Ⅰ型BMP受体介导的BMP信号在骨重塑中的作用及机理研究

发布时间：2018-08-07 08:24

【摘要】：骨骼是人体内最重要的器官之一,其功能包括运动、支持和保护身体、造血、储存矿物质等。然而,在衰老、骨折、骨质疏松,以及由骨肿瘤、炎症等各种原因引起的骨吸收破坏等情况,骨骼将丧失这些功能。因此维持骨的健康状态至关重要。骨组织通过骨重塑(bone remodeling)过程,即成骨细胞性骨形成和破骨细胞性骨吸收来维持骨量。因此,明确骨重塑的分子机理与影响因素对维持骨的健康状态具有重要的理论意义。骨形成蛋白(bone morphogenetic proteins,BMPs)最初因其能诱导异位骨和软骨的形成而被发现。虽然美国食品药品管理局(Food and Drug Administration,FDA)已在2002年批准了重组人BMP-2(recombinant human bone morphogenetic protein 2,rh BMP-2)和rh BMP-7在临床上应用于长骨开放性骨折、骨折不愈合以及脊柱融合的治疗,然而其有效性不显著,通常导致骨吸收、假性关节形成以及局部炎症反应等。因此,阐明BMP信号对骨形成和骨吸收的作用及其分子机理,有望为改善骨的健康状态提供新的视角;并通过进一步调控BMP信号通路下游分子,提高应用BMPs促骨再生的疗效,这又对防治骨吸收破坏性疾病具有潜在的实际意义。在第1章中,我们通过文献复习,综述了骨重塑的生理和病理过程,介绍了BMP配体和受体及其介导的BMP信号通路,简述了BMP信号在骨重塑中的作用。为了研究BMP信号在骨重塑中的作用及其分子机理,我们靶向于Ⅰ型BMP受体。我们利用了基因修饰动物模型进行研究,其中包括条件性基因敲除和全基因敲除,在本章中又介绍了本研究中所利用的基因敲除小鼠的相关内容,尤其是条件性基因敲除小鼠模型。在第2章中,基于骨量和骨质量是决定骨的生物特性和机械性能的重要因素,我们旨在探讨由ACVR1或BMPR1A介导的BMP信号对骨量和骨质量的影响。我们通过在小鼠体内早期分化的成骨细胞前体细胞中分别敲除两种Ⅰ型BMP受体,Acvr1和Bmpr1a,采用micro-CT观察骨量,采用组织形态测量学观察骨小梁、成骨细胞和破骨细胞的改变,采用拉曼光谱分析骨组织成分的改变。我们发现成骨细胞特异性Acvr1或Bmpr1a基因敲除后,小鼠体内骨量增多,尤其以松质骨为著;与Acvr1基因敲除相比,Bmpr1a基因敲除导致更显著的松质骨的骨量升高。我们还发现这些基因敲除小鼠的松质骨的矿物质结晶度增高(crystallinity),胶原有序性增加(collagen order/disorder),然而密质骨的组织矿物质密度(tissue mineral density)和矿物质-基质比例(mineral-matrix ratio)下降。该研究提示了在成骨细胞中由ACVR1和BMPR1A介导的BMP信号是决定骨量和骨质量的重要因素。在第3章中,基于第2章的实验结果ACVR1和BMPR1A介导的BMP信号负性调控骨量,我们旨在探讨另外一种Ⅰ型BMP受体BMPR1B介导的BMP信号对骨重塑的调控作用。我们通过在小鼠体内敲除Bmpr1b基因,研究了其对成骨细胞和破骨细胞的影响。结果发现Bmpr1b基因敲除导致8w龄雄性小鼠骨量下降,此表型是一过性的,并且是性别特异性的。然而减少的骨量不是由成骨细胞性骨形成和破骨细胞性骨吸收的改变引起的。在体外研究中,Bmpr1b基因敲除的破骨细胞分化增加但是骨吸收功能降低;Bmpr1b基因敲除的颅骨前成骨细胞的分化没有改变,然而却表现出BMP刺激后BMP-SMAD信号的上调;与颅骨前成骨细胞不同的是,Bmpr1b基因敲除的骨髓间充质细胞的分化降低,这可能是引起Bmpr1b基因敲除小鼠骨量下降的原因。BMPR1B对不同细胞的不用作用可能是由于该受体在不同组织细胞中的表达模式引起的。结合以往对Ⅰ型BMP受体在骨重塑中作用的研究,我们还提出了BMP受体介导的BMP信号在成骨细胞分化过程中的作用模式,说明不同受体在不同阶段所发挥的作用不同。该研究提示了BMPR1B在维持骨量和转导BMP信号中的作用与BMPR1A或ACVR1不同。在第4章中,基于我们在第3章中提出的BMP受体介导的BMP信号在成骨细胞分化过程中的作用模式,结合破骨细胞-成骨细胞相互作用在骨重塑过程中的重要作用,以及BMPR1A在转导BMP信号中的必要作用,我们旨在探讨破骨细胞中由BMPR1A介导的BMP信号是如何影响破骨细胞-成骨细胞相互作用的。已知在分化过程中的破骨细胞中特异性敲除Bmpr1a基因导致小鼠体内成骨细胞性骨形成增加。我们推测在破骨细胞中由BMPR1A介导的BMP信号调控了破骨细胞所产生的细胞膜结合的蛋白质或者分泌的分子,进而调控了成骨细胞的分化。为了验证这个假设,通过在体外将成骨细胞和破骨细胞共培养,我们发现Bmpr1a基因敲除的破骨细胞能促进成骨细胞的矿化。此外,我们发现Bmpr1a基因敲除的破骨细胞表达的Cx43/Gja1上升,这个基因编码了connexin43/缝隙连接蛋白α1(gap junction proteinα1,GJA1),是缝隙连接蛋白之一。我们进一步通过在Bmpr1a基因敲除的破骨细胞中敲低Cx43/Gja1的表达,发现成骨细胞-破骨细胞共培养后成骨细胞的矿化降低了。该研究提示了Cx43/GJA1可能是破骨细胞中BMP信号通路下游的靶点之一,并介导了骨重塑过程中破骨细胞-成骨细胞相互作用。
[Abstract]:Bone is one of the most important organs in the body. Its functions include exercise, support and protection of the body, hematopoiesis, and mineral storage. However, bone will lose these functions in aging, fracture, osteoporosis, bone absorption and destruction caused by bone tumors, inflammation and so on. Therefore, it is essential to maintain the health of the bone. Tissue can maintain bone mass through bone remodeling (bone remodeling), osteoblastic bone formation and osteoclast absorption. Therefore, it is important to clarify the molecular mechanism and influencing factors of bone remodeling to maintain the healthy state of bone. Bone morphogenetic protein (bone morphogenetic proteins, BMPs) was