摘要
机械力学刺激是骨代谢的重要调节因素,适宜的牵张应力刺激可以在正畸牙移动、牵张成骨以及骨折修复等许多临床治疗中刺激骨组织自身的生长与重建,达到形成新骨的目的。成骨细胞是一种对应力敏感的细胞,来自多潜能的间充质干细胞,与骨基质形成及钙化密切相关,在骨形成的过程中起主要作用。因此,深入研究成骨细胞的力学信号转导机制对于阐明正畸矫治中骨改建的机理非常重要,但目前机械牵张应力刺激成骨细胞的力学信号转导机制尚不清楚,有待进一步研究。
研究发现,周期性的力学刺激比持续性的力学刺激更能促进成骨细胞的增殖与分化,而碱性磷酸酶(ALP)、骨桥蛋白(OPN)、骨钙蛋白(OC)等的合成以及矿化结节的形成都是成骨细胞分化的经典标志物,它们共同增进细胞间的连接和信息的传递,促进成骨细胞的成熟。最近有研究表明,Wnt信号转导通路的受体Lrp5缺失会引起骨质疏松,而获得性Lrp5突变会导致骨硬化症,即Wnt信号通路对于骨骼发育以及骨量的维持和调控都非常重要。β-catenin是该通路的核心因子,它的表达与变化受到细胞内外许多激素和蛋白的调节,来共同发挥与成骨相关的信号转导的作用。C3H1OT1/2细胞系过度表达β-catenin蛋白会引起成骨细胞的早期分化标志ALP的表达及活性增加,这说明β-catenin在成骨前体细胞及成骨细胞增殖分化过程中发挥着重要作用,而给Lrp5转基因小鼠的长骨进行四点弯曲加载可以上调Wnt/β-catenin通路目标基因的表达,这提示Wnt/β-catenin信号通路在成骨细胞对牵张应力加载的反应过程中可能发挥着重要作用。
近年来,蛋白质组学的研究方法在对蛋白表达谱的检测和定量、翻译后修饰的识别,以及蛋白质之间相互作用等方面取得了重要进展,而机械应力对成骨细胞的影响正是通过多因子多通路的相互作用构成的网络来控制的,传统的研究手段只能针对某一种因子或蛋白质,无法全面地呈现参与这个过程的所有蛋白质,因此,蛋白质组学为我们提供了一个新的研究成骨细胞力学信号转导机制的平台。
本研究通过Flexcell牵张应力加载系统,对体外培养的人成骨样细胞系Saos-2细胞加载机械牵张力,拟采用免疫组化、RT-PCR、免疫印迹、蛋白质双向电泳、生物质谱等方法观察机械牵张应力对成骨细胞细胞增殖及骨特异性分化指标的影响;研究机械牵张应力对成骨细胞Wnt/β-catenin信号通路的影响以及β-catenin在力学信号转导过程中的定位和作用;获取机械牵张应力刺激成骨细胞后的蛋白质双向凝胶电泳图谱,并对差异蛋白质点进行质谱鉴定。研究内容如下:
1.机械牵张应力对成骨细胞增殖和分化的影响
目的:研究机械牵张应力对成骨细胞增殖和分化能力的影响。方法:人成骨样细胞系Saos-2购自美国培养物保存中心(ATCC号:HTB-85),采用Flexcell牵张应力加载系统,分别用不同大小应变值的张应力(0%,6%,12%,24%)以及不同的刺激时间(0h,4h,8h,12h,24h)对成骨样细胞Saos-2进行力学刺激。用MTT法检测细胞受力后的增殖变化;用ALP试剂盒检测细胞受力后ALP活性的变化;用半定量RT-PCR检测骨钙蛋白(OC)、骨桥蛋白(OPN)的mRNA的表达;在这个过程中进行力学刺激,分别在第10天和第20天,用茜素红染色观察矿化结节形成。结果:当应变值为12%时,力学刺激对成骨样细胞Saos-2增殖以及ALP活性的促进作用最强(P<0.01)。应变值为12%的力学刺激呈时间依赖性明显上调了成骨样细胞Saos-2的增殖、ALP活性以及骨桥蛋白和骨钙蛋白mRNA的表达(P<0.01);茜素红染色结果显示,牵张应力刺激组较对照组更加容易形成矿化结节。结论:大小适宜的牵张应力可以促进成骨细胞的增殖以及分化指标如ALP活性、骨桥蛋白和骨钙蛋白mRNA的转录水平、矿化结节的形成等,说明机械牵张应力对于成骨细胞的增殖和分化发挥着重要的调控作用。
2.机械牵张应力对成骨细胞Wnt/β-catenin信号通路的影响
