沉默Piezo1机械敏感型离子通道对MC3T3-E1成骨细胞迁移的影响

目的研究Piezo1机械敏感型离子蛋白在小鼠MC3T3-E1成骨细胞迁移中的作用。方法取第5～10代小鼠MC3T3-E1成骨细胞,分为Piezo1-小干扰RNA(small interfering RNA,siRNA)转染组(A组)、阴性对照组(B组)和空白对照组(C组)。A、B组分别采用siRNA转染试剂将Piezo1-siRNA或阴性对照siRNA转染入小鼠MC3T3-E1成骨细胞,C组仅加入siRNA转染试剂,在倒置相差显微镜及荧光显微镜下观察细胞形态并计算转染效率。采用免疫荧光染色和Western blot检测各组Piezo1蛋白表达水平;采用Transwell细胞迁移实验和细胞划痕实验检测Piezo1-siRNA转染后MC3T3-E1成骨细胞迁移能力的变化。结果转染48 h后,A组可见细胞体积较未转染细胞略有增大,细胞突变长增粗,但细胞集落有所减少,悬浮细胞较未转染增多,细胞碎片增多。荧光显微镜下可见B组小鼠MC3T3-E1成骨细胞中出现绿色荧光,转染效率为68.56%±4.12%。免疫荧光染色及Western blot检测示,A组细胞内Piezo1蛋白表达水平均显著低于B、C组,差异有统计学意义(P0.05);B、C组间差异无统计学意义(P0.05)。Transwell细胞迁移实验及细胞划痕实验检测示,A组每孔迁移细胞数及培养1～4 d的细胞划痕愈合率均明显低于B、C组,差异有统计学意义(P0.05);B、C组间差异无统计学意义(P0.05)。结论沉默Piezo1基因能显著抑制小鼠MC3T3-E1成骨细胞的迁移能力。     

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。     

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。     

ERK5信号通路介导流体剪切力对MC3T3-E1成骨细胞MMPs、TIMPs表达的影响

目的观察流体剪切力(fluid shear stress,FSS)作用下,MC3T3-E1成骨细胞中细胞基质金属蛋白酶(matrix metalloproteinases,MMPs)和基质金属蛋白酶抑制剂(tissue inhibitors of metalloproteinases,TIMPs)的表达情况,并探讨ERK5信号通路在其中的作用。方法对MC3T3-E1成骨细胞进行不同的处理,分为正常组、XMD8-92组、FSS组和FSS+XMD8-92组,对FSS组施加12 dyn/cm~2流体剪切力,采用蛋白免疫印迹法分别检测P-ERK5、ERK5、MMPs和TIMPs蛋白水平的变化。结果生理强度(12 dyn/cm~2)的流体剪切力作用于MC3T3-E1成骨细胞45 min后能显著上调MMPs的表达,下调TIMPs的表达,但此效应可被ERK5高选择性抑制剂XMD8-92阻断。结论 ERK5信号通路调控流体剪切力对成骨细胞MMPs、TIMPs蛋白的表达。     

痩素对小鼠成骨细胞MC3T3-E1增殖及OPG/RANKL mRNA表达的影响

目的初步探讨瘦素对小鼠成骨细胞MC3T3-E1细胞增殖及骨保护蛋白、核因子κB激活受体配体mRNA表达的影响。方法以小鼠成骨细胞MC3T3-E1为体外实验模型,用MTT法检测瘦素作用MC3T3-E1细胞后的增殖情况,RT-PCR方法检测OPG、RANKL mRNA表达。结果瘦素（10、20、40、80和160ng.mL-1）作用于MC3T3-E1细胞24h后,可促进其增殖,OD值显著增加（P〈0.01）,增加骨保护蛋白mRNA表达,同时降低核因子κB激活受体配体mRNA表达,且呈浓度依赖性。结论瘦素促进MC3T3-E1细胞增殖,同时通过调节骨保护蛋白mRNA和核因子κB激活受体配体mRNA的表达,从而促进骨形成,抑制骨吸收。     

