【作者】 景达；

【导师】 罗二平； 郭向东； 申广浩；

【作者基本信息】 第四军医大学 ， 生物医学工程， 2013， 博士

【摘要】 骨质疏松症（OP）是最为常见的临床疾病之一，以骨量下降和骨的微细结构破坏为特征，表现为骨的脆性增加和骨折风险的大大提高。OP的诱发因素众多，以衰老、性激素缺乏、应力刺激不足、营养不良、糖尿病所诱发的OP最为常见。随着我国人口老龄化速度的加剧，OP给我国带来的经济和社会负担也逐年加重。因此，只有对骨骼及其内部细胞的生物学特征进行更全面而深入的认识，才能进一步深入了解OP的发生机制，为临床治疗OP提供更科学而有力的依据。应力和电磁场是骨骼在日常活动中所承受的最常见的两种物理因子，它们对于机体的骨骼代谢及骨健康状态有着重要的影响。首先，骨骼是机体的应力承载系统，它能够调整和改变自身的内部结构以适应外界应力刺激的变化（Wolff定律）。而骨骼上的应力负荷不足或骨骼自身应力响应能力的衰退是诱发OP的主要原因之一。但是，骨适应原理的具体机制目前尚不明确。骨是以何种形式将外界的应力信号传递至骨骼内部的细胞的，骨骼内的细胞又是如何感应这些刺激并将其转导为细胞内部的生物化学信号的，这些骨力学信号转导研究领域的重要的疑问目前仍不明确。其次，骨骼处于复杂的电磁微环境中，而外界应力也会在骨骼内产生电磁信号的改变（骨骼“压电效应”），Bassett于1977年首次将外源性的脉冲电磁场（PEMF）引入临床成功的治疗骨不连，并预测外源性的电磁刺激可能作为一种经济、无创、副作用小的物理治疗方法用于临床OP的治疗。但是PEMF作用的骨骼内的靶细胞是什么，其具体作用的信号通路传导机制是什么，目前仍不明确。本研究首先以细胞中最重要的第二信使分子—钙信号作为突破口，发现了应力刺激下体外骨细胞（OCY）网络具有独特的多钙尖峰钙信号响应特性，并且OCY网络的应力敏感性要远高于成骨细胞（OB），这一发现为证实OCY网络是骨骼中感受应力刺激的核心组件提供了有力的依据。其次，我们对分离培养的胫骨组织进行动态载荷刺激，观察原位OCY网络的钙信号动力学特征，我们在国际上首次发现了原位OCY响应应力刺激的多钙尖峰特性，而骨陷窝—小管系统中的组织液流动是诱发OCY信号响应的主要内在驱动力，而OCY网络正是通过组织液流动所诱发的钙信号来响应和处理外界的应力刺激的。第三，我们分别构建去势（高骨转换）及胰岛素依赖型糖尿病（低骨转换）的OP动物模型，施加外源性的PEMF刺激以观察其作用效果及相关的机制。PEMF对于两种因素诱发的骨丢失都有很显著的缓解作用，PEMF对于骨形成的促进作用明显，而对于骨吸收的作用不显著，而OB的Wnt/LRP5/β-catenin信号通路则是PEMF发挥其作用的主要信号通路。该研究不仅极大的丰富了我们对骨骼力学信号转导和电磁信号转导及其机制的认识，并且为应力和电磁刺激在临床抗OP的应用提供了重要的实验依据。整个研究分为以下三个部分：第一部分：流体剪切力诱发的体外骨细胞网络钙信号响应时空动力学特征研究背景：OCY位于充满组织液的骨陷窝—小管系统（LCS）中，它们凭借独特的细胞突触彼此相连形成骨骼内广阔的OCY网络，科学家们推测这个骨基质内广阔的OCY网络是响应外界力学刺激的主要组件。但是，目前仍未见对于OCY和骨骼中其它细胞（如OB）力学敏感性的系统的比较和研究。方法：采用微接触压印和自组装单分子表面化学技术构建体外OCY和OB细胞网络，细胞表面施加流体剪切力，实时观察两种细胞网络的钙信号响应；开发基于独立成分分析（ICA）的细胞网络钙信号自动提取算法提取两种细胞网络钙信号，并基于该算法分析细胞网络钙信号时空动力学特征。结果：流体剪切力诱发OCY细胞网络产生独特的多钙尖峰钙信号响应，而大多数OB主要表现为单一的钙主峰；同等强度剪切力作用下，OB的钙响应尖峰数量显著低于OCY；OCY和OB的钙信号时空特征参数随应力强度的增加线性增加；OCY细胞网络与OB细胞网络在响应和处理应力刺激所表现的钙信号时空动力学特征显著不同，OCY细胞网络比OB细胞网络具有更显著的时间域钙信号振荡特征和更高的空间域钙信号相关性和钙尖峰同步性，且其空间同步性依赖于细胞的间隔距离；相比传统的手动钙信号分析法，我们开发的基于ICA的自动分析算法节省了巨大的工作量，且准确性更高，该算法具有在钙信号分析研究领域广阔的应用前景。结论：OCY细胞网络比OB细胞网络具有更高的应力响应敏感性，最直接的证实了OCY细胞网络是响应外界力学刺激的核心组件。第二部分：小鼠胫骨周期载荷作用下原位骨细胞钙信号响应及其作用的相关机制背景：OCY位于独特的LCS微环境中，骨是如何将外界应力转为细胞水平的应力信号的，OCY是如何感受力学信号并将其转导为细胞内的生物化学信号的，目前仍不得而知。深入探究这些问题，对于更全面、深入的了解OCY力学信号转导和骨适应的机制具有重要的意义。方法：离体培养小鼠胫骨，使用自行开发的应力加载系统施加生理水平应力，采用激光扫描共聚焦采集和应力加载同步技术实时观测胫骨内OCY钙信号响应，有限元分析结合微应变片测量技术测定骨表面的应变变化，荧光漂泊恢复技术结合计算模型分析骨陷窝—小管系统中液体流速，使用钙信号通路拮抗剂分析OCY钙响应的作用机制。结果：OB具有自发的钙信号响应，OCY基本没有自发的钙响应。应力刺激诱发原位OCY产生多钙尖峰的钙信号响应，OB对应力作用不敏感；骨LCS中的液体流速随应力线性增加，且细胞突触流速显著高于细胞体；OCY钙信号特征参数与组织间隙液流速线性相关；细胞内质网钙库和ATP相关的P2R/PLC/IP3信号通路在OCY钙信号响应中发挥主要作用。结论：外界应力以骨LCS中的间隙组织液流动的形式作用于OCY，OCY网络通过间隙组织液流动诱发的独特的多钙尖峰钙信号来响应和处理外界的应力刺激，细胞外ATP及其相关的细胞内P2R/PLC/IP3通路是OCY钙响应最重要的信号通路。该研究揭开了原位OCY力学信号转导的研究序幕，为证实“OCY是骨骼中最主要的应力信号响应元件”这一猜想提供了最直接而有力的证据。第三部分：脉冲电磁场对骨质疏松模型大鼠骨丢失的抑制作用及机制研究背景：骨中细胞的生物学特征受到周围电磁环境的影响，而外源性PEMF刺激可以显著促进骨生成。一些临床研究表明PEMF能够抑制OP患者的骨丢失。但PEMF对于OP的作用效果仍缺乏系统的研究，PEMF在骨中的作用靶点及其相关的信号传导机制目前仍不清楚，这些限制了其科学而广泛的临床应用。方法：设计一套多波形输出的、“3-HelmHolz线圈”电磁场发生系统，分别构建去势和胰岛素依赖型糖尿病大鼠OP模型，分别评价大鼠体质量、反映骨代谢的血清生物化学指标、骨生物力学强度、骨微观结构、骨骼中Wnt、Lrp5、β-catenin、RANKL和RANK的mRNA的表达。结果：PEMF对去势及糖尿病大鼠骨量丢失及小梁骨微结构衰退具有显著的抑制作用，并能改善骨的生物力学性能。血清生化指标分析表明，PEMF能够调控去势及糖尿病大鼠的骨生成行为，但对骨吸收的作用不明显。同时，PEMF能够显著提高其骨骼中的Wnt1，LRP5及β-catenin的mRNA水平，但是对RANKL和RANK表达的影响不明显。结论：PEMF作为一种经济、无创、副作用小的物理治疗方法能够有效的抑制雌激素缺乏及胰岛素缺乏诱发两种机体代谢性失常诱发的骨量丢失，其主要的作用靶点极有可能是OB的Wnt/Lrp5/β-catenin信号通路。更多还原

