不同温度下成骨细胞增殖及其OPG/RANKL mRNA表达的影响

目的:探讨不同温度下对小鼠成骨细胞MC3T3-E1的增殖以及OPG/RANKL表达水平的影响。方法:1.以小鼠成骨细胞MC3T3-E1为体外实验模型,MTT法检检测细胞的增殖情况。2.RT-PCR方法检测MC3T3-E1OPG/RANKL mRNA的表达水平。结果:设定对照组为37℃,高于对照组(38℃-39℃-40℃-41℃-42℃)分别作用于MC3T3-E1细胞1小时/天,连续1周,可刺激细胞增殖,OD值显著增加(P<0.05)。同时可增加OPG mRNA表达,降低RANKL mRNA表达,呈温度梯度依赖性。结论:热刺激促进MC3T3-E1细胞增殖,同时通过调节OPG/RANKL mRNA的表达,直接促进骨形成,抑制骨吸收。     

联合补充维生素D、胶原肽和钙对成骨细胞发育的影响研究

目的：观察联合补充维生素D、胶原肽和钙对MC3T3-E1成骨细胞增殖及对骨保护素细胞内核因子κB受体活化因子配体（receptor activator of NF-κB ligand,RANKL）、骨保护素（Osteoprotegerin,OPG）基因表达的影响。方法：α-MEM培养基培养MC3T3-E1细胞。检测Ca2+（20 μg/mL）、维生素D（0、10-12、10-11mol/L）、胶原肽（0、50、100μg/mL）三者交互作用剂量对MC3T3-E1细胞增殖的作用及RANKL、OPG mRNA表达。结果：维生素D、胶原肽和钙交互剂量作用下细胞增殖水平无明显差异。维生素D联合钙能够明显降低RANKL mRNA表达水平,提高OPG mRNA表达水平,降低RANKL/OPG比值。而胶原肽联合钙对RANKL以及 OPG mRNA表达无明显影响。结论：维生素D联合钙可通过抑制小鼠成骨细胞RANKL mRNA表达、促进OPG mRNA表达,从而促进骨的形成,抑制骨的吸收。维生素D和钙联合补充胶原肽,对成骨细胞RANKL,OPG mRNA表达并无明显影响。     

甲状旁腺激素对成骨细胞中ClC-3 氯通道表达及成骨分化影响的研究

目的:观察甲状旁腺激素(PTH)对成骨细胞中Cl C-3氯通道表达及成骨分化影响,初步探索Cl C-3介导PTH在细胞成骨分化中的作用。方法:采用10-8M、10-9M、10-10M PTH持续刺激和间断刺激MC3T3-E1细胞72 h后,通过CCK-8试剂盒法检测MC3T3-E1细胞的增殖情况,Real-Time PCR法检测MC3T3-E1细胞中Clcn3及成骨相关基因Alp、Runx2的表达情况,免疫荧光法检测10-9M PTH不同给药方式下对Cl C-3蛋白表达的影响。结果 :经不同浓度PTH连续和间断处理72 h后,结果显示10-9 M PTH间断刺激的MC3T3-E1细胞的增殖能力最强,且其Alp、Runx2 m RNA表达均高于10-8 M组和10-10 M组(P<0.05),而相同浓度间断刺激的MC3T3-E1细胞成骨相关基因的表达均高于持续刺激组,以10-9M间断刺激组差异最显著(P<0.05),而10-8 M和10-10M均无统计学差异(P>0.05),10-9 M PTH刺激的MC3T3-E1细胞中Cl C-3蛋白表达也显著增加(P<0.05)。结论 :成骨细胞的Cl C-3氯通道能够响应PTH的刺激发生变化,并伴随着成骨相关基因Alp、Runx2表达的增强。     

