Runx2、Osterix转录因子过表达驱动内皮细胞成骨分化的机制探讨

目的:探讨Runx2和Osterix (OSX)过表达对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)成骨分化的调控作用.方法:通过慢病毒载体,将Runx2和Osterix基因分别转染入HUVECs.通过碱性磷酸酶(ALP)染色、半定量活性检测,探讨过表达Runx2和Osterix对HUVECs成骨分化的影响.通过RT-PCR、蛋白免疫印迹、免疫荧光染色检测成骨相关标志物Runx2、OSX、ALP、骨涎蛋白(BSP)、骨桥蛋白(OPN)、骨钙蛋白(OCN)在HUVECs中的表达.采用GraphPad Prism 6.01软件包对数据进行统计学分析.结果:Runx2过表达有利于HUVECs的成骨分化,而Osterix过表达则无此作用.HUVECs转染Runx2过表达慢病毒后,成骨相关基因Runx2、OSX、ALP、BSP、OPN及OCN的转录水平上调,同时Runx2、OSX、OPN及OCN的蛋白表达水平亦有所上调.结论:Runx2过表达可促进HUVECs的成骨分化.     

成骨特异转录因子 Runx2 在去势大鼠正畸牙移动牙周组织中的表达

目的：研究成骨特异转录因子Runx2在去势大鼠正畸牙移动过程中牙周组织内的表达规律。方法：选用3月龄雌性未孕SD大鼠50只,随机分为去势组（OVX）与假手术组（Sham）各25只。全麻下行大鼠去势术或假手术,术后饲养3个月。构建大鼠上颌第一磨牙正畸牙移动模型,并分别于牙移动0d、1d、3d、7d与15d处死去势组大鼠和假手术组大鼠各5只,取上颌第一磨牙周围牙周组织,行H-E染色和免疫组织化学染色后观察正畸牙移动牙周组织的改建。结果：去势大鼠与假手术大鼠正畸牙移动过程中牙周组织的改建基本相似,即张力区以成骨为主,压力区以破骨为主。去势组大鼠牙移动15d张力区成骨细胞及压力区破骨细胞均显著多于假手术组大鼠（尸〈0．05）。大鼠正畸牙移动过程中牙周组织Runx2表达先升后降。牙移动1d、3d、7d张力区与压力区Runx2表达均显著高于0d（P〈0．05）,15d与0d则无显著差异。去势组大鼠牙移动牙周组织Runx2表达变化与假手术组大鼠基本一致,但是去势组1d压力区和7d张力区Runx2表达均显著高于假手术组大鼠（P〈0．05）。结论：去势大鼠正畸牙移动过程中的牙周组织改建符合正畸牙移动的基本规律,但其牙周组织的改建更为活跃。去势大鼠正畸牙移动牙周组织内Runx2的表达增强,这可能与活跃的牙周组织改建相关,其机制需进一步研究。     

Runx2/cbfal在人牙胚发育过程中的表达

观察核心因子Runx2/cbfal在人牙胚发育过程中的表达,探讨cbfal在牙齿发育过程中的作用.方法：制备人牙胚发育各期标本,免疫荧光法观察cbfal在人牙胚发育过程中的表达情况.结果：在牙胚发育的不同时期均可见不同的时空表达模式.蕾状期表达比较广泛;帽状期内外釉上皮细胞和牙囊中呈强阳性表达,中间层也为强阳性,牙乳头为弱阳性;钟状早期,前成牙本质细胞处有强阳性表达;钟状中后期,成釉细胞为强阳性,成牙本质细胞为阴性,而牙囊部位仍为强阳性.结论：Runx2/cbfal转录因子在人牙胚整个发育过程中的表达状况,提示该因子可能在牙胚各期的发育起一定作用,参与了各种成牙相关细胞的分化和硬组织的形成.     

Runx2/cbfa1在人牙胚发育过程中的表达

观察核心因子Runx2/cbfa1在人牙胚发育过程中的表达,探讨cbfa1在牙齿发育过程中的作用。方法:制备人牙胚发育各期标本,免疫荧光法观察cbfa1在人牙胚发育过程中的表达情况。结果:在牙胚发育的不同时期均可见不同的时空表达模式。蕾状期表达比较广泛;帽状期内外釉上皮细胞和牙囊中呈强阳性表达,中间层也为强阳性,牙乳头为弱阳性;钟状早期,前成牙本质细胞处有强阳性表达;钟状中后期,成釉细胞为强阳性,成牙本质细胞为阴性,而牙囊部位仍为强阳性。结论:Runx2/cbfa1转录因子在人牙胚整个发育过程中的表达状况,提示该因子可能在牙胚各期的发育起一定作用,参与了各种成牙相关细胞的分化和硬组织的形成。     

