8-异戊烯基柑橘素促进体外培养成骨细胞成熟矿化的研究

研究8-异戊烯基柑橘素对体外培养大鼠颅骨成骨细胞(rat skull osteoblasts,ROB)的分化成熟及生物矿化的影响.取新生大鼠颅骨多次酶消化法得到成骨细胞,培养于含10%FBS的MEM培养液中,3天后首次换液,待细胞铺满皿底传代培养.以碱性磷酸酶(alkaline phosphatase,ALP)为检测指标,96孔板梯度筛选作用最佳浓度,在最佳浓度作用并成骨性诱导培养的第3、6、9、12天测ALP活性、钙盐沉积量;第12天进行ALP和钙化结节组织化学染色及计数;成骨性诱导后不同时间点提取Total RNA,RT real-time PCR法检测成纤维细胞生长因子(bFGF)、胰岛素样生长因子-1(IGF-1)、成骨相关转录因子Osterix、Runx-2和骨形态发生蛋白-2(BMP-2)的基因表达情况;成骨性诱导的第4、8、12天裂解获得细胞总蛋白,蛋白质印迹法检测人Ⅰ型胶原蛋白(COL-Ⅰ)的蛋白质表达量.研究结果表明:1×10-6 mol/L能显著促进成骨细胞的成熟分化,表现为提高ROB的ALP活性、促进钙盐沉积、增加钙化结节数量;提高bFGF、IGF-1、Osterix、Runx-2和BMP-2 mRNA表达水平;促进COL-Ⅰ的合成.由此可知终浓度为1×10-6 mol/L 8-异戊烯基柑橘素能显著促进ROB的分化成熟及生物矿化,证明8-异戊烯基柑橘素能促进成骨细胞的分化成熟及生物矿化,作为促进骨修复和抗骨质疏松的有效成分具有较大的药用价值.     

外源性β-NGF调节骨改建过程中的可能作用机制

目的探讨外源性的β-NGF在骨缺损愈合过程中对骨改建可能作用机制。方法利用外科手术方法切取SD大鼠颅骨顶部左右两侧的骨质,建立配对大鼠颅骨标准骨缺损局部持续灌注β-NGF给药模型。通过免疫组织化学技术检测骨缺损区组织中BMP-2的表达水平以及特殊染色技术(TRAP染色)检测骨缺损区组织中抗酒石酸碱性磷酸酶的表达水平,通过图像处理软件IPP6.0分别测定两者积分光密度值(IOD),以探讨BMP-2与破骨细胞活性在β-NGF调节新骨形成和改建过程中的可能作用机制。结果实验组BMP-2免疫组化染色阳性表达水平在14 d时IOD值明显高于对照组,且差异有显著性(P0.05);TRAP染色结果显示:实验组骨吸收的活性在第7,21,28天三个时间点上明显低于对照组,且差异有显著性(P0.05)。结论外源性的β-NGF在骨缺损修复过程中具有重要的调节作用,其可能是通过促进BMP-2表达和抑制破骨细胞的活性以抑制骨吸收。     

染料木黄酮对成骨细胞增殖分化的影响

目的：研究染料木黄酮对体外培养乳鼠颅盖骨成骨细胞增殖分化的影响。方法：取乳鼠颅盖骨,采用胶原-胰蛋白酶消化法,进行颅骨成骨细胞培养,取第二代成骨细胞,添加10^-5～10^-7mol／L染料木黄酮,在CO2孵箱中培养48h和72h后MTT比色法测定细胞增殖,培养72h采用^3H-TdR和^H-Pro掺入实验测定DNA和胶原合成。用试剂盒检测细胞裂解液碱性磷酸酶(ALP)活性。结果：染料木黄酮明显增加成骨细胞MTT的吸光度值、^3H-TdR和^3H-Pro的掺入,增加成骨细胞碱性磷酸酶活性。结论：染料木黄酮促进体外培养的乳鼠颅盖骨成骨细胞DNA和胶原的合成,促进增殖和分化。     

改良组织块法培养小鼠成骨细胞的研究

目的 探讨简便、经济、高效的成骨细胞培养方法.方法 取新生乳小鼠的颅骨,用改良的组织块法分离培养成骨细胞;从细胞的形态学、增殖、碱性磷酸酶染色、矿化结节茜素红染色及骨钙素基因表达水平等方法鉴定、比较培养的成骨细胞.结果 分离培养的成骨细胞生长状态良好,纯度较高,具有典型的成骨细胞形态特征,碱性磷酸酶染色及矿化结节茜素红染色阳性,骨钙素基因表达水平明显高于阴性对照,差异有统计学意义(P＜0.05).比较1～4次分离培养的成骨细胞在形态、增殖及分化功能上差异均无统计学意义(P＞0.05).结论 改良的组织块法分离培养乳小鼠的颅骨可获得大量纯度较高的成骨细胞,节约经费、时间,操作简便.     

