主穹隆蛋白通过抑制破骨细胞分化在牙周炎中起骨保护作用

目的:通过建立小鼠实验性牙周炎模型及体外骨髓间充质干细胞(BMMSCs)破骨细胞向诱导,探讨主穹隆蛋白(MVP)在牙周炎骨吸收中的作用。方法:MVP基因敲除(MVP-/-)和野生型(WT)C57BL/6小鼠分别局部注射脂多糖(LPS)以建立实验性牙周炎模型,通过micro CT扫描、耐酒石酸酸性磷酸酶(TRAP)染色等方法检测骨吸收程度。同时,体外分离培养MVP-/-与WT C57BL/6小鼠的BMMSCs,并诱导其向破骨细胞分化,通过TRAP染色、麦胚凝集素(WGA)染色等方法观察MVP对BMMSCs破骨向分化及骨吸收活性的影响。结果:在LPS诱导的小鼠实验性牙周炎中,MVP-/-组小鼠牙周炎骨吸收更为明显,且在注射区域内可见更多破骨细胞;体外实验证明,MVP-/-组小鼠的BMMSCs分化形成更多的破骨细胞,且骨吸收更明显。结论:MVP可以抑制破骨细胞分化,在牙周炎中起骨保护作用。     

二膦酸盐对骨质疏松大鼠下颌牙槽骨牙本质基质蛋白1表达的影响

目的 :观察二膦酸盐对去卵巢骨质疏松大鼠下颌牙槽骨内破骨细胞以及牙本质基质蛋白1(dentin matrix protein1,DMP1)表达的影响。方法 :取6月龄SD大鼠30只,随机分为假手术组(Sham)、去卵巢模型组(OVX)和二膦酸盐药物治疗组(RIS),每组10只。Sham组大鼠仅术中暴露卵巢不切除,OVX组大鼠行"双侧卵巢切除术+生理盐水皮下注射",RIS组行"双侧卵巢切除术+利塞磷酸钠(2.4μg/kg)皮下注射"。术后3个月取各组大鼠下颌骨常规脱钙,甲苯胺兰染色观察下颌第一磨牙(M1)区域牙槽骨组织学结构,抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色观察M1区域牙槽纵隔破骨细胞的骨吸收情况,免疫组化染色观察各组M1牙槽骨DMP1分布表达情况,并进行相关半定量图像分析。结果:与Sham组相比,OVX组TRAP阳性细胞数增加;而较OVX组而言,RIS组TRAP阳性细胞数显著减少(P<0.05)。免疫组化染色显示,各组DMP1不仅表达在牙槽骨骨细胞内,牙槽间隔的松质骨骨基质、牙周韧带中均可见其表达,OVX组M1牙槽骨DMP1表达较Sham组减少(P<0.05),而RIS组表达较OVX组有升高。结论:二膦酸盐能减少骨质疏松大鼠下颌牙槽骨破骨细胞数量,并上调DMP1表达,从而影响下颌牙槽骨矿化。     

破骨细胞过氧化物酶体增殖因子活化受体gamma敲除对腭中缝扩张后骨新生和骨重建的影响

目的 探讨破骨细胞过氧化物酶体增殖因子活化受体gamma(PPAR-g)敲除对腭中缝扩张(MSE)后骨新生和骨重建的影响.方法 将雄性对照野生型小鼠(WT)和破骨细胞PPAR-g敲除小鼠(KO)分为如下4组,WT+假手术,WT+ MSE,KO+假手术,KO+ MSE.术后14天处死,采用HE染色、抗酒石酸酸性磷酸酶(TRAP)染色、以及实时荧光定量PCR方法研究破骨细胞PPAR-g敲除对腭中缝扩张后骨新生和骨重建的影响.结果 KO+MSE组新骨形成面积较WT+ MSE组增加,细胞总数增多,但是破骨细胞计数较WT+ MSE组减少,破骨细胞分化相关基因(cFos,Calcr,Car2,Ctsk,和Mmp9)的表达显著下降.结论 破骨细胞PPAR-g敲除可以促进腭中缝扩张(MSE)后骨新生和骨重建.在腭中缝扩张后的骨重建过程中,PPAR-g是调控破骨细胞分化和骨新生的一个重要分子.     

