EphrinB2-EphB4信号通路在炎性骨缺损愈合中的作用

发布时间：2018-01-07 02:37

  本文关键词：EphrinB2-EphB4信号通路在炎性骨缺损愈合中的作用　出处：《山东大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 骨改建 ephrinB2-EphB4信号通路 TNF-α 炎性环境 siRNA

【摘要】：背景与目的目前,由炎症、损伤、畸形和肿瘤等原因引起的口腔牙槽骨吸收与颌骨缺损是口腔科许多疾病最常见的临床表现之一,严重影响人们的美观、咀嚼、发音以及进一步的义齿修复。因此,如何调控骨重塑、恢复组织功能成为临床和基础研究的重点[1]。我们口腔科的常见病多发病之一牙周病就是其中的一种以牙槽骨吸收、牙齿松动脱落为主要特征的炎性骨代谢疾病。在牙周病的进程中,骨改建的两大参与者成骨细胞和破骨细胞均浸浴于慢性炎性环境中,其功能都会受到不同程度的影响。然而,虽然现有的研究对成骨细胞或者破骨细胞在炎性刺激过程中表现出的功能变化已有了较为清晰的认识,但这些研究大多是将成骨细胞(骨形成)或破骨细胞(骨吸收)各自作为独立的个体分离开进行研究,因此缺乏在炎性环境刺激下,成骨细胞和破骨细胞之间在信息交流和互相调控等方面是否存在失衡的准确信息[2,3]。关于成骨细胞和破骨细胞之间互相交流的分子信号通路,主要分为以下两大通路:RANKL-RANK-OPG通路和EphrinB2-EphB4信号通路。以往文献中报道最多的是RANKL-RANK-OPG信号通路,而对另一通路知之甚少,在口腔领域的研究更少。EphrinB2-EphB4信号通路是2006年由Matsuo等首先报道的骨改建过程中成骨细胞-破骨细胞间信号传递的一条新通路,这条信号通路是通过破骨细胞表面表达的ephrinB2和成骨细胞表达的EphB4跨膜蛋白受体间产生双向信号传导从而实现信息的传递和互相调控。其作用机理简言之,破骨细胞表面表达的NFATc1的靶基因ephrinB2和成骨细胞表面表达的EphB4受体相结合产生双向的信号传递,其中逆向信号由ephrinB2介导将信息传入破骨细胞,并通过抑制c-Fos-NFATc1的活性从而抑制破骨细胞前体细胞分化为破骨细胞;另一方面,正向信号由EphB4介导通过增强RhoA的活性诱导成骨细胞的分化。Zhao C的研究表明,通过在转基因小鼠的成骨细胞中过度表达EphB4,骨基质的沉积量被显著增加[2]。由EphB4-ephrinB2参与的信号通路在促进骨形成的同时限制了过度的骨吸收,因而对于维持骨改建的平衡起着至关重要的作用。TNF-α是目前公认的炎性微环境中最重要的一种内源性的炎性介质,在炎症过程中起着最基本的作用,也是牙周病发生发展过程中重要的介导因子。它的作用原理是通过激活靶细胞内:NF-kB通路和MAPKS通路如p38,ERK和JNK通路使细胞发生增殖、分化、炎症等应激反应[4,5,6]。RNA干扰(RNA interference,RNAi)是广泛存在于真菌、植物和动物等真核生物体内一种序列特异性基因沉默,而小干扰RNA(small interferingRNA,siRNA)是RNA干扰的中间产物,它是通过外源性或内源性的双链RNA(dsRNA),特异性结合了与其序列互补的m RNA,诱导该m RNA的降解,沉默该基因的表达,属于转录后水平的基因沉默[7,8]。慢病毒载体是常用的介导RNA干扰的病毒载体之一,其优点为既可以感染分裂期细胞,又可以感染非分裂期细胞,且不易诱发宿主免疫反应,转染效率高,表达的持续时间较长,因此是基因沉默的有效工具[9,10]。