【Abstract】 Osteoporosis is one of the most common diseases in clinics, characterized by significant bone mass loss and bone microarchitecture deterioration, leading to bone fragility and an increased risk of bone fractures. Lots of risk factors are able to cause the occurrence of osteoporosis, such as aging, deficiency of sex hormone, disuse, and diabetes mellitus etc. As the issue of aging populations is becoming even more serious in our country, the economic and social burdens resulted from osteoporosis are dramatically increased year by year. Therefore, understanding the etiology and exploring more effective therapeutic methods for osteoporosis carries great significance for both the economic and social development.Mechanical loading and electromagnetic fields are the most two common physical factors on the skeletal system, which play critical roles in mediating the bone metabolism and maintaining the bone health of the organism. First, the skeleton acts as the weight bearing organ of human body, and it can adjust and modify its structure in accordance with the stress placed upon it. This concept about bone adaptation was raised by Julius Wolff in1892. Either the insufficient loads or deteriorative response of skeleton to the mechanical stimulus can induce significant bone loss, which is also the major triggering factor for osteoporosis. However, the exact mechanism of bone adaptation is still elusive. Critical questions in bone mechanotransduction regarding how the external mechanical force signal is transformed into that at the cellular level, and how bone cells perceive the stimulation and transduce the physical signals into the intracellular biochemical signals are still unknown. Second, the skeleton is exposed to a complex electromagnetic environment, and the external changing stress can also induce electric and magnetic effects inside the bone microenvironment (piezoelectric effect of bone). In1977, an American scientist Andrew Bassett for the first time applied the exogenous pulsed electromagnetic fields (PEMF) in clinics for the treatment of the nonunion of human tibiae after bone fractures. The exciting and positive therapeutic effects of PEMF were found in his study, and he hypothesized that PEMF might become an easy and noninvasive physical methods for the treatment of osteoporosis in clinics. Although the inhibitive effects of PEMF on osteoporosis have been confirmed by several clinical and experimental studies, the exact mechanisms are still unclear. The major concerns about the target bone cells which PEMF functions and the related exact mechanism of the signal transduction are still unknown to date.In this thesis, we first found that calcium (Ca2+) signaling, a pivotal and ubiquitous second messenger regulating many downstream cellular activities, displayed unique Ca2+oscillations with multiple and robust Ca2+spikes in osteocyte-like MLO-Y4cell networks under fluid flow stimulation, which were more responsive and sensitive than osteoblast networks in response to the fluid shear. These findings provide novel and direct evidence that