ALK2基因敲除对小鼠股骨骨形成及骨吸收影响的研究

目的 探究激活素受体样激酶2(Alk2)功能缺陷对出生后小鼠股骨骨形成、骨吸收的影响,并探讨其相关机制.方法 首先用Cre-loxp重组酶系统培育出Alk2条件基因敲除小鼠,通过基因型鉴定筛选出实验组(基因型:Osx-Cre;Alk2fx/fx)和对照组(基因型:Osx-Cre;Alk2fx/+).于出生后21天取材、固定,HE染色观察股骨骨量的差异.抗酒石酸酸性磷酸酶(TRAP)染色,比较破骨细胞的差异.提取股骨组织的RNA,通过Real-Time PCR检测成骨相关基因Col1、Alp、Runx2、Bsp、Ocn基因的表达.结果 小鼠股骨HE染色结果显示,基因敲除组小鼠股骨的骨量明显大于对照组.TRAP染色结果提示,对照组中破骨细胞多于基因敲除组.股骨组织中提取RNA的Real-Time PCR结果,基因敲除组中Col1、Alp、Runx2、Bsp的表达升高,Ocn的表达明显降低.结论 Alk2基因敲除导致小鼠股骨骨量减少,这一作用是通过成骨细胞和破骨细胞双向介导的.     

破骨细胞在大鼠正畸牙移动性根吸收修复早期中的改变

目的初步研究大鼠正畸牙移动性根吸收修复早期中破骨细胞和骨保护因子(osteoprotegerin,OPG)的变化和规律,探讨破骨细胞在正畸导致的炎性牙根吸收修复中的作用。方法建立大鼠正畸牙移动性根吸收修复早期模型,免疫组化检测OPG的表达,TRAP染色观察破骨细胞的变化。结果实验第1天,OPG表达量下降,第3天以后逐渐恢复至生理水平。实验第1天起,TRAP染色阳性细胞逐渐减少,实验第7、10、14天,TRAP染色阳性数量已逐渐恢复至生理水平。结论施力终止后,大鼠牙根即停止吸收,并于1~3d后开始牙根修复,OPG的量与此过程密切相关。     

牙龈卟啉单胞菌脂多糖(Pg-LPS)对破骨细胞EphA2基因表达的调控

目的:研究小鼠单核巨噬细胞系RAW264.7在破骨分化过程中,Pg-LPS对破骨细胞EphA2表达的影响。方法:用终浓度10 mg/L的Pg-LPS 刺激RAW264.7 细胞后,分别在1、3、5 d,应用RT-PCR检测破骨细胞中EphA2基因和破骨细胞相关基因(破骨细胞内基质金属蛋白酶( MMP9)、ACP5、c-fos、组织蛋白酶K(CtsK)、NFATc1)的表达,并且通过酒石酸抗酸性磷酸酶(TRAP)染色观察实验组和对照组破骨细胞的分化成熟情况。结果:RT-PCR检测10 mg/L的Pg-LPS在第3天和5天,实验组比对照组EphA2基因表达分别增高2.4倍和1.2倍,两组之间存在显著差异(P<0.01);同时也能够促进破骨相关基因c-fos、NFATc1、CtsK、ACP5、MMP9的表达,实验组与对照组相比差异有显著性;TRAP染色结果显示:实验组比对照组的TRAP阳性多核细胞数目明显增多。结论:10 mg/L的Pg-LPS对小鼠单核巨噬细胞系RAW264.7,在破骨分化的中期和晚期均能够促进EphA2基因的表达,但是在破骨分化早期对EphA2基因的表达无明显作用。     

仿骨保护素小分子活性多肽的设计及其对破骨活动的影响

目的:设计并探究仿骨保护素(osteoprotegerin, OPG)小分子活性多肽是否具有抑制破骨细胞分化作用,及其抑制破骨的可能机制。方法:以OPG与核因子-κB受体活化因子配体(receptor activator of nuclear factor kappa-B ligand, RANKL)结合位点氨基酸序列为依据设计小分子活性多肽OPGL 1、2、3,以50μmol/L浓度加入RANKL诱导的破骨细胞前体细胞,通过抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase, TRAP)染色检测破骨细胞数目,实时荧光定量PCR检测破骨表型基因TRAP、CTSK和通路基因NFATC1表达情况,Western blot检测破骨表型蛋白TRAP和C-fos表达情况。结果:3条小分子活性多肽中,OPGL2有效抑制破骨分化,下调TRAP、CKST、NFATC1基因并抑制TRAP、C-fos蛋白表达。结论:仿OPG小分子活性多肽OPGL2可以有效抑制破骨向分化,其可能通路为NFATC1。     