降钙素相关肽诱导SD大鼠骨髓间充质干细胞成骨分化的机制

目的:研究外源性降钙素基因相关肽(calcitonin gene-relate peptide,CGRP)对SD大鼠骨髓来源间充质干细胞(BMSCs)增殖和成骨分化功能的影响。方法:采用贴壁法分离骨髓间充质干细胞,扩增传代至第三代,根据分组,培养体系中添加含不同浓度(10-11～10-6mol/L)CGRP的条件培养液,WST-1法检测细胞增殖能力;碱性磷酸酶染色及钙结节染色法观察CGRP诱导BMSCs向成骨细胞分化、矿化的效果。采用RT-PCR方法检测碱性磷酸酶(ALP)、I型胶原(COLL-I)、BMP-2、RunX2、骨粘连蛋白(Osteonectin,ON)等成骨相关细胞因子mRNA的表达。结果:增殖率测定CGRP组各浓度均较对照组增加,且呈剂量依赖关系,CGRP浓度大于1×10-10mol/L时差异有显著性(P<0.05);碱磷酶染色与钙结节染色结果显示,CGRP组均有阳性显色,对照组无显色或显色不明显。CGRP组的细胞因子表达较对照组显著升高(P<0.05)。结论:适当浓度的CGRP能够直接促进体外培养的BMSCs增殖,并可短期内诱导其在向成骨细胞分化。CGRP可能在骨修复及骨重建中发挥重要的作用...     

不同强度50 Hz脉冲电磁场促进大鼠颅骨成骨细胞矿化成熟最佳参数的筛选

为研究不同强度脉冲电磁场(pulse electromagnetic fields,PEMFs)对大鼠颅骨成骨细胞（rat skull osteoblasts,OB）增殖及成熟矿化的影响,将大鼠颅骨成骨细胞随机分为 7 组. 检测大鼠颅骨成骨细胞的增殖,细胞内碱性磷酸酶（ALP）活性变化,细胞沉积钙盐的情况,组织化学染色以及成骨细胞内标志性分子表达量的改变.结果显示,0.6 mT组促细胞增殖作用最强（P <0.01）;0.6 mT、1.8 mT、3.0 mT和3.6 mT均能提高ALP活性,其中0.6 mT ALP活性最高（P＜0.01）;在磁场处理4 ～12 d时细胞沉积钙盐逐渐增加,6种强度的脉冲电磁场均能促进钙盐沉积,尤以0.6 mT水平最高; ALP 染色、茜素红染色0.6 mT 组均显著高于对照组（P<0.01）;0.6 mT组 Bmp-2和Collagen-1 mRNA 的表达明显（P<0.01）高于对照组,磁场处理组Rankl mRNA 的表达均比对照组低. 0.6 mT 50 Hz 脉冲电磁场是促进成骨细胞增殖和矿化成熟的最佳参数,这为采用脉冲电磁场治疗骨质疏松症提供了治疗参数的基础支持.     

异补骨脂素加锌对大鼠成骨细胞相关细胞因子表达影响的实验研究

为探讨异补骨脂素加锌对体外培养新生大鼠颅骨成骨细胞相关基因表达的影响,用改良的组织块培养法分离培养新生大鼠颅骨成骨细胞,在成骨细胞体系中加入异补骨脂素与锌,以雌激素为阳性对照,空白组为阴性对照。结果显示:异补骨脂素加锌较单纯应用异补骨脂素或硫酸锌在48 h时促体外大鼠成骨细胞Ⅰ型胶原的表达;异补骨脂素加锌组可以明显上调大鼠成骨细胞TGF-β1的细胞信号转导因子Smad4 mRNA的表达(P0.01);能促进Runx2/Cbfa1 mRNA的表达(P0.05)。与空白对照组相比,异补骨脂素组,异补骨脂素加锌组均能增强Osterix mRNA的表达(P0.05)。与单纯应用异补骨脂素或者锌相比,异补骨脂素与锌联合应用能够协同增效,对促进体外培养的成骨细胞相关转录因子的表达更加明显,探讨了异补骨脂素及其与锌配伍调节骨代谢的分子机制,为提高骨质疏松症的临床疗效以及抗骨质疏松新药的开发提供了实验依据。     