osteocyte networks act as the major ’mechanical sensor’ in bone. Second, we developed a novel protocol for in situ Ca2+probing and imaging, synchronization technique between controlled mechanical loading and confocal imaging to enable the accurate and systematic investigation of osteocytic Ca2+signaling in mouse long bone under dynamic physiological loading. We for the first time found the load-induced unique repetitive spike-like Ca2+peaks in osteocytes. Our results also indicated that lacuna-canalicular system (LCS) fluid shear act as the major inner driving force to trigger the cellular activities in osteocytes. Our data also provide direct evidence that osteocyte networks possess high sensitivity in detecting and processing mechanical stimuli through LCS fluid shear induced calcium signaling. Third, we applied PEMF stimulations on ovariectomy-induced (high bone turnover osteoporosis model) and diabetes-induced (low bone turnover osteoporosis model) osteoporotic rats, respectively. PEMF exhibited significantly preventive effects on both estrogen-deficient and diabetic bone mass loss, and bone quality deterioration. Furthermore, PEMF mainly regulated the bone formation rather than bone resorption for both two animal models, and the Wnt/β-catenin signaling pathway in osteoblast was the major mechanism for PEMF mediation on bone loss. Taken together, our studies greatly enrich our basic knowledge to the mechanisms of signaling transduction in bone cells in response to the stimulations of physical factors, and also provide critical evidence for the application of mechanical and PEMF stimulations in clinics for inhibiting osteoporosis. The thesis includes three parts:Part I:The spatiotemporal dynamics of Ca2+signaling in osteocytic and osteoblastic networks under fluid shear stimulationBackgrounds. Osteocytes, residing in the LCS microenvironment and forming extensive cell networks in bone matrix, are regarded as the major mechanosensors in bone. However, few studies to date are able to provide direct evidence to confirm this hypothesis, and no study has ever systematically compared the mechanical sensitivity between osteocytes and other major cell types in bone(e.g. osteoblasts).Methods. A novel two-dimensional patterned bone cell network was constructed to mimic the elaborate in vivo osteocytic network topology using microcontact printing and self-assembled monolayers techniques. Osteocytic and osteoblastic cell networks were respectively stimulated under physiological related fluid shear (0.5-4Pa), and real-time Ca2+signals were recorded. A set of novel unsupervised Ca2+signaling analysis algorithm based on independent component analysis (ICA) was developed to extract the Ca2+signaling, and the spatiotemporal characteristics in bone cell networks were also systematically analyzed.Results. Osteocytes in the network displayed unique highly repetitive spikelike Ca2+peaks under fluid shear, whereas most osteoblasts in the network only exhibited one major Ca2+peaks at the onset of fluid shear. Ca2+spikes in osteocytes were more repetitive and robust than that in osteoblasts