initially induced to be heterotopic The formation of bone and cartilage was found. Although the Food and Drug Administration (FDA) approved the recombinant human BMP-2 (recombinant human bone morphogenetic protein 2, RH) and clinically applied to the treatment of open fracture of long bones, nonunion of fracture, and spinal fusion. Its effectiveness is not significant, which usually leads to bone absorption, pseudarthrosis and local inflammatory reactions. Therefore, it is expected that the role of BMP signal to bone formation and bone absorption and its molecular mechanism may provide a new perspective to improve the health of bone, and improve the application of BMPs to promote bone regeneration by further regulating the downstream molecules of the BMP signaling pathway. In the first chapter, we reviewed the physiological and pathological processes of bone remodeling, introduced the BMP ligand and receptor and its mediated BMP signaling pathway, and reviewed the role of BMP signals in bone remodeling. In order to study the role of BMP signals in bone remodeling, we reviewed the role of BMP signals in bone remodeling. Using its molecular mechanism, we targeted the type I BMP receptor. We used the gene modified animal model to study, including conditional knockout and full gene knockout. In this chapter, the related contents of gene knockout mice used in this study, especially the conditioned gene knockout mice model, were introduced in this chapter. In the second chapter, the basis of gene knockout mice was introduced. Bone mass and bone mass are important factors in determining the biological and mechanical properties of bone. We aim to explore the effect of BMP signals mediated by ACVR1 or BMPR1A on bone mass and bone mass. We have knocked out two types of BMP receptors, Acvr1 and Bmpr1a, by micro-CT view in the early differentiated osteoblast cells of the mice. The changes in bone trabecula, osteoblasts and osteoclasts were observed by histomorphometry. The changes in bone tissue were analyzed by Raman spectroscopy. We found that after the osteoblast specific Acvr1 or Bmpr1a gene knockout, the bone mass increased in mice, especially in the cancellous bone; compared with the Acvr1 knockout, the Bmpr1a gene knockout. We also found that the mineral crystallinity of the cancellous bone in these knockout mice increased (crystallinity), and the order of collagen increased (collagen order/disorder), but the mineral density (tissue mineral density) and the mineral matrix ratio (mineral-matrix ratio) of the dense bone (mineral-matrix ratio). The study suggests that the BMP signal mediated by ACVR1 and BMPR1A in osteoblasts is an important factor in determining bone mass and bone mass. In the third chapter, the negative regulation of bone mass based on the results of ACVR1 and BMPR1A mediated BMP signals based on the results of the second chapter is to explore the modulation of another type I BMP receptor BMPR1B mediated BMP signal to bone remodeling. Control. We studied the effect