骨形态发生蛋白2介导甲状旁腺素促进成骨细胞分化的实验研究

目的 探讨骨形态发生蛋白2(BMP2)在甲状旁腺素(PTH)促进成骨细胞分化过程中的重要介导作用.方法培养MC3T3-E1细胞,分为4组:1)盐水对照组;2)PTH组;3)6-[4-[2-(1-哌啶基)乙氧基]苯基]-3-(4-吡啶基)吡唑并[1,5-a]嘧啶 (Dorsomorphin) 组;4) PTH+Dorsomorphin组.Real-time PCR法和Westernblot方法检测细胞BMP2、BMP2下游基因和成骨因子的表达,碱性磷酸酶(ALP)染色方法检测细胞ALP的活性;双荧光素酶报告基因检测方法检测12xSBE-OC荧光素酶的活性.结果:PTH组BMP-2、成骨因子的表达及其12xSBE-OC荧光素酶的活性,明显高于盐水对照组.Dorsomorphin组和PTH+Dorsomorphin组BMP-2、BMP-2下游基因和成骨因子的表达,均明显低于盐水对照组;但其表达于两组间无明显差别.结论 BMP2介导PTH促进成骨细胞的分化,PTH可通过上调BMP2的表达,提高其功能,促进成骨细胞的成熟分化.     

Statin类药物促成骨细胞BMP-2表达机理

骨形态形成蛋白因子Ⅱ(BMP-2)属转化生长因子TGF-β超家族成员,具有活性最高和能单独诱导骨形成的特性。BMP-2能促进成骨细胞分化和增加成骨细胞标志基因的表达,这在成骨细胞分化过程中起非常关键的作用,最终促进骨形成。因此BMP-2可作为治疗骨质疏松症药物的新作用靶点。目前,已证明可上调BMP-2表达的上调剂多为Statin类药物。它们多通过抑制Rho和Rho激酶的活性;增加游离的有活性的eNOS;抑制HMG-CoA还原酶的活性等方面促骨形成。研究Statin类药物促BMP-2表达上调的机理,可为进一步寻找更有效的治疗骨质疏松症药物提供新靶点和新思路。     

异补骨脂素加锌对大鼠成骨细胞增殖与分化的影响

为探讨异补骨脂素加锌对体外培养新生大鼠颅骨成骨细胞增殖与分化作用的影响,用改良的组织块培养法分离培养新生大鼠颅骨成骨细胞,在成骨细胞体系中以不同浓度加入异补骨脂素与锌,MTT法检测加药后不同时间成骨细胞的增殖情况;用对硝基苯二钠基质动力学法(PNPP)测定细胞内碱性磷酸酶的活性,用改良的Lowry法测蛋白含量.结果显示:异补骨脂素加锌较单纯应用异补骨脂素或硫酸锌在24和48 h时促体外大鼠成骨细胞增殖的作用更加明显;在48和72 h时能促进成骨细胞碱性磷酸酶活性(ALP),其中ALP的测定在72h的活性更为显著.与单纯应用异补骨脂素或者锌相比,异补骨脂素与锌联合应用能够协同增效,对体外培养的成骨细胞的增殖与分化作用更加显著.     

Modulating osteogenesis of mesenchymal stem cells by modifying growth factor availability

Growth factors control the proliferation and differentiation of osteoprogenitor cells. This study explores the effects of modulating growth factors (VEGF, IGF-1, FGF-2 and BMP-2) on osteogenesis of mesenchymal stem cells (MSCs) in vitro. Constant and profiled delivery protocols, in accordance with protein expression in vitro, were applied to deliver or neutralize growth factors. Cell number, alkaline phosphatase (ALP-2) and osteocalcin (OC) expression, and mineralization were measured as outcome variables. Profiled addition of VEGF increased MSC proliferation. Constant and profiled application of FGF-2 and neutralization of IGF-1 and BMP-2 decreased ALP-2 levels. Profiled addition of BMP-2 vastly increased OC release from MSCs, but constant addition of IGF-1, constant and profiled neutralization of IGF-1 and FGF-2 reduced OC levels. Constant addition of IGF-1 and FGF-2, as well as profiled loading of FGF-2 decreased mineralization of MSCs. This study indicated that endogenous IGF-1 and FGF-2 are essential to osteogenesis; excess IGF-1 and FGF-2 were inhibitory to bone formation. Selective, temporally specific addition of growth factors, such as BMP-2 and VEGF appears to be an important strategy to enhance osteogenesis.     