in response to fluid flow. The Ca2+oscillatory nature in osteocytes and osteoblasts was highly positively correlated with the fluid flow levels. The present study also revealed a dramatic spatiotemporal difference in Ca2+signaling for osteocytic and osteoblastic cell networks in processing the mechanical stimulus. Osteocytes exhibited dramatically higher intracellular Ca2+oscillatory behaviors and intercellular Ca2+coordination than osteoblasts. The spatial intercellular synchronous activities of Ca2+signaling in osteocyte cell networks were also negatively correlated with the intercellular distance. Also, the ICA-based technology yield higher signal fidelity and save much more human efforts than the manual region of interest (ROI) method, with the potential to be widely employed in the Ca2+signaling studies.Conclusion. Osteocytes possess much higher mechanical sensitivity than osteoblasts in detecting and processing the external mechanical loading signals. Osteocytes’ unique living microenvironment and high sensitive nature to fluid shear endow the capacity of them to act as the major mechanosensing system in bone. This study provides evidence that osteocytes are qualified as a critical coordinator in bone modeling and remodeling process.Part Ⅱ:Ex vivo Ca2+oscillations in live osteocytes in intact mouse long bones under dynamic mechanical loading and the related mechanismsBackgrounds. Osteocytes are encapsulated in a fluid-filled mineralized bone matrix. Questions pertaining to how external mechanical signals are transformed into the cell levels and how osteocytes decode these signals and transduce them into the intracellular biochemical signals are still unknown. Systematic and comprehensive investigations for the mechanisms of oseocyte mechanotransduction and bone adaptation carry great scientific and clinical significance.Methods. We designed an ex vivo murine tibia mechanical loading system. Mouse tibiae were sterilely dissected and cultured. A synchronization technique between controlled mechanical loading and confocal imaging was developed to avoid the focus shift during mechanical loading. The combination with both strain gauge and μCT based finite element analysis enables accurate measurement of load-induced resultant strain on the bone surface. The shear stress on osteocytes in the lacuna-canalicular system (LCS) was measured and estimated via a combination of fluorescence recovery after photobleaching (FRAP) imaging and LCS modeling techenique. Furthermore, various pathway inhibitors essential for Ca2+signaling were employed to evaluate the exact mechanisms of in situ osteoycte Ca2+oscillations.Results:Autonomous Ca2+responses with1-2robust spikes were found in13%osteoblasts, but only in1.3%osteocytes. Mechanical loading didn’t induce significant changes in osteoblasts. However, osteocytes reacted to the mechanical loading with unique Ca2+oscillations with multiple and robust Ca2+spikes. Cyclic mechanical loading could induce fluid shear through the bone LCS, which was also highly positively correlated with the tissue strains. The load-induced fluid