Runt相关基因2/核心结合因子a1在小鼠牙周组织发育中的免疫组化定位研究

目的探讨Runt相关基因2/核心结合因子a1(Runx2/Cbfa1)在小鼠牙周组织发育过程中的时空表达及意义。方法建立BALB/c小鼠牙周发育动物模型,采用免疫组化方法检测Runx2/Cbfa1在小鼠出生后各期牙周组织发育中的表达及特点。结果Runx2/Cbfa1在牙齿发育过程中的表达具有时空特异性,在牙根开始发育之前,仅在牙槽骨及成骨细胞中表达;当牙根开始发育即从第11天以后各期,在根部牙周膜细胞、成牙骨质细胞及成骨细胞中均呈阳性表达,但牙槽骨呈阴性表达。结论Runx2/Cbfa1在成牙骨质细胞及成骨细胞的分化及牙植骨的形成具有重要作用,可能在牙周组织的发育中发挥作用。     

Runx2/Cbfa1在出生后小鼠牙髓牙本质复合体形成中的作用研究

目的　探讨Runx2/Cbfa1在出生后小鼠成牙本质细胞分化及牙本质形成中时间和空间的分布特点及意 义。方法　采用免疫组化SABC法检测Runx2/Cbfa1在出生后BALB/c小鼠牙本质、成牙本质细胞及牙髓细胞中不 同发育时期的表达及特点。结果　Runx2/Cbfa1在成牙本质细胞的发育成熟过程中具有时空特异性,在牙根尚未发 育之前的牙胚钟状晚期,Runx2/Cbfa1只在前期牙本质中有表达,在牙髓细胞及成牙本质细胞中皆为阴性;约从第11 天开始,随着牙根的发育,Runx2/Cbfa1在根部成牙本质细胞、牙髓细胞中表达为阳性,在冠部成牙本质细胞中表达 为阴性。结论　Runx2/Cbfa1参与牙本质的形成和矿化及成牙本质细胞和牙髓细胞的发育,可能对它们起重要作 用。     

Sequential induction of marrow stromal cells by FGF2 and BMP2 improves their growth and differentiation potential in vivo

Background

Repairing bone loss by autologous grafting requires that a patient's marrow stromal cells (MSCs) be collected and cultured until the number of cells is adequate for implantation. Currently used techniques allow a slow proliferation rate and produce a culture that contains only small amounts of pluripotent stem cells that will become osteoblasts in culture.

Objective

To develop culture conditions that permit a rapid increase in the number of MSCs while retaining or improving their potential for complete differentiation in vivo.

Results

Sequential applications of low doses of basic fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 2 (BMP2) improved the growth and differentiation potential of MSCs. FGF2 also elevated sensitivity of the cells to BMP2. BMP2 increased the syntheses of alkaline phosphatase (ALP), collagen type I and bone sialoprotein, while FGF2 increased the expression of osteocalcin (OC). Full induction as determined by the formation of mineralised nodules in vitro was observed within 7 days. Seeding the induced cells onto scaffolds and then implanting them into nude mice resulted in newly formed bone 4 weeks later. The results of real-time polymerase chain reaction (PCR) and Western blotting suggested that FGF2 increased the pool of committed osteoblasts by up-regulating the Cbfa1/Runx2 gene. The later stages of bone formation seemed to be induced by Cbfa1/Runx2-downstream factors such as BMP2, ALP, collagen type I, bone sialoprotein and OC.

Conclusion

The culture system that was developed increased both the proliferation of MSC and the proportion that developed into pre-osteoblasts.     

Roles of CCN2 as a mechano-sensing regulator of chondrocyte differentiation

Cellular communication network factor 2 (CCN2) is a cysteine-rich secreted matricellular protein that regulates various cellular functions including cell differentiation. CCN2 is highly expressed under several types of mechanical stress, such as stretch, compression, and shear stress, in mesenchymal cells including chondrocytes, osteoblasts, and fibroblasts. In particular, CCN2 not only promotes cell proliferation and differentiation of various cells but also regulates the stability of mRNA of TRPV4, a mechanosensitive ion channel in chondrocytes. Of note, CCN2 behaves like a biomarker to sense suitable mechanical stress, because CCN2 expression is down-regulated when chondrocytes are subjected to excessive mechanical stress. These findings suggest that CCN2 is a mechano-sensing regulator. CCN2 expression is regulated by the activation of various mechano-sensing signaling pathways, e.g., mechanosensitive ion channels, integrin-focal adhesion-actin dynamics, Rho GTPase family members, Hippo-YAP signaling, and G protein-coupled receptors. This review summarizes the characterization of mechanoreceptors involved in CCN2 gene regulation and discusses the role of CCN2 as a mechano-sensing regulator of mesenchymal cell differentiation, with particular focus on chondrocytes.     

Runx2在牙釉质形成中的作用研究

目的:研究小鼠成釉细胞中Runx2对釉成熟蛋白(Amelotin,AMTN)、成釉蛋白(Ameloblas-tin,AMBN)、牙成釉细胞相关蛋白(odontogenic ameloblast-asssociated protein,ODAM)基因表达的调控作用,为研究Runx 2在牙釉质形成中的作用奠定基础.方法:通...     