骨样细胞MLO-Y4与成骨样细胞MC3T3-E1生物学特性的比较

分别采用倒置显微镜观察法、细胞计数法、RT-PCR法、磷酸对硝基苯酚法(PNPP法)和ELISA法来比较小鼠骨样细胞MLO-Y4与小鼠成骨样细胞MC3T3-E1的细胞形态、增殖、相关基因的表达和分泌功能的差异。结果显示MC3T3-E1细胞呈长梭形,具有少量短的突触;而MLO-Y4细胞呈星状或树枝状且具有很多长的突触。MC3T3-E1细胞的增殖能力强于MLO-Y4细胞,两者的倍增时间分别是18 h和20 h。MC3T3-E1细胞中原癌基因c-fos和骨桥蛋白基因OPNmRNA的表达明显高于MLO-Y4细胞,而骨钙素基因OCmRNA的表达则是MC3T3-E1细胞远低于MLO-Y4细胞,白细胞分化抗原44基因CD44 mRNA在两种细胞中的表达差异不明显。ALP的分泌在MC3T3-E1细胞中高于MLO-Y4细胞,NO的分泌在两种细胞中没有显著性差异,M-CSF在MLO-Y4细胞中的分泌较高。由此可见骨样细胞MLO-Y4与成骨样细胞MC3T3-E1在形态、ALP和MCSF分泌及c-fos、OPN和OCmRNA表达方面差异明显。     

小鼠前成骨细胞MC3T3-E1在自组装多肽水凝胶支架中的成骨分化

目的研究MC3T3-E1细胞在自组装多肽水凝胶支架上的生长和成骨分化.方法在多肽水凝胶支架RADA16上接种MC3T3-E1细胞,荧光染色观察细胞形态和存活情况;组织化学染色检测MC3T3-E1细胞碱性磷酸酶活性以及细胞外钙质沉积;RT-PCR分析成骨特异性基因的表达.结果 MC3T3-E1细胞在水凝胶支架RADA16上粘附铺展良好,呈纺锤样形态.诱导培养后支架上的细胞有较高水平的碱性磷酸酶表达和矿化基质沉积.此外,骨分化特异性基因骨桥蛋白和骨涎蛋白也有表达,且表达量随培养时间的延长而增多.结论 在自组装水凝胶内MC3T3-E1细胞可向成骨方向分化,并能在凝胶内产生矿化的细胞外基质.     

模拟微重力通过调节AKT磷酸化抑制前成骨细胞的增殖

目的:研究模拟微重力对前成骨细胞的增殖作用及其分子机制。方法:利用2D回转器模拟失重条件培养前成骨细胞MC3T3-E1 24小时;将p-AKT激活剂SC79加入细胞培养基后模拟失重条件下培养前成骨细胞MC3T3-E1 24小时;利用Western blot技术分别检测细胞增殖相关蛋白PCNA以及AKT、p-AKT的表达变化情况。结果:(1)与对照组相比,模拟失重组前成骨细胞增殖受到抑制(P0.01),p-AKT/AKT比值减小(P0.01);(2)加入SC79组与对照组相比,p-AKT/AKT比值显著增加;(3)加入SC79的模拟失重组(MG+SC79)与模拟失重组相比(MG),p-AKT/AKT比值增加,PCNA蛋白表达增加(P0.01),成骨细胞增殖有所恢复。结论:模拟微重力可能通过抑制AKT的磷酸化形式抑制前成骨细胞的增殖,加入p-AKT激活剂可部分恢复前成骨细胞的增殖。     

NMDA型谷氨酸受体在骨祖细胞和成骨细胞表达并介导力学信号转导

目的：鉴定骨髓间充质干细胞D1和成骨细胞MC3T3-E1、MC3T3-E1亚克隆14是否表达NMDA型谷氨酸受体（NMDAR）,并初步探讨NMDAR是否参与力学信号转导。方法：采用RT—PCR技术、免疫荧光染色技术、蛋白免疫杂交技术和体外细胞循环拉伸装置,鉴定了上述3个细胞系中NMDAR的表达情况,并初步探讨了在MC3T3-E1亚克隆14细胞系中,NMDAR在力学信号转导中的可能作用。结果：3个细胞系均表达NMDA受体亚基1（NR1）和不同的亚基2（NR2A—NR2D）。细胞微丝骨架的破坏并没有阻断力学应变诱导的c-jun、C—fos的表达上调;而阻断NMDAR后,却抑制了力学应变诱导的c-jun、C—fos和成骨细胞特异的转录因子Cbfa1／Runx2的表达上调。结论：NMDAR在骨中表达并发挥功能,参与了骨的力学信号转导过程。     