shear on the cell dendrites was significantly higher than that on the cell body. All Ca2+dynamic parameters were highly positively correlated with the tissue strains, as well as with the LCS shear stress. Pathway studies showed that the intracellular Ca2+store ER and ATP-related signaling pathway played major roles in osteocytic Ca2+signaling.Conclusion:Interstitial fluid flow act as the inner driving force by converting the external load signals to the intracellular signaling responses in osteocytes. Osteocyte networks process mechanical stimuli through fluid flow induced uniqe Ca2+signaling with multiple Ca2+spikes. Extracellular ATP and its related P2R/PLC/IP3signaling pathway play major roles in regulating the Ca2+signaling in osteocytes.Part III:In vivo investigation for the inhibitive effects of pulsed electromagnetic fields on bone loss in osteoporotic rats and the related mechanismsBackgrounds. Bone cells are exposed to a complex electric and magnetic environment, and their biological functions are also regulated by the electromganetic fields. Substantial and growing evidences have proven that exogenous PEMF stimulation was able to promote osteogenesis both in vivo and in vitro. Several clinical trials also demonstrated that PEMF could significantly inhibit bone loss in patients with OP. However, systematic and scientific investigations for the effects of PEMF on OP are still lacking, and the therapeutic target of PEMF on bone cells and its related mechanism of the signaling pathway transduction is still unknown. These issues all limit the widely and scientific applications of PEMF in clinics for the treatment of OP.Methods. We designed a PEMF exposure system, which was composed of a signal generator together with a Helmholz coil assembly with three-coil array. Ovariectomy-induced and type I diabetes-induced OP models were established, respectively. The parameters including the body mass, serum biochemical indices reflecting bone turnover, bone mechanical strength, microstructure of trabecular and cortical bone, and mRNA expression of Wntl, Lrp5, β-catenin, RANK and RANKL were analyzed to eveluate the inhibitive effects of PEMF on estrogen-deficient and diabetic OP.Results:PEMF was capable of significanly inhibiting bone mass loss and trabecular microstructure deterioration induced by either ovariectomy or type I diabetes in rats, as well as improving the mechanical strength of bone. The serum biochemical analysis results showed that PEMF was able to regulate the bone formation activities in both of bone loss animal models, whereas showed minor mediation roles in the bone resorption. Furthermore, PEMF dramatically increased the gene expression of Wntl, LRP5and β-catenin in the rat tibia, and showed no obvious effects on RANKL and RANK levels in both OP animal models.Conclusion:PEMF, as an easy, safe and non-invasive physical treatment method, has demonstrated its great potential to reverse both the estrogen-deficient and insulin-deficient bone loss. The major regulatory target of PEMF in bone is probably the Wnt/Lrp5/β-catenin signaling pathway in OB.更多还原