CYR61 Regulates BMP-2-dependent Osteoblast Differentiation through the αvβ3 Integrin/Integrin-linked Kinase/ERK Pathway

Osteoporosis is one of the most common bone pathologies. A number of novel molecules have been reported to increase bone formation including cysteine-rich protein 61 (CYR61), a ligand of integrin receptor, but mechanisms remain unclear. It is known that bone morphogenetic proteins (BMPs), especially BMP-2, are crucial regulators of osteogenesis. However, the interaction between CYR61 and BMP-2 is unclear. We found that CYR61 significantly increases proliferation and osteoblastic differentiation in MC3T3-E1 osteoblasts and primary cultured osteoblasts. CYR61 enhances mRNA and protein expression of BMP-2 in a time- and dose-dependent manner. Moreover, CYR61-mediated proliferation and osteoblastic differentiation are significantly decreased by knockdown of BMP-2 expression or inhibition of BMP-2 activity. In this study we found integrin α_vβ₃ is critical for CYR61-mediated BMP-2 expression and osteoblastic differentiation. We also found that integrin-linked kinase, which is downstream of the α_vβ₃ receptor, is involved in CYR61-induced BMP-2 expression and subsequent osteoblastic differentiation through an ERK-dependent pathway. Taken together, our results show that CYR61 up-regulates BMP-2 mRNA and protein expression, resulting in enhanced cell proliferation and osteoblastic differentiation through activation of the α_vβ₃ integrin/integrin-linked kinase/ERK signaling pathway.     

Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein-2

We have examined the effects of BMP-2 on the expression of bone matrix proteins in both human bone marrow stromal cells (HBMSC) and human osteoblasts (HOB) and their proliferation and mineralization. Both HBMSC and HOB express BMP-2/-4 type I and type II receptors. Treatment of these two cell types with BMP-2 for 4 weeks in the presence of β-glycerophosphate and ascorbic acid results in mineralization of their matrix. BMP-2 increases the mRNA level and activities of alkaline phosphatase and elevates the mRNA levels and protein synthesis of osteopontin, bone sialoprotein, osteocalcin, and α1(I) collagen in both cell types. Whereas the mRNA level of decorin is increased, the mRNA concentration of biglycan is not altered by BMP-2. No effect on osteonectin is observed. The effect of BMP-2 on bone matrix protein expression is dose dependent from 25 to 100 ng/ml and is evident after 1–7 days treatment. In the presence of BMP-2, proliferation of HBMSC and HOB is decreased under either serum-free condition or in the presence of serum. Thus, BMP-2 has profound effects on the proliferation, expression of most of the bone matrix proteins and the mineralization of both relatively immature human bone marrow stromal preosteoblasts and mature human osteoblasts. J. Cell. Biochem. 67:386–398, 1997. © 1997 Wiley-Liss, Inc.     

Effect of TAK1 on osteogenic differentiation of mesenchymal stem cells by regulating BMP-2 via Wnt/β-catenin and MAPK pathway

Mesenchymal stem cells (MSCs) have the ability to differentiate into osteoblasts and chondrocytes. In vitro osteogenic differentiation is critical but the molecular mechanism has yet to be further clarified. The role of TGF-β activated kinase 1 (TAK1) in MSCs osteogenesis differentiation has not been reported. By adding si-TAK1 and rhTAK1, the osteogenic differentiation of MSCs was measured. Expression levels of the osteoblastic marker genes during osteogenic differentiation of MSCs were checked. As well as molecules involved in BMP and Wnt/β-catenin signaling pathways. The phosphorylation of p38 and JNK was also checked. TAK1 is essential for mineralization of MSCs at low concentration, but excessive rhTAK1 inhibits mineralization of MSCs. It up regulates the expression levels of bone sialoprotein (BSP), osteocalcin (OSC), Alkaline phosphatase (ALP), and RUNX2 during osteogenic differentiation of MSCs. It can also promote TGF-β/BMP-2 gene expression and β-catenin expression, and down regulate GSK-3β expression. Meanwhile, TAK1 promotes the phosphorylation of p38 and JNK. Additionally, TAK1 up regulates the expression of BMP-2 at all concentration under the inhibition of p38 and JNK. Our results suggested that TAK1 is essential in MSCs osteogenesis differentiation, and functions as a double-edged sword, probably through regulation of β-catenin and p38/JNK.     