3D水凝胶压应力模型建立及压应力对成骨细胞的影响

目的:通过建立3D水凝胶细胞模型,采用不同强度、频率、时间对MC3T3-E1细胞进行机械加压干预,进而探讨促进成骨细胞生长的适宜压应力方案。方法:设计不同强度、频率、时间的压应力梯度方案对成骨细胞MC3T3-E1进行干预。加压干预结束后即刻收集细胞,对样本中的ATF4、ALP、Runx2、Osteocalcin、RANKL和RANK mRNA进行定量检测。结果:经统计学分析后发现不同强度和频率对ALP(P0.05)、Runx2(P0.01)交互作用显著。此外,4h加压Runx2的表达量比12h加压的高(P0.05);4h加压RANKL的表达量比12h加压的低(P0.05)。结论:确定了压力强度和频率后,对3D细胞水凝胶模型施加一段时间的压应力发现,以1%强度、0.5Hz频率、4h的压应力干预方案能够促进成骨细胞系的生长。     

锌协同三羟异黄酮对成骨细胞MC3T3-E1的影响

目的:观察锌协同三羟异黄酮对成骨细胞MC3T3-E1增殖、细胞中碱性磷酸酶(ALP)含量、骨形成蛋白-2(BMP-2)表达的影响,探讨锌协同三羟异黄酮对骨质疏松的防治作用.方法:采用四甲基偶氮噻唑蓝比色法检测(1×10-7)mol/L、(1×10-6)mol/L、(1x 10-5)mol/L、(1×10-4)mol/L的三羟异黄酮以及与(1×10-5)mol/L锌联合作用时对MC3T3-E1增殖的作用;应用Western blot法检测三羟异黄酮与锌联合作用前后,成骨细胞中BMP-2蛋白的表达水平,用比色法检测MC3T3-E1中ALP的含量.结果:锌与三羟异黄酮单独作用或协同作用于MC3T3-E1细胞,其增殖率随着三羟异黄酮浓度的增加和作用时间的延长而升高,(1×10-5)mol/L的三羟异黄酮协同(1×10-5)mol/L的锌作用72h,其细胞增殖率为(160.1±14.3)%.细胞中的LP含量及BMP-2的表达也随着三羟异黄酮浓度的增加及作用时间的延长而增加.三羟异黄酮和锌联合作用后,对ALP活性的增强、BMP-2表达的增加作用均较各自单独作用时更为明显(P＜0.05).结论:三羟异黄酮与锌协同作用表现出雌激素效应,可通过促进骨形成蛋白的合成从而促进成骨细胞的增殖、增加骨量.     

三维回转骨细胞条件培养基对成骨细胞功能的调节作用

骨细胞是骨组织中主要的力学感受器.研究失重条件下骨细胞对效应细胞的调控作用对于揭示失重引起的骨丢失机制具有重要意义.本研究拟采用三维回转器模拟失重,探讨模拟失重骨细胞条件培养基(RCM)对成骨功能的调节作用.小鼠骨细胞系MLO-Y4三维回转培养72h后,收集回转条件培养基(RCM)和未回转对照组的条件培养基(CCM),用四甲基偶氮唑盐比色(MTT)法、对硝基苯磷酸(pNPP)法和流式细胞术(FCM)分别检测RCM对小鼠成骨样细胞系MC3T3-E1增殖、周期及细胞分泌碱性磷酸酶(ALP)活性的影响.采用RT-PCR方法检测RCM对MC3T3-E1成骨相关基因表达的影响.结果显示,三维随机回转72h后的MLO-Y4RCM可促进MC3T3-E1增殖:条件培养基培养MC3T3-E124h和48h后,50%RCM组比CCM组分别增加了1.62和1.60倍,差异显著(*P0.05),培养72h后,100%RCM组比CCM组增加了1.69倍,差异显著(*P0.05);细胞周期检测结果表明,条件培养基培养24、48和72h后,RCM组部分恢复CCM引起的MC3T3-E1细胞周期阻滞;MC3T3-E1的ALP活性在RCM组和CCM组之间无差异;RT-PCR检测结果表明,100%MLO-Y4条件培养基培养MC3T3-E148h后,降低了成骨相关基因ALP、Runx2、OPN、OC的表达.差异显著(*P0.05,**P0.01,***P0.001).实验结果表明,三维随机回转模拟失重培养骨细胞72h后的条件培养基促进了成骨细胞增殖,抑制了成骨相关基因表达.     