目的:研究机械牵张应力对成骨细胞Wnt/β-catenin信号通路的影响以及β-catenin在力学信号转导过程中的定位和作用。方法:采用Flexcell牵张应力加载系统,用12%大小的应力值对成骨样细胞Saos-2进行力学刺激。作用1h、2h和4h后,用免疫荧光的方法检测β-catenin的表达和在细胞上定位的变化;用半定量RT-PCR检测β-catenin mRNA表达的变化;用质粒转染的方法检测荧光素酶报告基因Tcf转录的情况;用半定量RT-PCR检测Wnt/β-catenin通路下游基因COX-2,cyclinD1, c-fos, c-Jun mRNA表达的情况;用免疫共沉淀的方法检测β-catenin和E-Cadherin的结合情况。结果:对照组的β-catenin主要表达于胞膜,在牵拉刺激1h、2h和4h后β-catenin大量聚集于胞核,核内β-catenin荧光强度明显强于对照组,而各实验组间无显著性差异;实验组与对照组之间β-catenin mRNA表达的水平没有显著性差异(P>0.05);细胞受力后细胞Tcf报告基因的转录活性与对照组相比显著提高(P<0.01);实验组的四种下游基因COX-2,cyclinD1, c-fos, c-Jun的mRNA表达水平均较对照组有显著提高(P<0.01);实验组β-catenin和E-Cadherin的蛋白质水平与对照组相比都没有显著改变(P>0.05),但是β-catenin与E-Cadherin的结合水平显著降低(P<0.01)。结论:12%牵张应力加载会引起成骨细胞内β-catenin的核移位,刺激核内转录因子Tcf的表达,激活Wnt/β-catenin信号通路,促进下游目的基因转录水平的提高,成骨细胞胞膜上的β-catenin/E-Cadherin复合体在牵张力的作用下解聚并造成β-catenin入核则可能是机械信号转导的内在机制之一。
3.机械牵张应力刺激成骨细胞的蛋白质双向凝胶电泳
目的:研究机械牵张应力刺激成骨细胞后蛋白质双向凝胶电泳表达的差异。方法:采用Flexcell牵张应力加载系统,用12%大小的应力值对成骨样细胞Saos-2进行力学刺激24h后,进行细胞裂解,Bradford法蛋白质定量,蛋白质双向凝胶电泳,凝胶硝酸银染色,并使用凝胶分析软件ImageMaster 2D对蛋白点进行分析。结果:对照组和加力组分别得到1031±41和928±25个蛋白质点。总蛋白质点在细胞受到牵张力后有所减少。两组蛋白电泳基本类似,蛋白点集中分布于pI 6 -10间的区域,通过软件对差异蛋白点的光密度、点大小以及三维观察等作图像分析,比值大于2或小于0.5被认为是差异蛋白点,共检测出30个差异表达蛋白点。结论:通过蛋白质双向凝胶技术结合硝酸银染色可以得到分辨率较高,可重复性较好的成骨细胞牵张应力加载的差异表达蛋白质图谱,为进一步进行质谱鉴定奠定了良好的基础。
4.机械牵张应力刺激成骨细胞差异蛋白质点的质谱鉴定与分析
目的:获得差异蛋白点的肽质量指纹图谱后进行质谱鉴定,根据鉴定结果推测这些差异蛋白质在成骨细胞受到机械牵张应力后所发挥的作用。方法:蛋白质原位酶解后制备样品,样品脱盐,使用MALDI-TOF质谱仪进行分析,将获得的蛋白质肽质量指纹数据通过Mascot搜索引擎在NCBI数据库中进行检索,并确定出蛋白质的各种信息。结果:送检30个差异蛋白点经MALDI-TOF质谱获得了肽质量指纹图,进入蛋白数据库鉴定出26个相应蛋白质,按照功能分类大致与以下生物活动相关:应激反应,能量代谢,细胞增殖,细胞骨架重建,信号转导及成骨分化。结论:成骨细胞对机械牵张应力的反应是一个非常复杂的过程,牵张应力会引起成骨细胞应激反应,能量代谢,细胞增殖,细胞骨架重建,信号转导及成骨分化等许多方面的改变,每一种生物学功能的实现都涉及到很多种蛋白质的参与和调控。这些鉴定结果为以后研究这些差异蛋白在正畸骨组织改建中的生物学功能确定了目标。
The skeleton is a very complex tissue which can regulate its mass and architecture to adapt to functional environment. Orthodontic tooth movement results from alveolar bone remodeling process induced by mechanical strain. Osteoblasts, osteocytes and osteoclasts are specialized cells that orchestrate the process. As the osteoblast is highly sensitive to mechanical stimuli,it plays the leading role in the bone remodeling process. However,the mechanism of how these mechanical load is translated into intracellular signals is not very clear.