Gene profiling on mixed embryonic stem cell populations reveals a biphasic role for beta-catenin in osteogenic differentiation

The differentiation of embryonic stem cells (ESCs) into osteoblasts is enhanced to 60% when exposed to vitamin D3 (VD3) but leaves a remainder of one half of the cell population unidentified. To increase differentiation outcome, the known osteoinducers retinoic acid (RA) and bone morphogenetic protein-2 (BMP-2) were evaluated. Initial studies using RA and BMP-2 during early osteogenesis in addition to VD3 increased osteogenic yield in the case of RA, but surprisingly decreased osteogenesis when BMP-2 was administered together with VD3 or RA. This paper describes a comprehensive microarray study examining the gene expression profile of differentiating osteoblasts in these mixed ESC populations. In addition to five other families of signaling molecules (insulin growth factors, prostaglandin, follistatin, TGFbeta2, and Wnt molecules), we identified an endogenous expression pattern for BMPs and RA that differed from our previous exogenous administration of these molecules. By mimicking the change in expression of the RA and BMP-2 families with exogenous supplementation at the correct time, it was then possible to increase the number of ESC-derived osteoblasts to 90%. This effect was mediated through alteration in beta-catenin (CatnB) expression levels and nuclear CatnB activity, both of which are modulated by VD3, RA, and BMP-2. Our results suggest that blockage of CatnB activity by VD3 and RA is opposed by induction of CatnB activity through BMP-2 when administered together. Hence, osteoinduction, in vitro, is an intricate process involving both temporal and quantitative changes in gene expression and CatnB activity.     

Comparative secretome analysis of human bone marrow‐derived mesenchymal stem cells during osteogenesis

Osteogenesis is a tightly regulated process that involves coordinated extracellular signals from autocrine and paracrine loops. Secretory proteins during osteogenesis can inhibit cell proliferation and activate cell differentiation toward mature osteoblasts, which are characterized by mineralization. In this study, we attempted to identify these secretory proteins during osteogenesis using LC–MS/MS analysis. We compared the secretome between undifferentiated human bone marrow‐derived mesenchymal stem cells (hBMSCs) and differentiated osteoblasts. Among 315 proteins that were identified, 177 proteins were present at increased levels in osteoblasts, whereas 88 proteins were present at decreased levels. Among the identified proteins, several were validated by quantitative RT‐PCR and immunoblot analysis. Of particular interest, calcium homeostasis‐related proteins were upregulated, whereas stem cell proliferation‐related proteins and other lineage‐related proteins were downregulated during osteogenesis. These findings provide information about the dynamic changes in the expression and secretion of proteins during osteogenesis and suggest the putative role of secretory proteins in osteogenesis. J. Cell. Physiol. 228: 216–224, 2013. © 2012 Wiley Periodicals, Inc.     

川芎嗪抑制血管紧张素Ⅱ诱导的平滑肌细胞NF-κB激活和骨形成蛋白-2表达降低

本文旨在观察血管紧张素Ⅱ（angiotensinⅡ,AngⅡ）对血管平滑肌细胞核转录因子-κB（nuclear factor-κB,NF-κB）的活性及骨形成蛋白-2（bone morphogenetic protein-2,BMP-2）表达的影响,以探讨AngⅡ参与动脉粥样硬化的机制,并探讨川芎嗪是否能抑制AngⅡ的促动脉粥样硬化作用。采用Western blot、免疫组化和原位杂交等方法分别检测AngⅡ刺激和川芎嗪干预后NF-κB活性、BMP-2蛋白和mRNA表达的变化。结果显示：（1）AngⅡ刺激激活NF-κB。AngⅡ刺激15min即有NF-κB p65核转移,30min达高峰（P〈0．01）,1h后减退。川芎嗪抑制AngⅡ诱导的NF-κB激活,与AngⅡ组比较,川芎嗪＋AngⅡ组NF-κB活性显著降低（P〈0．01）。（2）AngⅡ刺激6h时BMP-2表达增强（P〈0．05）,12h时减弱（P〈0．01）,24h时更弱（P〈0．01）。川芎嗪＋AngⅡ组中,川芎嗪干预6h时BMP-2表达亦增强,12与24h时保持正常水平。（3）川芎嗪对正常细胞的NF-κB活性和BMP-2表达无影响。以上结果表明,AngⅡ刺激后激活NF-κB并最终使生长抑制因子BMP-2表达下降,这可能是其参与动脉粥样硬化发生的机制之一。BMP-2一过性增高可能不依赖NF-κB通路的激活。川芎嗪可抑制AngⅡ诱导的NF-κB激活与BMP-2表达降低,提示它在抗动脉粥样硬化形成中起重要作用。