本研究采用慢病毒干扰载体降低小鼠体内目的基因ephrinB2和EphB4的表达,研究ephrinB2和EphB4基因沉默后骨缺损处骨形成和骨吸收活动的变化,并进一步探讨了 ephrinB2-EphB4信号通路对炎性骨缺损愈合的作用及意义,以期为临床研发新的牙周治疗方法和牙周病新药的研发开辟一条新思路,并为牙周病新药的研发提供新的药物作用靶点,因而具有重要的临床意义和科研价值。材料与方法第一部分小鼠炎性微环境模型的构建及对骨缺损愈合的影响方法雄性C57BL/6小鼠30只,体重18-20g,随机分为3组:0.5μg/kg组(下颌骨缺损,隔日腹腔注射TNF-α 0.5μg/kg)、3μg/kg组(下颌骨缺损,隔日腹腔注射TNF-α3μg/kg)和5μg/kg组(下颌骨缺损,隔日腹腔注射TNF-α5μg/kg)。于术后当天、3天、7天、10天、14天通过小鼠的内眦静脉采血,酶联免疫吸附实验检测血液中TNF-α的水平;36只雄性C57BL/6小鼠体内建立下颌骨实验性骨缺损,手术当天开始腹腔注射0、0.5、3或5μg/kg的TNF-α,隔天注射一次。于术后3天、7天、14天取缺损侧的下颌骨组织,HE染色观察骨缺损处新骨形成的情况。结果Elisa(酶联免疫吸附实验)结果显示:0.5μg/kg腹腔注射组3天时血清中TNF-α的含量上升至1.50±0.25 ng/ml,14天时又降低至0.31±0.35 ng/ml,呈先上升后下降的趋势;而3μg/kg组呈先上升后下降的趋势,10天时小鼠血清中TNF-α的含量最高,但14天时又有所下降;5μg/kg组血清中TNF-α的含量随时间呈现升高的趋势,7天后达到稳定的血药浓度。因此,为了在小鼠体内形成稳定的炎性环境,我们选择了 5μg/kgTNF-α腹腔注射,隔天注射一次的方法用于后续的实验。HE染色结果发现,术后3天,0.5μg/kg组、3μg/kg组和5μg/kg组新生骨量低于对照组(0μg/kg组),但四者无统计学差异(p0.05);术后7天和14天时,3μg/kg组和5μg/kg组缺损处的新生骨量显著低于对照组(p0.05),但0.5μg/kg组与对照组相比,新生骨量无统计学差异(p0.05)。第二部分EphrinB2和EphB4小干扰RNA慢病毒载体的构建和包装方法1.包含目的基因的siRNA干扰载体的构建委托ThermoFisher Scientific公司合成含设计好的针对ephrinB2和EphB4干扰序列的单链DNA(single strand DNA,ssDNA)oligo,共8对,然后退火延伸形成互补双链。用特异限制性内切酶酶切干扰序列两端所含酶切位点后,使用DNA连接酶接入酶切后的pcDNA6.2TM-GW/EmGFP-miR载体上,将产物转染进入细菌感受态细胞,测序验证。PCR反应检测ephrinB2和EphB4的mRNA水平,筛选干扰效率最高的两组干扰载体和慢病毒目的载体pLenti6.3/V5-DEST重组,以获得含干扰序列的慢病毒表达载体。2.慢病毒载体的包装和滴度测定扩增构建完成的含有目的基因干扰序列的慢病毒表达载体,将其与三种包装载体质粒pLP1,pLP2和pLP/VSVG共转染HEK293T细胞,包装慢病毒并收集病毒原液,超速离心浓缩后,梯度稀释法测定病毒的滴度,分装保存备用。结果1.包含目的基因的siRNA干扰载体的构建DNA序列测定结果证明所插入的片段序列与合成序列完全一致,表明已成功构建了针对ephrinB2和EphB4基因的干扰载体。转染48小时后,PCR筛选结果显示,针对目的基因ephrinB2,1#干扰载体的基因沉默效果最佳(87%),针对目的基因EphB4,2#干扰载体的基因沉默效果最佳(77%)。所以我们选择了ephrinB2 siRNA1#和 EphB4 siRNA 2#载体和慢病毒目的载体 pLenti6.3/V5-DEST进行重组,获得含靶基因干扰序列的慢病毒表达载体。2.