on osteoblasts and osteoclasts by knocking the Bmpr1b gene in mice. The results showed that the Bmpr1b gene knockout led to a decrease in bone mass in the male mice of 8W age. This phenotype was one over sex and sex specific. However, the reduced bone mass was not caused by osteoblastic formation and osteoclast. The changes in bone resorption were caused. In the study in vitro, the osteoclast differentiation of the Bmpr1b gene knockout increased but the bone absorption function decreased; the differentiation of the Bmpr1b gene knockout anterior osteoblasts was not changed, but the BMP-SMAD signal was up-regulated after the BMP stimulation; the Bmpr1b gene knockout bone was different from the anterior osteoblasts of the skull. The differentiation of intramedullary mesenchymal cells may be caused by the decrease in bone mass of Bmpr1b gene knockout mice. The effect of.BMPR1B on different cells may be due to the receptor expression patterns in different tissue cells. Combined with previous studies on the role of type I BMP receptor in bone remodeling, we also proposed a BMP receptor. The role model of BMP signal in osteoblast differentiation shows that different receptors play different roles at different stages. This study suggests that the role of BMPR1B in maintaining bone mass and transduction of BMP signals is different from that of BMPR1A or ACVR1. In the fourth chapter, the BMP receptor mediated BMP signal in the third chapter is bone fine In the process of cell differentiation, combined with the important role of osteoclast - osteoblast interaction in the process of bone remodeling and the essential role of BMPR1A in transduction of BMP signals, we aim to explore how the BMPR1A mediated BMP signals in osteoclasts affect the interaction of osteoclast - osteoblasts. The specific knockout of Bmpr1a gene in osteoclasts in the course of osteoclasts increase the osteogenic bone formation in mice. We speculate that the BMPR1A mediated BMP signal in osteoclasts regulates the cell membrane binding proteins or secreted molecules produced by osteoclasts, and then regulates the differentiation of osteoblasts. Set, by co culture of osteoblasts and osteoclasts in vitro, we found that the Bmpr1a gene knockout osteoclasts could promote mineralization of osteoblasts. In addition, we found that the expression of Bmpr1a gene knockout osteoclasts increased, and this gene encodes the connexin43/ slit connexin alpha 1 (gap junction protein alpha 1, GJA1). One of the gap junctional proteins. We further found that the mineralization of osteoblasts in osteoblasts - osteoclasts co culture was reduced by knocking down the expression of low Cx43/Gja1 in the osteoclasts of the Bmpr1a gene knockout. This study suggests that Cx43/GJA1 may be one of the targets in the BMP signaling pathway in osteoclasts and mediates the bone remodeling process. Osteoclasts and osteoblasts interact with each other.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R68

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