Notch信号促进核因子κB受体活化因子配体诱导的破骨细胞分化的体外研究

目的 探讨Notch信号抑制对核因子κB受体活化因子配体（RANKL）诱导的小鼠RAW264.7细胞向破骨细胞分化的影响。方法 建立RANKL诱导的小鼠RAW264.7细胞体外分化模型,实时定量聚合酶链反应（real-time PCR）检测Notch信号成分（Notch1、Notch2、Delta1、Jagged1）、下游靶基因Hes1以及破骨细胞标志基因抗酒石酸酸性磷酸酶（TRAP）和Cathepsin K在诱导前后mRNA的表达。在诱导体系中加入不同浓度的γ分泌酶抑制剂（GSI）,抑制Notch受体的表达,TRAP染色检测破骨细胞分化的变化情况。结果 50 ng•mL-1 RANKL诱导小鼠RAW264.7细胞3 d,Notch1、Notch2、Delta1、Jagged1及Hes1的mRNA表达均有不同程度的提高,其中以Notch2、Jagged1增高最明显;破骨细胞标志基因表达显著增高。在RANKL诱导的同时加入不同浓度GSI,抑制Notch的表达,可致Notch下游靶基因Hes1表达下降,同时TRAP阳性细胞计数显著减少,且呈剂量依赖性。结论 Notch信号可促进RANKL诱导的RAW264.7细胞向破骨细胞分化。     

骨折愈合中牙本质基质蛋白1与破骨细胞表达时间效应的观察

目的探讨骨折愈合过程中牙本质基质蛋白1（dentin matrix protein 1，DMP1）与破骨细胞的时间效应。为研究DMP1在体内矿化重建中的作用提供参考。方法将40只成年Wistar雄性大鼠左侧下颌支骨折，建立下颌骨骨折模型。骨折后5、7、14、21d处死大鼠，取骨痂和对侧正常骨组织（对照组），分别采用HE染色、TRAP染色和免疫组织化学链霉抗生物素．蛋白过氧化物酶（streptavidin perosidase，sp）法染色切片检测。结果在对照组正常下颌支组织中没有DMP1的表达，偶见破骨细胞；实验组在骨折后14～21d是破骨细胞活动高峰。结论DMP1与破骨细胞在骨折愈合过程中具有一定的时间效应。     

破骨细胞骨吸收上清液对成骨活动影响的实验研究

目的 研究破骨细胞骨吸收活动中的分泌产物对成骨细胞增殖、分化及成骨功能的影响,以了解活化的破骨细胞在体外对成骨细胞的影响.方法 诱导小鼠RAW264.7细胞为破骨细胞,用抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色和破骨细胞特异性基因检测鉴定.破骨细胞与牛骨磨片共培养,甲苯胺蓝染色.收集共培养上清液作用于小鼠MC3T3-E1细胞,用甲基噻唑基四唑法(methyl thiazolyl tetrazolium,MTT)法、酶联免疫吸附测定法、茜素红染色及反转录聚合酶链反应检测细胞的增殖、骨钙素水平、矿化及成骨特征基因的表达.结果 TRAP 染色、破骨细胞特异性基因检测及甲苯胺蓝染色表明RAW264.7细胞可分化为具有骨吸收能力的破骨细胞;破骨细胞骨吸收上清液作用后,MC3T3-E1细胞增殖受抑制(A值在0.062±0.004和0.405±0.033之间,P＜0.05);骨钙素水平增高[第10天上清液组的骨钙素质量浓度为(2.965±0.047) μg/L],钙化结节增多,碱性磷酸酶和Runt相关转录因子2的转录增强.结论 RAW264.7破骨细胞骨吸收上清液具有抑制成骨样细胞增殖、促进分化和钙化成骨的作用.     