蓇密钙片与马鹿角多肽对成骨细胞增殖分化的研究

目的观察蓇密钙片及马鹿角多肽对鼠胚成骨细胞MC3T3-E1的增殖分化作用及细胞内OPG、RANKL mRNA的表达。方法采用MEM(10%FBS)培养液培养细胞,细胞计数法描绘成骨细胞生长曲线;MTT法检测蓇密钙片(0、50、100、500、1000μg/ml)、马鹿角多肽(0、5、50、500μg/ml)和钙尔奇D对照组对鼠胚成骨细胞MC3T3-E1增殖的作用;蓇密钙片(0、50、100、500、1000μg/ml)、马鹿角多肽(0、5、50、500μg/ml)和钙尔奇D对照组干预鼠胚成骨细胞MC3T3-E1 72 h后,比色法检测细胞内碱性磷酸酶(alkaline phosphatase,ALP)的含量,RT-PCR法检测骨保护蛋白(osteoprotegerin,OPG)、核因子КB激活受体配体(receptor activator of NK-КB ligand,RANKL)mRNA的表达。结果蓇密钙片及马鹿角多肽作用于MC3T3-E1成骨细胞72 h后,可促进其增殖,OD值增加(P0.05),增加ALP的含量,促进OPG mRNA表达,同时抑制RANKL mRNA表达。结论蓇密钙片及马鹿角多肽促进MC3T3-E1成骨细胞增殖分化,可能与促进成骨细胞OPG mRNA表达、抑制RANKL mRNA表达有关,从而促进骨形成,抑制骨吸收。     

STIM1调控Ras信号通路促进成骨细胞增殖的研究

目的成骨细胞增殖的抑制导致成骨细胞数量减少是骨质疏松症发生发展的重要原因。之前的研究发现介导受体依赖性钙离子内流的钙通道蛋白Orai1在调控成骨细胞增殖、分化、凋亡等细胞功能有非常重要的作用。STIM1是一类可激活Orai1的重要蛋白。然而,至今还未见STIM1是否参与调控成骨细胞增殖相关报道。本研究旨在明确STIM1是否参与调控成骨细胞增殖。方法应用靶向STIM1的siRNA在MC3T3-E1成骨细胞中沉默STIM1的表达,然后用MTT法检测细胞增殖变化、流式细胞仪检测细胞周期变化、Real-Time PCR检测CyclinD1、CyclinE、CDK4、CDK6的转录水平,并用Western Blot检测Ras信号通路活性。结果我们转染靶向STIM1的siRNA后,MC3T3-E1细胞中STIM1的表达和转录均明显降低(P0.05);接着,我们发现在MC3T3-E1细胞中沉默STIM1的表达后,MC3T3-E1细胞增殖抑制并且细胞周期阻滞在G0/G1期,并且调控细胞增殖的细胞周期蛋白CyclinD1的转录水平也显著下降(P0.05);进一步研究发现,在MC3T3-E1细胞沉默STIM1的表达后,明显抑制了钙离子依赖的Ras信号通路的活性(P0.05)。应用ARS-853抑制Ras信号通路的活性后,明显抑制了STIM1促进成骨细胞增殖的作用(P0.05)。结论 STIM1激活Ras信号通路发挥促进成骨细胞增殖的作用。     

续苓健骨汤含药血清对MC3T3-E1细胞分化及增殖的影响

目的探讨续苓健骨汤含药血清对MC3T3-E1成骨细胞分化及增殖的影响。方法制备续苓健骨汤含药血清,实验分为空白对照组、含药血清低剂量组、中剂量组和高剂量组。采用CCK-8法和流式细胞术检测续苓健骨汤含药血清对MC3T3-E1细胞增殖和细胞周期的影响;碱性磷酸酶(ALP)活性测定MC3T3-E1细胞的成骨分化能力;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、OC、Bmp2、Col1a1mRNA水平。结果与空白对照组比较,中、高剂量续苓健骨汤含药血清能促进MC3T3-E1细胞增殖、S期细胞比率和细胞增殖指数,并且呈现一定的剂量依赖性;同时中高剂量续苓健骨汤含药血清组能明显提高MC3T3-E1细胞ALP活性(P0.01)和钙化能力(P0.01),促进Runx2、OC、Bmp2、Col1a1 mRNA的表达(P0.05)。结论续苓健骨汤含药血清能促进成骨细胞MC3T3-E1的增殖,并通过上调骨形成相关基因Runx2、OC、BMP2、Col1a1的表达水平,提高MC3T3-E1细胞的成骨能力。     