In order to better understand the mechanotransdution process , we investigated proliferation and biological characteristics of osteoblasts under mechanical strain. Osteoblasts secrete alkaline phosphatase (ALP), osteopotin(OPN) and osteocalcin (OC) which involve in bone modeling. Recently, Wnt/β-catenin signaling has been shown to play a particularly crucial role in bone formation, bone growth and remodeling. Many studies have shown canonical Wnt/β-catenin signaling could promote osteoblast differentiation, proliferation, and mineralization. The central player of Wnt signaling isβ-catenin, which is a transcription cofactor with Tcf/Lef in the Wnt pathway and a structural adaptor protein linking cadherins to the actin cytoskeleton in cell-cell adhesion. The cytoplasmic accumulation ofβ-catenin leads toβ-catenin entering the nucleus and heterodimerizing with Lef/Tcf transcription factors to regulate Wnt target genes including COX-2, c-myc, cyclin D1 and so on. Previous studies have shown that Wnt/β-catenin signaling is a component of osteoblastic cell early responses to load-bearing.
On the other hand, a proteomics approach,in which entire proteins in tissue or cells are identified and quantified directly,has been shown to be a valuable way to bring insight into the molecular basis of signal transduction. Compare the proteins of the osteoblasts in different conditions: mechanical strained and unstrained group,which will help us to find the key signal transduction protein and understand the responsive mechanism of the osteoblasts under the mechanical strain. Therefore in the study of this part, we quantitatively and qualitatively analyzed the differentially expressed proteins in Saos-2 osteoblastic cells of 24h after mechanical strain loading by two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF-MS), computer informatics and computer network communications technology. The aim of our study was to elucidate the molecular mechanisms of osteoblast signal transduction under mechanical strain.
In the present study, Saos-2 osteoblastic cells were subjected to mechanical strain using Flexcell strain loading system and observed the effects of mechanical strain on cellular proliferation, differentiation. In addition, Wnt/β-catenin signal transduction pathway that mediated cellular responses to mechanical strain was investigated using different biological techniques. Furthermore, proteins from samples in the Saos-2 osteoblastic cells obtained under mechanical strain for 24h were subjected to two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. The research works are as follows:
1. Effects of mechanical strain on proliferation and differentiation of Saos-2 osteoblastic cells
Objective: To investigate the effects of mechanical strain on proliferation and differentiation of Saos-2 osteoblastic cells. Methods: Saos-2 osteoblastic cells were subjected to 0%,6%,12%,24% elongation for 0h,4h,8h,12h,24h by using Flexcell strain loading system simultaneously. MTT colorimetric method was used to assess cellular proliferation;Biochemical analysis was used to study the effects of mechanical strain on alkaline phosphatase (ALP) activity;semi quatative RT-PCR was used to examine mRNA level of osteocalcin and osteopotin. Alizarin Red-S method was used to study the mineralization of Saos-2 osteoblastic cells. Results: The proliferation and ALP activity of Saos-2 osteoblastic cells were significantly increased after mechanical strain treatment,12% elongation rate showed the strongest stimulatory effects (P<0.01). The expression of OC,OPN mRNA and the formation of mineralization nodules in Saos-2 osteoblastic cells were promoted by 12% mechanical strain elongation rate. Conclusion: Our data indicated that mechanical strain stimulation increased the proliferation and differentiation markers such as ALP activity , mRNA expression level of OC and OPN,and the formation of mineralization nodules in Saos-2 osteoblastic cells. Mechanical strain may play a very important role in proliferation and differentiation of osteoblasts.