Glycosylation of DMP1 maintains cranial sutures in mice

Craniosynostosis, a severe craniofacial developmental disease, can only be treated with surgery currently. Recent studies have shown that proteoglycans are involved in the suture development. For the bone matrix protein, dentin matrix protein 1 (DMP1), glycosylation on the N-terminal of it could generate a functional proteoglycan form of DMP1 during osteogenesis. We identified that the proteoglycan form of DMP1 (DMP1-PG) is highly expressed in mineralisation front of suture. But, the potential role of DMP1-PG in suture fusion remain unclear. To investigate the role of DMP1-PG in cranial suture fusion and craniofacial bone development. By using a DMP1 glycosylation site mutation mouse model, DMP1-S89G mice, we compared the suture development in it with control mice. We compared the suture phenotypes, bone formation rate, expression levels of bone formation markers in vivo between DMP1-S89G mice and wild-type mice. Meanwhile, cell culture and organ culture were performed to detect the differences in cell differentiation and suture fusion in vitro. Finally, chondroitin sulphate (CHS), as functional component of DMP1-PG, was employed to test whether it could delay the premature suture fusion and the abnormal differentiation of bone mesenchymal stem cells (BMSCs) of DMP1-PG mice. DMP1-S89G mice had premature closure of suture and shorter skull size. Lack of DMP1-PG accelerated bone formation in cranial suture. DMP1-PG maintained the essential stemness of BMSCs in suture through blocking the premature differentiation of BMSCs to osteoblasts. Finally, chondroitin sulphate, a major component of DMP1-PG, successfully delayed the premature suture fusion by organ culture of skull in vitro. DMP1-PG could inhibit premature fusion of cranial suture and maintain the suture through regulating the osteogenic differentiation of BMSCs.     

Exploration of the role of the subodontoblastic layer in odontoblast-like cell differentiation after tooth drilling using Nestin-enhanced green fluorescent protein transgenic mice

ObjectivesOriginal odontoblasts and regenerated odontoblast-like cells (OBLCs) may differently regulate Nestin expression. This study aimed to investigate the role of the subodontoblastic layer (SOBL) using green fluorescent protein (GFP) reactivity in the process of OBLC differentiation after tooth drilling in Nestin-enhanced GFP transgenic mice.MethodsA groove-shaped cavity was prepared on the mesial surface of the maxillary first molars of 5- or 6-week-old mice under deep anesthesia. Immunohistochemical staining for Nestin and GFP and Nestin in situ hybridization were conducted on the sections obtained at 1–14 days postoperative.ResultsOdontoblasts showed intense endogenous Nestin protein and mRNA expression, whereas the coronal SOBL cells showed a Nestin-GFP–positive reaction in the control groups. The injured odontoblasts had significantly decreased Nestin immunoreactivity as well as decreased expression of Nestin mRNA 1–2 days after the injury; subsequently, newly differentiated OBLCs were arranged along the pulp–dentin border, with significantly increased Nestin expression as well as increased expression of Nestin mRNA on days 3–5 to form reparative dentin. Nestin-GFP–positive cells at the pulp–dentin border significantly increased in number on days 1 and 2. GFP(+)/Nestin(+) and GFP(?)/Nestin(+) cells were intermingled in the newly differentiated OBLCs.ConclusionsThe commitment of Nestin-GFP–positive cells into Nestin-positive OBLCs suggests that the restriction of endogenous Nestin protein and mRNA expression in the static SOBL cells was removed by exogenous stimuli, resulting in their migration along the pulp–dentin border and their differentiation into OBLCs.     

Osteopontin antisense deoxyoligonucleotides inhibit bone resorption by mouse osteoclasts in vitro