抑制核纤层蛋白A/C表达对牵张刺激下成骨细胞增殖及凋亡的影响

目的 观察小干扰RNA技术(siRNA)抑制核纤层蛋白A/C(lamin A/C)表达对牵张刺激下小鼠前成骨细胞MC3T3-E1增殖及凋亡的影响.方法 化学合成3条针对lamin A/C靶点的siRNA序列,脂质体转染MC3T3-E1细胞,实时定量聚合酶链反应(PCR)和Western blot检测lamin A/C mRNA和蛋白变化.细胞转染72 h后行牵张刺激6 h,Hoechst 33258检测DNA含量来反映细胞增殖,流式细胞术检测细胞周期及凋亡.结果 筛选出的siRNA-2显著抑制lamin A/C mRNA和蛋白产物表达(P＜0.01).未转染细胞经牵张刺激,DNA合成随时间推移而增加;细胞G0/G1期比例为65.19%;S期为22.57%,细胞凋亡率为11.49%;转染细胞经牵张刺激后,DNA合成较未转染细胞显著性减少(P＜0.01);G0/G1期比例提高至85.82%;S期降低至11.37%,凋亡率达19.32%(P＜0.01).结论 抑制lamin A/C表达会导致牵张刺激诱导的促增殖作用减弱,细胞周期阻滞于G0/G1期,促进细胞的凋亡.     

BMP receptor ALK3 controls collecting system development

The molecular signals that regulate growth and branching of the ureteric bud during formation of the renal collecting system are largely undefined. Members of the bone morphogenetic protein (BMP) family signal through the type I BMP receptor ALK3 to inhibit ureteric bud and collecting duct cell morphogenesis in vitro. We investigated the function of the BMP signaling pathway in vivo by generating a murine model of ALK3 deficiency restricted to the ureteric bud lineage (Alk3(UB-/-) mice). At the onset of branching morphogenesis, Alk3(UB-/-) kidneys are characterized by an abnormal primary (1 degrees ) ureteric bud branch pattern and an increased number of ureteric bud branches. However, during later stages of renal development, Alk3(UB-/-) kidneys have fewer ureteric bud branches and collecting ducts than wild-type kidneys. Postnatal Alk3(UB-/-) mice exhibit a dysplastic renal phenotype characterized by hypoplasia of the renal medulla, a decreased number of medullary collecting ducts, and abnormal expression of beta-catenin and c-MYC in medullary tubules. In summary, normal kidney development requires ALK3-dependent BMP signaling, which controls ureteric bud branching.     

Wachstumshormone und BMP

BMP (bone morphogenetic proteins) belong to the TGFβ superfamily and appear to play a central role in fracture healing. Two growth factors have been in use since 2001 as an approved therapy option for the delayed healing of fractures or open lower leg fractures: BMP-7 for delayed fracture healing and BMP-2 for open lower leg fractures. The aim of the present study was to examine the effectiveness of the BMP in fracture healing and compare fracture healing without the use of bone growth factors. Patients who were treated with BMP-7 following repeated attempts to treat atrophic pseudoarthrosis of the tibia were compared with a large standardized collective of patients with first-time spongiosa plastic. Patients receiving BMP-7 showed significantly better bone fusion in the fracture area at radiological examination at four months. In order to prove the advantages of this procedure, as well as to be able to recommend it as a treatment modality, further studies on larger patient collectives and prospective control groups are needed.     

Smad3-deficient chondrocytes have enhanced BMP signaling and accelerated differentiation.

Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF-beta signaling factors, but BMP-related gene expression is increased. Smad2 or Smad3 overexpression and BMP blockade abrogate accelerated maturation in Smad3-/- chondrocytes. BMP signaling is increased in TGF-beta deficiency and is required for accelerated chondrocyte maturation. INTRODUCTION: Disruption of TGF-beta signaling results in accelerated chondrocyte maturation and leads to postnatal dwarfism and premature osteoarthritis. The mechanisms involved in this process were studied using in vitro murine chondrocyte cultures. MATERIALS AND METHODS: Primary chondrocytes were isolated from the sterna of neonatal wildtype and Smad3-/- mice. Expressions of maturational markers, as well as genes involved in TGF-beta and BMP signaling were examined. Chondrocytes were treated with TGF-beta and BMP-2, and effects on maturation-related genes and BMP/TGF-beta responsive reporters were examined. Recombinant noggin or retroviral vectors expressing Smad2 or Smad3 were added to the cultures. RESULTS: Expression of colX and other maturational markers was markedly increased in Smad3-/- chondrocytes. Smad3-/- chondrocytes lacked compensatory increases in Smad2, Smad4, TGFRII, Sno, or Smurf2 and had reduced expression of TGF-beta1 and TGFRI. In contrast, Smad1, Smad5, BMP2, and BMP6 expression was increased, suggesting a shift from TGF-beta toward BMP signaling. In Smad3-/- chondrocytes, alternative TGF-beta signaling pathways remained responsive, as shown by luciferase assays. These non-Smad3-dependent TGF-beta pathways reduced colX expression and alkaline phosphatase activity in TGF-beta-treated Smad3-/- cultures, but only partially. In contrast, Smad3-/- chondrocytes were more responsive to BMP-2 treatment and had increased colX expression, phosphoSmads 1, 5, and 8 levels, and luciferase reporter activity. Overexpression of both Smad2 and Smad3 blocked spontaneous maturation in Smad3-deficient chondrocytes. Maturation was also abrogated by the addition of noggin, an extracellular BMP inhibitor. CONCLUSIONS: These findings show a key role for BMP signaling during the chondrocyte maturation, occurring with loss of TGF-beta signaling with important implications for osteoarthritis and cartilage diseases.     