Runx3在唾液腺腺样囊性癌中的表达及意义

目的:探讨Runx3在唾液腺腺样囊性癌(SACC)中的表达及其临床意义.方法:采用RT-PCR和蛋白分析检测Runx3在4例SACC组织及癌旁组织样品中的表达.免疫组化分析65例SACC病理组织切片中 Runx3的蛋白表达采用SPSS 17.0软件包分析病理诊断与临床资料之间的关系.结果:mRNA和蛋白水平检测结果显示,Runx3在SACC组织中的表达与癌旁组织相比明显下调.免疫组化结果显示,Runx3在SACC中的表达与淋巴结转移相关.结论:Runx3表达下调与SACC发病进程相关,可能是临床诊断和治疗SACC的重要生物标志物.     

不同下颌前伸力对髁突Runx2和X型胶原表达的影响

目的:探讨不同作用方式的下颌前伸力对髁突软骨内成骨的影响.方法:将28只5周龄雄性wistar大鼠随机分配到周期性、静止性、功能性下颌前伸组及阴性对照组(n=7).各组按照设定的相关参数接受下颌前伸力刺激.实验结束时取标本,免疫组织化学染色,对Runx2、X型胶原的表达进行半定量分析.结果:Runx2和X型胶原表达动态组最高(与对照组比P＜0.01),静态组和功能组较低(与对照组比P＜0.01).静态组X型胶原表达高于功能组(P＜0.01),Runx2表达静态组与功能组差异无显著性(P＞0.05).结论:不同作用方式及作用时间的下颌前伸力差异性调节髁突软骨细胞Runx2及X型胶原的表达水平.     

Alteration of BMP-4 and Runx2 expression patterns in mouse temporomandibular joint after ovariectomy

OBJECTIVE: Temporomandibular disorder (TMD) includes a number of clinical conditions involving the masticatory musculature or the temporomandibular joint (TMJ) and associated structures. Previous studies have shown the presence of high-affinity estrogen receptors in the TMJ articular cartilage. The aim of this study was to evaluate the developmental changes in mouse TMJ under estrogen deficiency. MATERIALS AND METHODS: Four-month-old ovariectomized mice were killed after certain weeks. We examined the significant alterations of the expression patterns of bone morphogenetic protein (BMP)-4, Runx2, and bone sialoprotein (BSP) after ovariectomy. Results: In the control group, BMP-4, Runx2, and BSP expressions showed no definite difference at any stage. In the ovariectomy group, the intensity of BMP-4 and Runx2 expression increased after ovariectomy. BSP immunoreactivity, however, increased slightly at 2 weeks but then decreased gradually. CONCLUSIONS: Estrogen plays important roles in the metabolism and maintenance of TMJ via regulations of signaling molecules such as BMP-4, Runx2, and BSP. Our results suggest that estrogen deficiency is a candidate cause of TMD. This study revealed further osteogenetic properties of estrogen that may be useful in the clinical treatment and prevention of TMD.     

ISO对大鼠正畸牙移动效果及牙周组织中Runx2、ODF水平的影响

建立SD大鼠正畸牙模型,随机分为对照组与ISO组,各20只,术后当天及7、14、21 d分别测定大鼠牙齿移动距离,HE染色观察牙周组织变化,对破骨细胞数量计数,检测Runt相关转录因子2(Runx2)和破骨细胞分化因子(ODF)水平.结果表明:加力后两组大鼠的牙齿均明显移动,且ISO组大鼠移动距离显著长于对照组(P＜0...     

Delayed tooth eruption and suppressed osteoclast number in the eruption pathway of heterozygous Runx2/Cbfa1 knockout mice

Genetic studies have recently identified a mutation of one allele of runt-related gene 2 (RUNX2/CBFA1) as the cause for an autosomal-dominant skeletal disorder, cleidocranial dysplasia (CCD), which is characterised by hypoplasia of the clavicles and calvariae and widened sutures and fontanelles. In addition, CCD is frequently affected with multiple supernumerary teeth and the impaction and delayed eruption of teeth, the causes of all these dental abnormalities are still unknown. To clarify the cellular mechanism of the delayed tooth eruption in CCD, the process of tooth eruption was examined in heterozygous Runx2/Cbfa1 (mouse homolog of RUNX2/CBFA1) knockout mice, known to mimic most of the bone abnormalities of CCD. The timing of the appearance of maxillary and mandibular teeth into the oral cavity was significantly delayed in heterozygous mutant mice compared with wild-type mice. From postnatal days 8 to 10, an active alveolar bone resorption and a marked increase of the osteoclast surfaces was observed in the eruption pathway of both genotypes, but this increase was significantly suppressed in the mutant mice. In contrast, the osteoclast surfaces did not show a significant difference between the two genotypes in the future cortical area of femora. These results suggest that haploinsufficiency of Runx2/Cbfa1 does not effect the femoral bone remodelling but is insufficient for the active alveolar bone resorption essential for the prompt timing of tooth eruption. These results also suggest the possibility that impaired recruitment of osteoclasts is one of the cellular mechanisms of delayed tooth eruption in CCD patients.