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NRAGE role in regulating bone metabolism and the mechanisms

Author: XuLiJuan
Tutor: LiuMei
School: Nanjing Normal University
Course: Developmental Biology
Keywords: NRAGE Knockout mice Osteoporosis Osteoclasts
CLC: R580
Type: Master's thesis
Year: 2011
Downloads: 10
Quote: 0
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Abstract

Bone occur including bone formation (bone formation) absorption and bone tissue (bone resorption) two basic processes. Bone formation after a person's life through bone remodeling of bone formation and resorption (bone remodeling) process to maintain bone update. Bone formation by osteoblasts regulation, the regulation of bone resorption by osteoclasts. The bone metabolism Steady dependent on the balance of the coupling between these two cell types and precise regulation of this balance is broken, will result in the occurrence of osteoporosis as a representative of a variety of metabolic bone diseases. NRAGE (neurotrophin receptor-interacting MAGE homolog) is also known as MAGE-D1, or Dlxin-1 is one of the MAGE family. Pre about the function of the gene is more concentrated in the areas of apoptosis, cell cycle and cell differentiation. No NRAGE bone development or bone metabolism relationship. But through the relevant literature in the field of analysis, we speculated: NRAGE may be correlated with bone development and regulation of bone metabolism. NRAGE gene expression profiles prompted the genes expressed in the attached limb bones and skull rich; situ hybridization experiments also show that, in the process of the formation of finger / toe, the strong expression of the gene in the cell layer around the cartilage model. Further, NRAGE Dlx5, Msx2, Ror2 Rora binding, while these receptors or transcription factors have been shown to play an important role in bone development or bone metabolic processes. In addition, NRAGE can affect the activity of the NF-κB family of NF-κB p50 and p52 subunits while knockout mice, osteoclast differentiation blocked showed severe bone sclerosis. In view of the above analysis, the subject of the use of NRAGE knockout mouse model, in vitro and in vivo experiments to explore the NRAGE role in the regulation of bone metabolism, and initially were possible mechanism to explore. The main contents include two parts: 1. Clear NRAGE role in the regulation of bone metabolism; 2. Explore NRAGE the mature osteoclast differentiation and bone resorption function and its possible molecular mechanisms involved. The main experimental content and methods are as follows: The first part: NRAGE gene knockout mice clearly NRAGE role in regulating bone metabolism 1) the use of X-ray imaging, combined with the the mouse hindlimb comparison of the length of the femur and tibia, analysis NRAGE gene knock In addition to the after bone morphogenetic; 2) separation of 10-week-old and 16-month-old NRAGE knockout mice and wild-type mice femur, small animal in vivo imaging system in the X-ray machine and dual-energy X-ray absorptiometry instrument mouse femur bone mineral density (DXA) testing; 3) Micro-CT technology to detect the 16-month-old NRAGE gene knockout and wild-type mice femur ultrastructural changes; 4) by three-point bending experiments detect NRAGE gene knock In addition to mice after biomechanical changes. Part II: explore NRAGE differentiation and maturation of osteoclasts and resorption and its possible molecular mechanisms involved 1) separation of 16-month-old knockout mice and wild mice femur, decalcified paraffin sections preparation and osteoclast marker enzyme tartrate-resistant acid phosphatase (Tartrate-resistant acid phosphatase, TRAP) staining, from the organizational level detection NRAGE osteoclast; 2) separation of 10-week-old knockout mice and wild-type small mouse bone marrow mononuclear cells, induced by M-CSF and RANKL osteoclast differentiation and bone resorption experiment by osteoclasts TRAP staining of osteoclast cell area, toluidine blue staining and scanning electron microscopy of bone resorption lacunae detecting osteoclast differentiation and bone resorption activity; osteoclast RNA extraction induced culture 8d by qPCR, detecting osteoclast flag factor trap, CTR and cathepsinK the expression level; 3) separation of the 10-week-old NRAGE knockout bone marrow mononuclear cells of mice and wild-type mice, the use of M-CSF and RANKL to induce differentiation of osteoclasts, extracting the induction time for Omin, 5min, 15min, and 30min osteoclasts total protein, through the detection of NF-kappaB core signaling pathway proteins of IKK phosphorylation levels of IκB and NF-κB judgment NRAGE osteoclast whether through NF-κB signaling pathway. Main experimental results: the first part: NRAGE gene knockout leads to osteoporosis in mice occurrence of X-ray imaging, the mouse limb bone length NRAGE, gene knockout mouse bone morphogenetic normal; bone mineral density testing found, NRAGE The bone mineral density of the knockout mice was significantly lower than the wild-type mice (P lt; 0.01). Micro-CT analysis NRAGE, gene knock significantly reduced (P lt; 0.01) In addition to the mouse femur cancellous bone mineral density and bone mineral content, bone surface area and volume ratio increased (P lt; 0.01) decline in bone volume fraction ( P lt; 0.05), while the decrease in the number of trabecular bone connection reduced thickness decreases, the gap increases trabecular structure morphology rod, the ultrastructural degenerative changes, significant bone type of osteoporosis table. Part II: NRAGE gene knock slice TRAP color in addition to promoting the differentiation and maturation of osteoclasts and bone resorption activity organization NRAGE, knockout mice osteoclast number and activity was significantly enhanced; osteoclasts in vitro M-CSF and RANKL-induced differentiation experiments found NRAGE, gene knock in addition to result in the number of osteoclasts significantly increased (P lt; 0.01), volume significantly with increased (P lt; 0.01), a piece of bone resorption trap nest more and the area is large (P lt; 0.01 ). Molecular level detected and NRAGE, gene knockout mouse osteoclast marker genes TRAP, CTR, and cathepsinK the expression was significantly increased (P lt; 0.01, P lt; 0.05 and P lt; 0.05). These results suggest that: NRAGE, gene knock In addition to promoting the differentiation and maturation of osteoclasts and bone resorption activity. Vitro cultured osteoclasts extracted proteins at different time points, found that the induction time of 5min of IKK in the NF-κB signaling pathway. IκB and NF-κB phosphorylation levels reached a peak, and NRAGE knockout group higher than the wild-type group. The main conclusions: 1. NRAGE, gene knockout lead to the mice bone metabolism disorder; NRAGE, gene knock In addition to promoting the differentiation and maturation of osteoclasts and bone resorption activity.

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