BMP—2在成纤维细胞对BMP—3表达的调节作用

目的：观察在BMP－2作用下,成纤维细胞表达BMP－3的情况,探讨BMP－2促进成纤维细胞成骨表型表达的机制。方法：向培养的成纤维细胞内添加BMP－2,当细胞生长到60％时免疫组化观察正常与BMP－2作用下的成纤维细胞内表达BMP－3的情况。结果：下成纤维细胞内无BMP－3表达,在BMP－2作用下成纤维细胞出现BMP－3表达。结论：在成纤维细胞,BMP－2可促进BMP－3的表达。     

兔胫骨干骺端截骨延长延长区BMP、TGF—β1的蛋白表达

目的：研究肢体延长过程中延长区内源性BMP、TGF－β1蛋白表达情况。方法：采用兔胫骨干骺端截骨延长模型,通过免疫组化方法研究延长区内源性BMP、TGF－β1蛋白表达情况。结果：BMP、TGF－β1主要表达在软骨细胞、成骨细胞、成纤维细胞中。BMP在开始延长后前2周高度表达,2周后表达逐渐降低;停止延长1周时在延长区中央见到少量的阳性表达细胞,停止延长2周时,随着延长中央区域纤维组织消失,BMP阳性表达细胞也消失。TGF－β1在延长前2周表达逐渐增强,到2周时最强,2周后逐渐减弱,并在延长及停止延长期间均见阳性表达细胞。结论：肢体延长中延长区内源性BMP、TGF－β1持续表达。     

骨缺损中内源性BMP的分布及其作用

了解内源性ＢＭＰ的浓度及分布，对提高ＢＭＰ在骨再生中的作用有帮助。本实验在兔桡骨１０ｍｍ骨缺损模型上，利用ＢＭＰ免疫组化技术及真彩色计算机定量分析系统观察了内源性ＢＭＰ在骨缺损中的分布及对骨再生的作用。术后３日，骨端可见“空穴”，骨缺损间血肿ＢＭＰ染色阳性。来自骨内外膜的间质细胞，成软骨细胞。成骨细胞ＢＭＰ阳性。ＢＭＰ分布定量研究表明，骨缺损区存在ＢＭＰ浓度梯度，即距骨端愈远ＢＭＰ量渐渐下降。术后１周，ＢＭＰ值最大。提示内源性ＢＭＰ有骨端坏死，吸收及间质细胞分泌两种来源。据此，作者提出了内源性ＢＭＰ有效量的概念。提高内源性ＢＭＰ浓度，改善其分布，将是骨缺损治疗的有效途径。     

Mechanical Loading Stimulates BMP7, But Not BMP2, Production by Osteocytes

Bone mechanical adaptation is a cellular process that allows bones to adapt their mass and structure to mechanical loading. This process is governed by the osteocytes, which in response to mechanical loading produce signaling molecules that affect osteoblasts and osteoclasts. Bone morphogenic proteins (BMPs) are excellent candidates as signaling molecules, but it is unknown whether mechanically stimulated osteocytes affect bone adaptation through BMP production. Therefore, the aim of this study was to assess whether osteocytes produce BMPs in response to mechanical loading. In addition, since BMP7 has a vitamin D receptor (VDR) response element in the promoter region, we also investigated whether VDR is involved in the BMP7 response to mechanical loading. Human or VDR^−/− mouse primary bone cells were submitted in vitro to 1 h pulsating fluid flow (PFF) and postincubated without PFF (PI) for 1–24 h, and gene and protein expression of BMP2 and BMP7 were quantified. In human bone cells, PFF did not change BMP2 gene expression, but it upregulated BMP7 gene expression by 4.4- to 5.6-fold at 1–3 h PI and stimulated BMP7 protein expression by 2.4-fold at 6 h PI. PFF did not stimulate BMP7 gene expression in VDR^−/− mouse bone cells. These results show for the first time that mechanical loading upregulates BMP7, likely via the VDR, but not BMP2, gene and protein expression in osteocytes in vitro. Since BMP7 plays a major role in bone development and remodeling, these data might contribute to a better understanding of the mechanism leading to the mechanical adaptation of bone.