2. The role of Wnt/β-catenin signaling in mechanotransduction of Saos-2 osteoblastic cells
Objective:To investigate the effects of mechanical strain onβ-catenin in Saos-2 osteoblastic cells and to examine whether mechanical stimuli modulate Wnt/β-catenin signaling and its mechanism. Method:Saos-2 osteoblastic cells were subjected to 12% elongation by using Flexcell strain loading system. Cells were immediately fixed and processed for immuno?uorescence and tested for translocation ofβ-catenin.β-catenin and target genes of Wnt/β-catenin signaling COX-2, cyclinD1, c-fos, c-Jun mRNA expression were examined by RT-PCR. Saos-2 osteoblastic cells were co-transfected with a wild type or mutant Tcf luciferase reporter gene and Renilla luciferase plasmid. Posttransfection cells were subjected to 1 hour of 12% elongation. Luciferase activities were then assayed.β-catenin and E-Cadherin activities were determined by Western Blot analysis. The interaction betweenβ-catenin and E-Cadherin was determined by Co-immunoprecipitation. Result: In cells subjected to mechanical strain, there appeared to be more nuclearβ-catenin than in cells held in static culture. Mechanical strain induced a significant increase in Tcf reporter gene activity (P<0.01). The expression of target genes COX-2, cyclinD1, c-fos, c-Jun mRNA in Saos-2 osteoblastic cells were promoted by 12% mechanical strain elongation (P<0.01). Co-immunoprecipitation analysis revealed less E-cadherin associated withβ-catenin in cells subjected to mechanical strain (P<0.01). Conclusion:Our data indicate that 12% elongation mechanical strain can activate Wnt/β-catenin signaling and decreases the interaction betweenβ-catenin and E-Cadherin in Saos-2 osteoblastic cells. Wnt/β-catenin signaling plays an important role in mechanotransduction of osteblasts.
3. Two-dimensional electrophoresis analysis of Saos-2 osteoblastic cells stimulated by mechanical strain
Objective: To establish two-dimensional eletrophoresis (2-DE) images for Saos-2 osteoblastic cells stimulated by mechanical strain. Methods: Saos-2 osteoblastic cells were subjected to 12% elongation for 24 hours by using Flexcell strain loading system. Cells were immediately lysated and the total protein of the Saos-2 osteoblastic cells was extracted. Total protein content was determined in cell cultures lysates using Bradford protein assay. 2-DE was carried out according to procedures. The silver stained 2-DE were scanned and analyzed with ImageMaster 2D software. Results: Saos-2 osteoblastic cells stimulated by mechanical strain showed significant difference in a 2-DE system compared with control group. A total of 1031±41or 928±25 protein spots were resolved by 2-DE of controls or experimental groups extractions,respectively. We found 30 statistically significant change amount of protein compared with controls samples. Conclusion: The Saos-2 osteoblastic cells stimulated by mechanical strain could express the differentially expressed proteins, and 2-DE technique is effective to investigate mechanics of osteoblasts in bone remodeling.
4. MALDI-TOF-MS analysis of differential expression protein in Saos-2 osteoblastic cells stimulated by mechanical strain
Objective: To identify the differentially expressed proteins of Saos-2 osteoblastic cells under mechanical strain loading and to clarify the major proteins involved in the molecular mechanism of osteoblasts under mechanical strain loading. Methods: Matrix-assisted laser-desorprtion ionization time of flight mass spectrotmetry (MALDI-TOF-MS) were used to separate and identify proteins in the whole cell lysate and peptide mass fingerprinting (PMF).The proteins involved in Saos-2 osteoblastic cells under mechanical strain loading were identified and searched with proteome NCBI database. Result: The PMFs of 30 proteins were obtained through MALDI-TOF-MS analysis and 26 proteins were further analyzed with the protein and peptide databases. These bone remodeling associated proteins fell into 6 groups, including Stress reaction (cyclophilin, Chain A, Dopamine Quinone Conjugation To Dj-1), energy metabolism(mitochondrial ATP synthase, Alpha-enolase), cell proliferation( NUMA1, peroxiredoxin 1), reconstruction of cytoskeleton(Cofilin 1), signaling(Protein phosphatase 1, Nuclear receptor interacting protein 1, Proteasome Activator) and osteogenesis(Elongation factor 2, eukaryotic initiation factor 2, Annexin I). Conclusion: Our study provided fundamental information on the differential expression of proteins of osteoblast under mechanical strain. These proteins described here may play important roles in mechanisms of biological bone remodeling process.
引文
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