Ⅰ型BMP受體介導的BMP信號在骨重塑中的作用及機理研究
發(fā)布時間：2018-08-07 08:24
【摘要】：骨骼是人體內(nèi)最重要的器官之一,其功能包括運動、支持和保護身體、造血、儲存礦物質(zhì)等。然而,在衰老、骨折、骨質(zhì)疏松,以及由骨腫瘤、炎癥等各種原因引起的骨吸收破壞等情況,骨骼將喪失這些功能。因此維持骨的健康狀態(tài)至關(guān)重要。骨組織通過骨重塑(bone remodeling)過程,即成骨細胞性骨形成和破骨細胞性骨吸收來維持骨量。因此,明確骨重塑的分子機理與影響因素對維持骨的健康狀態(tài)具有重要的理論意義。骨形成蛋白(bone morphogenetic proteins,BMPs)最初因其能誘導異位骨和軟骨的形成而被發(fā)現(xiàn)。雖然美國食品藥品管理局(Food and Drug Administration,FDA)已在2002年批準了重組人BMP-2(recombinant human bone morphogenetic protein 2,rh BMP-2)和rh BMP-7在臨床上應用于長骨開放性骨折、骨折不愈合以及脊柱融合的治療,然而其有效性不顯著,通常導致骨吸收、假性關(guān)節(jié)形成以及局部炎癥反應等。因此,闡明BMP信號對骨形成和骨吸收的作用及其分子機理,有望為改善骨的健康狀態(tài)提供新的視角;并通過進一步調(diào)控BMP信號通路下游分子,提高應用BMPs促骨再生的療效,這又對防治骨吸收破壞性疾病具有潛在的實際意義。在第1章中,我們通過文獻復習,綜述了骨重塑的生理和病理過程,介紹了BMP配體和受體及其介導的BMP信號通路,簡述了BMP信號在骨重塑中的作用。為了研究BMP信號在骨重塑中的作用及其分子機理,我們靶向于Ⅰ型BMP受體。我們利用了基因修飾動物模型進行研究,其中包括條件性基因敲除和全基因敲除,在本章中又介紹了本研究中所利用的基因敲除小鼠的相關(guān)內(nèi)容,尤其是條件性基因敲除小鼠模型。在第2章中,基于骨量和骨質(zhì)量是決定骨的生物特性和機械性能的重要因素,我們旨在探討由ACVR1或BMPR1A介導的BMP信號對骨量和骨質(zhì)量的影響。我們通過在小鼠體內(nèi)早期分化的成骨細胞前體細胞中分別敲除兩種Ⅰ型BMP受體,Acvr1和Bmpr1a,采用micro-CT觀察骨量,采用組織形態(tài)測量學觀察骨小梁、成骨細胞和破骨細胞的改變,采用拉曼光譜分析骨組織成分的改變。我們發(fā)現(xiàn)成骨細胞特異性Acvr1或Bmpr1a基因敲除后,小鼠體內(nèi)骨量增多,尤其以松質(zhì)骨為著;與Acvr1基因敲除相比,Bmpr1a基因敲除導致更顯著的松質(zhì)骨的骨量升高。我們還發(fā)現(xiàn)這些基因敲除小鼠的松質(zhì)骨的礦物質(zhì)結(jié)晶度增高(crystallinity),膠原有序性增加(collagen order/disorder),然而密質(zhì)骨的組織礦物質(zhì)密度(tissue mineral density)和礦物質(zhì)-基質(zhì)比例(mineral-matrix ratio)下降。該研究提示了在成骨細胞中由ACVR1和BMPR1A介導的BMP信號是決定骨量和骨質(zhì)量的重要因素。在第3章中,基于第2章的實驗結(jié)果ACVR1和BMPR1A介導的BMP信號負性調(diào)控骨量,我們旨在探討另外一種Ⅰ型BMP受體BMPR1B介導的BMP信號對骨重塑的調(diào)控作用。我們通過在小鼠體內(nèi)敲除Bmpr1b基因,研究了其對成骨細胞和破骨細胞的影響。結(jié)果發(fā)現(xiàn)Bmpr1b基因敲除導致8w齡雄性小鼠骨量下降,此表型是一過性的,并且是性別特異性的。然而減少的骨量不是由成骨細胞性骨形成和破骨細胞性骨吸收的改變引起的。在體外研究中,Bmpr1b基因敲除的破骨細胞分化增加但是骨吸收功能降低;Bmpr1b基因敲除的顱骨前成骨細胞的分化沒有改變,然而卻表現(xiàn)出BMP刺激后BMP-SMAD信號的上調(diào);與顱骨前成骨細胞不同的是,Bmpr1b基因敲除的骨髓間充質(zhì)細胞的分化降低,這可能是引起B(yǎng)mpr1b基因敲除小鼠骨量下降的原因。BMPR1B對不同細胞的不用作用可能是由于該受體在不同組織細胞中的表達模式引起的。結(jié)合以往對Ⅰ型BMP受體在骨重塑中作用的研究,我們還提出了BMP受體介導的BMP信號在成骨細胞分化過程中的作用模式,說明不同受體在不同階段所發(fā)揮的作用不同。該研究提示了BMPR1B在維持骨量和轉(zhuǎn)導BMP信號中的作用與BMPR1A或ACVR1不同。在第4章中,基于我們在第3章中提出的BMP受體介導的BMP信號在成骨細胞分化過程中的作用模式,結(jié)合破骨細胞-成骨細胞相互作用在骨重塑過程中的重要作用,以及BMPR1A在轉(zhuǎn)導BMP信號中的必要作用,我們旨在探討破骨細胞中由BMPR1A介導的BMP信號是如何影響破骨細胞-成骨細胞相互作用的。已知在分化過程中的破骨細胞中特異性敲除Bmpr1a基因?qū)е滦∈篌w內(nèi)成骨細胞性骨形成增加。我們推測在破骨細胞中由BMPR1A介導的BMP信號調(diào)控了破骨細胞所產(chǎn)生的細胞膜結(jié)合的蛋白質(zhì)或者分泌的分子,進而調(diào)控了成骨細胞的分化。為了驗證這個假設,通過在體外將成骨細胞和破骨細胞共培養(yǎng),我們發(fā)現(xiàn)Bmpr1a基因敲除的破骨細胞能促進成骨細胞的礦化。此外,我們發(fā)現(xiàn)Bmpr1a基因敲除的破骨細胞表達的Cx43/Gja1上升,這個基因編碼了connexin43/縫隙連接蛋白α1(gap junction proteinα1,GJA1),是縫隙連接蛋白之一。我們進一步通過在Bmpr1a基因敲除的破骨細胞中敲低Cx43/Gja1的表達,發(fā)現(xiàn)成骨細胞-破骨細胞共培養(yǎng)后成骨細胞的礦化降低了。該研究提示了Cx43/GJA1可能是破骨細胞中BMP信號通路下游的靶點之一,并介導了骨重塑過程中破骨細胞-成骨細胞相互作用。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R68
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R68
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