Smad4促进成骨分化的机制研究
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摘要
目的探讨Smad4基因促进成骨分化的作用机制。方法采用条件性基因敲除技术Cre/loxp,制备骨细胞特异性敲除Smad4小鼠(Smad4~(ot)cko),小鼠胚胎骨骼透明染色分析胚胎期小鼠长骨生长状况;待小鼠成长至1月龄,X-ray检测突变小鼠与对照组小鼠的骨密度差异;静态骨组织形态学分析检测突变鼠及对照鼠的骨量变化、成骨细胞数量变化等差异;实时荧光定量PCR检测Smad4突变鼠股骨成骨细胞相关因子Runx2、ALP、OSX及OCN;qPCR检测突变鼠股骨破骨吸收标志基因RANKL、OPG,并计算RANKL/OPG比率。结果 Smad4基因敲除小鼠在胚胎期未出现骨生长异常。X线结果显示,1月龄时,与对照组小鼠相比,Smad4突变鼠的骨密度降低(P<0.05),静态骨组织形态学分析表明突变鼠松质骨减少,皮质骨变薄,骨小梁数量减少(P<0.05);Smad4突变鼠成骨细胞标志基因表达量显著降低,成骨细胞的数量明显减少(P<0.05);RANKL作为破骨吸收标志物表达上调、作为其拮抗剂的OPG表达量下调,RANKL/OPG比率增高(P<0.05)。结论 Smad4基因通过促进成骨分化维持骨稳态。
Objective To determine the mechanism of promoting osteogenic differentiation by osteocyte-specific gene Smad4. Methods Conditional gene knockout technique Cre/loxp was used to produce osteocytic Smad4 specific knockout mice(Smad4~(ot)cko). Embryonic mouse skeletal growth was shown by whole mount skeletal staining of new pups. The bone mineral density was detected in one-month-old mice with radiography. Changes in bone mass, trabecular number, and osteoblast number were analyzed by static bone histomorphology. The osteoblastic makers ALP, Runx2, OSX, OCN, and osteoclastic markers RANKL, OPG were detected by qPCR. The RANKL/OPG ratio was calculated. Results Smad4~(ot)cko mice displayed no gross skeletal abnormalities at birth. X-ray radiography showed reduced bone mineral density, bone mass, and osteoblast number compared with controls(P<0.05). Similarly, the expression of osteoblastic differentiation marker genes were decreased. The expression of RANKL increased while OPG decreased. The radio of RANKL/OPG increased significantly(P<0.05). Conclusion The Smad4 gene in the osteocytes regulates bone homeostasis by promoting osteoblastic differentiation and possibly by down-regulation of osteoclastic differentiation.
Objective To determine the mechanism of promoting osteogenic differentiation by osteocyte-specific gene Smad4. Methods Conditional gene knockout technique Cre/loxp was used to produce osteocytic Smad4 specific knockout mice(Smad4~(ot)cko). Embryonic mouse skeletal growth was shown by whole mount skeletal staining of new pups. The bone mineral density was detected in one-month-old mice with radiography. Changes in bone mass, trabecular number, and osteoblast number were analyzed by static bone histomorphology. The osteoblastic makers ALP, Runx2, OSX, OCN, and osteoclastic markers RANKL, OPG were detected by qPCR. The RANKL/OPG ratio was calculated. Results Smad4~(ot)cko mice displayed no gross skeletal abnormalities at birth. X-ray radiography showed reduced bone mineral density, bone mass, and osteoblast number compared with controls(P<0.05). Similarly, the expression of osteoblastic differentiation marker genes were decreased. The expression of RANKL increased while OPG decreased. The radio of RANKL/OPG increased significantly(P<0.05). Conclusion The Smad4 gene in the osteocytes regulates bone homeostasis by promoting osteoblastic differentiation and possibly by down-regulation of osteoclastic differentiation.
引文
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[3] Iqbal J,Yuen T,Kim SM,et al.Opening windows for bone remodeling through a SLIT[J].J Clin Invest,2018,128(4):1255-1257.
[4] Masi L,Simonini G,Piscitelli E,et al.Osteoprotegerin (OPG)/RANK-L system in juvenile idiopathic arthritis:is there a potential modulating role for OPG/RANK-L in bone injury?[J].J Rheumatol,2004,31(5):986-991.
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[6] Herlofson BB,Kjolle GK,Westgaard KL,et al.Osteonecrosis of the jaw in a patient with bone metastatic prostate cancer after long-term bisphosphonate treatment with severe deterioration following radium-223[J].Clin Genitourin Cancer,2018,16(5):328-331.
[7] Bonewald LF.The amazing osteocyte[J].J Bone Miner Res,2011,26(2):229-238.
[8] Bellido T.Osteocyte-driven bone remodeling[J].Calcif Tissue Int,2014,94(1):25-34.
[9] Tu X,Delgado-Calle J,Condon KW,et al.Osteocytes mediate the anabolic actions of canonical Wnt/beta-catenin signaling in bone[J].Proc Natl Acad Sci U S A,2015,112(5):E478-486.
[10] Kode A,Manavalan JS,Mosialou I,et al.Leukaemogenesis induced by an activating beta-catenin mutation in osteoblasts[J].Nature,2014,506(7487):240-244.
[11] 任磊,代光明,林枭,等.骨细胞Wnt/β-Catenin 通过Notch 信号促进BMSCs成骨分化[J].中国骨质疏松杂志,2018,24(5):600-605.
[12] Razzouk S,Sarkis R.BMP-2:biological challenges to its clinical use[J].N Y State Dent J,2012,78(5):37-39.
[13] Kim HY,Lee JH,Lee HAR,et al.Sustained release of BMP-2 from porous particles with leaf-stacked structure for bone regeneration[J].ACS Appl Mater Interfaces,2018,10(25):21091-21102.
[14] Smoljanovic T,Caric D,Bojanic I.RE:Clinical applications of bone morphogenetic proteins:current evidence.Kanakaris,NK;Giannoudis,PV;JSOA 17(3):133-46,2008[J].J Surg Orthop Adv,2010,19(2):135-137.
[15] Asparuhova MB,Caballe-Serrano J,Buser D,et al.Bone-conditioned medium contributes to initiation and progression of osteogenesis by exhibiting synergistic TGF-beta1/BMP-2 activity[J].Int J Oral Sci,2018,10(2):20.
[16] Bivi N,Condon KW,Allen MR,et al.Cell autonomous requirement of connexin 43 for osteocyte survival:consequences for endocortical resorption and periosteal bone formation[J].J Bone Miner Res,2012,27(2):374-389.
[17] Rodan GA,Martin TJ.Role of osteoblasts in hormonal control of bone resorption - a hypothesis[J].Calcif Tissue Int,1982,34(3):311.
[18] Dempster DW.Standardized nomenclature,symbols,and units for bone histomorphometry:2012 update of the report of the ASBM Histomorphometry Nomenclature Committee[J].J Bone Miner Res,2013,28(1):2-17.
[19] Moon YJ,Yun CY,Choi H,et al.Smad4 controls bone homeostasis through regulation of osteoblast/osteocyte viability[J].Exp Mol Med,2016,48(9):e256.
[20] Sirard C,de la Pompa JL,Elia A,et al.The tumor suppressor gene Smad4/Dpc4 is required for gastrulation and later for anterior development of the mouse embryo[J].Genes Dev,1998,12(1):107-119.