Osteopontin (OPN) is an acidic phosphoprotein synthesized by osteoblasts and osteoclastic cells that are localized in the mineralized phase of bone matrix. OPN is thought to bind to the vitronection receptor on the osteoclast membrane and regulates bone resorption by the osteoclast. In this study, we investigated whether or not OPN can relate to osteoclast differentiation and bone resorption in a co-culture system. When C57Black/6N mouse bone marrow cells suspended on ivory slices coated with collagen were inoculated onto a MC3T3-G2/PA6 cell layer, colonies containing TRAP(+) mononuclear and multinuclear cells were formed in the presence of 1α, 25-dihydroxyvitamin D₃ and dexamethasone. At the end of the culture period the number of TRAP(+) osteoclast-like cells were counted and the resorption pits were evaluated by reflected light microscopy. The mRNA of OPN was detected by in situ hybridization. Osteoclast-like cells expressed OPN mRNA. The addition of an OPN antisense oligomer(5'AAT CAC TGC CAA TCT CAT 3') at the start of the co-culture period decreased the number of TRAP(+) cells present after 7 d (30.3 ± 3.4 vs 56.9 ± 12.4), and the ratio of mononuclear and multinucleated cells was changed (77.6:23.2 vs 60.8:39.3). The total area of pits per ivory slice was also decreased by adding the OPN antisense oligomer(246813 vs 303139 μm2). These results showed that OPN can be an important mechanism for regulating differentiation and bone resorption.     

DMP1 processing is essential to dentin and jaw formation

Dentin matrix protein 1 (DMP1), an acidic protein that is essential to the mineralization of bone and dentin, exists as proteolytically processed fragments in the mineralized tissues. In this study, we characterized the tooth and jaw phenotypes in transgenic mice containing no wild-type DMP1, but expressing a mutant DMP1 in which Asp(213), a residue at one cleavage site, was replaced by Ala(213) (named "Dmp1-KO/D213A-Tg" mice). The teeth and mandible of Dmp1-KO/D213A-Tg mice were compared with those of wild-type, Dmp1-knockout (Dmp1-KO), and Dmp1-KO mice expressing the normal Dmp1 transgene. The results showed that D213A-DMP1 was not cleaved in dentin, and the expression of D213A-DMP1 failed to rescue the defects in the dentin, cementum, and alveolar bones in the Dmp1-KO mice. These findings indicate that the proteolytic processing of DMP1 is essential to the formation and mineralization of dentin, cementum, and jaw bones.     

High extracellular calcium affects osteoclastogenesis in mouse bone marrow cell culture

The effects of high extracellular calcium (high Ca) in the local microenvironment on osteoclasts, osteoclast progenitors and stromal cells are not fully understood. We examined high Ca effect on osteoclastogenesis in mouse bone marrow cell culture. Mouse bone marrow cells were cultured for up to 6 days in the medium supplemented with 1, 25(OH)2 vitamin D3 (D3). High Ca treatment at the early stage of culture (the initial 24 hours) reduced the number of tartrate resistant acid phosphatase-positive multinuclear cells (TRAP(+)MNCs). This treatment slightly up-regulated the mRNA expressions of receptor activator of NF-(B ligand (RANKL), RANK and osteoprotegerin (OPG). This inhibitory effect on the formation of TRAP(+)MNCs was recovered by RANKL. In contrast, high Ca treatment at the later stage of osteoclastogenesis (the last 2 days of culture) stimulated the formation of TRAP(+)MNCs, increased RANKL and RANK mRNA expressions and decreased OPG mRNA. High Ca at neither the early nor the later stage of culture affected the total number of adherent cells and the mRNA expression of alkaline phosphatase and osteopontin. In conclusion, high Ca affects osteoclastogenesis in a manner depending on the stage of osteoclastogenesis, which is partly mediated via the RANKL-RANK-OPG regulatory system.     

DMP1、Runx2在小鼠下颌第一磨牙萌出过程中的免疫学定位

目的 研究小鼠下颌第一磨牙萌出过程中牙本质基质蛋白1（DMP1）和Runt相关转录因子2（RUNX2）的表达, 并探索其表达与牙萌出之间的关系。 方法 分离出生后1 d至14 d小鼠的下颌骨,连续切片,偶氮卡红苯胺蓝染色显示磨牙萌出过程骨胶原形成情况,切片原位杂交法分析DMP1及RUNX2 mRNA在牙齿及周围组织的表达与分布,免疫组化法显示DMP1和RUNX2蛋白的表达与分布。结果 冠方骨组织中骨胶原形成在P5 d出现高峰,以后呈逐渐减少的趋势;根方骨胶原形成在整个萌出过程中一直呈活跃状态,在P9 d出现高峰。DMP1表达越丰富,骨胶原形成越多,成骨活动越活跃。结论 下颌第一磨牙萌出过程中,根方成骨活动一直很活跃,DMP1的表达与根方成骨活动参与了小鼠第一磨牙的萌出过程。