated surfaces. The number of attached cells (30 and 60 min) was significantly higher on the bombarded surface. The cell proliferation after 3 and 7 days was also significantly higher on the ion‐bombarded surface. In addition, the ALP activity and expression of osteocalcin were higher in cells grown on the treated surface. The results showed that bombardment with argon ions increased the roughness and the wettability of the Ti surface, promoting a significant increase in the adhesion, proliferation, and differentiation of preosteoblasts.