Leptin effect on RANKL and OPG expression in MC3T3-E1 osteoblasts

Recent studies have suggested that leptin hormone may play a pivotal role on bone remodeling through a direct effect by modulating positively the OPG/RANKL balance. Here, we investigate the effect of leptin hormone on RANKL and OPG expression in MC3T3-E1 osteoblasts using RT-PCR and ELISA measurements. We have at first identified the expression of Ob-Rb and Ob-Ra leptin receptor isoforms in MC3T3-E1 and observed that these cells respond to mrleptin treatments. We then investigated the effect of mrleptin on RANKL and OPG expression. We show that mrleptin dose-dependently regulated the expression of RANKL mRNA with complete inhibition observed at concentrations higher than 12 ng/ml. This effect was confirmed with sRANKL protein measurements. However, the exposure of MC3T3-E1 to mrleptin had no effect on OPG mRNA. Taken together, these results suggest that leptin modulates positively OPG/RANKL balance by inhibiting the expression of RANKL gene.     

Fluid shear stress induction of COX-2 protein and prostaglandin release in cultured MC3T3-E1 osteoblasts does not require intact microfilaments or microtubules.

Cultured osteoblasts express three major types of cytoskeleton: actin microfilaments, microtubules, and intermediate filaments. The cytoskeletal network is thought to play an important role in the transmission and conversion of a mechanical stimulus into a biochemical response. To examine a role for the three different cytoskeletal networks in fluid shear stress-induced signaling in osteoblasts, we individually disrupted actin microfilaments, micro-tubules, and intermediate filaments in MC3T3-E1 osteoblasts with multiple pharmacological agents. We subjected these cells to 90 min of laminar fluid shear stress (10 dyn/cm(2)) and compared the PGE(2) and PGI(2) release and induction of cyclooxygenase-2 protein to control cells with intact cytoskeletons. Disruption of actin microfilaments, microtubules, or intermediate filaments in MC3T3-E1 cells did not prevent a significant fluid shear stress-induced release of PGE(2) or PGI(2). Furthermore, disruption of actin microfilaments or microtubules did not prevent a significant fluid shear stress-induced increase in cyclooxygenase-2 protein levels. Disruption of intermediate filaments with acrylamide did prevent the fluid shear stress-induced increase in cyclooxygenase-2 but also prevented a PGE(2)-induced increase in cyclooxygenase-2. Thus none of the three major cytoskeletal networks are required for fluid shear stress-induced prostaglandin release. Furthermore, although neither actin microfilaments nor microtubules are required for fluid shear stress-induced increase in cyclooxygenase-2 levels, the role of intermediate filaments in regulation of cyclooxygenase-2 expression is less clear.     

Single Bout Short Duration Fluid Shear Stress Induces Osteogenic Differentiation of MC3T3-E1 Cells via Integrin β1 and BMP2 Signaling Cross-Talk

Fluid shear stress plays an important role in bone osteogenic differentiation. It is traditionally believed that pulsed and continuous stress load is more favorable for fracture recovery and bone homeostasis. However, according to our clinical practice, we notice that one single stress load is also sufficient to trigger osteogenic differentiation. In the present study, we subject osteoblast MC3T3-E1 cells to single bout short duration fluid shear stress by using a parallel plate flow system. The results show that 1 hour of fluid shear stress at 12 dyn/cm² promotes terminal osteogenic differentiation, including rearrangement of F-actin stress fiber, up-regulation of osteogenic genes expression, elevation of alkaline phosphatase activity, secretion of type I collagen and osteoid nodule formation. Moreover, collaboration of BMP2 and integrin β1 pathways plays a significant role in such differentiation processes. Our findings provide further experimental evidence to support the notion that single bout short duration fluid shear stress can promote osteogenic differentiation.     

Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts

Osteoblasts subjected to fluid shearincrease the expression of the early response gene, c-fos, andthe inducible isoform of cyclooxygenase, COX-2, two proteins linked tothe anabolic response of bone to mechanical stimulation, in vivo. Theseincreases in gene expression are dependent on shear-induced actinstress fiber formation. Here, we demonstrate that MC3T3-E1osteoblast-like cells respond to shear with a rapid increase inintracellular Ca²⁺ concentration([Ca²⁺]_i) that wepostulate is important to subsequent cellular responses to shear. Totest this hypothesis, MC3T3-E1 cells were grown on glass slides coatedwith fibronectin and subjected to laminar fluid flow (12 dyn/cm²). Before application of shear, cells were treatedwith two Ca²⁺ channel inhibitors or various blockers ofintracellular Ca²⁺ release for 0.5-1 h. Althoughgadolinium, a mechanosensitive channel blocker, significantly reducedthe [Ca²⁺]_i response, neithergadolinium nor nifedipine, an L-type channel Ca²⁺ channelblocker, were able to block shear-induced stress fiber formation andincrease in c-fos and COX-2 in MC3T3-E1 cells. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid-AM, an intracellular Ca²⁺ chelator, or thapsigargin,which empties intracellular Ca²⁺ stores, completelyinhibited stress fiber formation and c-fos/COX-2 production in shearedosteoblasts. Neomycin or U-73122 inhibition of phospholipase C, whichmediates D-myo-inositol 1,4,5-trisphosphate (IP₃)-induced intracellular Ca²⁺ release, alsocompletely suppressed actin reorganization and c-fos/COX-2 production.Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform ofU-73122, did not inhibit these shear-induced responses. These resultssuggest that IP₃-mediated intracellular Ca²⁺release is required for modulating flow-induced responses in MC3T3-E1 cells.

     

Fluid shear stress remodels expression and function of junctional proteins in cultured bone cells

We tested the hypothesis that fluidshear stress () modifies the expression, function, and distributionof junctional proteins [connexin (Cx)43, Cx45, and zona occludens(ZO)-1] in cultured bone cells. Cell lines with osteoblastic (MC3T3-E1cells) and osteocytic (MLO-Y4 cells) phenotypes were exposed to-values of 5 or 20 dyn/cm² for 1-3 h.Immunostaining indicated that at 5 dyn/cm², thedistribution of Cx43, Cx45, and ZO-1 was moderately disrupted at cellmembranes; at 20 dyn/cm², disruption was more severe.Intercellular coupling was significantly decreased at both shear stresslevels. Western blots showed the downregulation of membrane-bound Cx43and ZO-1 and the upregulation of cytosolic Cx43 and Cx45 at differentlevels of shear stress. Similarly, Northern blots revealed thatexpression of Cx43, Cx45, and ZO-1 was selectively up- anddownregulated in response to different shear stress levels. Theseresults indicate that in cultured bone cells, fluid shear stressdisrupts junctional communication, rearranges junctional proteins, anddetermines de novo synthesis of specific connexins to an extent thatdepends on the magnitude of the shear stress. Such disconnection fromthe bone cell network may provide part of the signal whereby thedisconnected cells or the remaining network initiate focal bone remodeling.

     

Synergism of biochemical and mechanical stimuli in the differentiation of human placenta-derived multipotent cells into endothelial cells

There have been intensive studies on the differentiation of endothelial progenitor cells (EPCs) into endothelial cells. We investigated the endothelial differentiation of placenta-derived multipotent cells (PDMCs), a population of CD34(-)/CD133(-)/Flk-1(-) cells. PDMCs were cultured in basal media or media containing endothelial growth factors (EGM), including vascular endothelial growth factor (VEGF), for 3 days and then subjected to shear stress of 6 or 12dyn/cm(2) for 24h. Culture of PDMCs in EGM under static conditions resulted in significant increases in VEGF receptor-1 (Flt-1) and receptor-2 (Flk-1) expression. Application of shear stress at 12dyn/cm(2) to these cells led to significant increases in their expression of von Willebrand Factor and platelet-endothelial cell adhesion molecule-1 at both the gene and protein levels. Shear stress at 6dyn/cm(2) had lesser effects. Uptakes of acetylated low-density lipoproteins as well as formation of tube-like structures on Matrigel were significantly increased after subjecting to shear stress of 12dyn/cm(2) for 24h. Our findings suggest that the combined use of endothelial growth factors and high shear stress is synergistic for the endothelial differentiation of PDMCs.