慢病毒载体的包装和滴度测定将Lenti-E2 1#和Lenti-E4 2#慢病毒感染HEK293T细胞,96小时后梯度稀释法测定病毒的滴度,经计算得知,Lenti-E2 1#病毒的滴度为3.5*108TU/ml,Lenti-E42#病毒的滴度为2.5*108TU/ml,分别命名为pLenti6.3-efnb2siRNA和pLenti6.3-ephb4siRNA,将慢病毒液稀释至1*108TU/ml,分装保存于-80度备用。第三部分EphrinB2-EphB4信号通路对炎性环境下骨缺损愈合的影响方法选用6-8周龄健康雄性C57BL/6小鼠108只,随机分成pLenti6.3-ctrl、pLenti6.3-efnb2siRNA 和 pLenti6.3-ephb4siRNA 3 组,建立下颌骨骨缺损,各组缺损内分别注射 5μl pLenti6.3-ctrl,pLenti6.3-efnb2siRNA 和pLenti6.3-ephb4siRNA,隔日腹腔注射 TNF-α 5μg/kg,分别于术后 7、14 和 21天取缺损侧的小鼠下颌骨标本,Real-time PCR检测骨缺损处ephrinB2和EphB4两种因子以及成骨因子Runx2,Osterix,OC,ALP,BSP和骨吸收因子Nfatc1 mRNA的表达;Western blot检测Runx2和BSP蛋白的表达;HE染色观察骨缺损处新骨形成的情况;免疫组化观察Runx2和OC在骨缺损处的定位和蛋白表达情况,并进行光密度值分析;TRAP染色观察缺损处骨吸收情况并进行破骨细胞计数。结果Realtime-PCR 结果显示,实验组(pLenti6.3-efnb2siRNA 组和pLenti6.3-ephb4siRNA组)目的基因ephrinB2和EphB4的表达水平相对于对照组pLenti6.3-ctrl组在三个时间点(7天、14天和21天)均是显著降低的(p0.05);7天和14天时,pLenti6.3-ephb4组Runx2和Osterix mRNA表达显著低于对照组(分别p0.01,p0.05),而ALPmRNA的表达量在三个时间点均显著低于对照组,晚期成骨因子OC和BSP mRNA的表达量在14天和21天时亦显著低于对照组(分别p0.01,p0.05),而Nfatc1(7天和14天)mRNA的表达显著高于对照组(分别p0.01,p0.05);Western blot结果显示,Runx2和BSP蛋白表达的结果与PCR分析相一致;HE染色发现,14天时,pLenti6.3-ephb4组新生骨量显著低于对照组(p0.01);免疫组化结果显示:Runx2和OC在小鼠的成骨细胞、骨细胞和成纤维细胞中均有广泛的表达,光密度分析显示Runx2和OC蛋白表达的结果与PCR分析相一致;TRAP染色结果表明:7天和14天时,pLenti6.3-ephb4siRNA组TRAP(+)破骨细胞数显著高于对照组(分别p0.01,p0.05),而pLenti6.3-efnb2siRNA组骨形成因子和骨吸收因子、缺损处的新生骨量以及破骨细胞计数与对照组相比均无统计学差异(p0.05)。结论1.采用5μg/kgTNF-α腹腔注射,隔天注射一次的方式,成功构建了小鼠体内稳定的炎性环境模型。2.成功构建了针对目的基因ephrinB2和EphB4的稳定高效的慢病毒载体:pLenti6.3-efnb2siRNA 和 pLenti6.3-ephb4siRNA,基因沉默效果分别为 87%和87%,可用于后续的体内实验。3.通过下调EphrinB2和EphB4的表达发现:EphB4表达降低,骨形成抑制,同时伴有成骨相关标志物mRNA和蛋白表达的下降,破骨细胞数及破骨标志物表达的升高;而ephrinB2表达降低,上述各指标无明显影响,推测可能由于体内EphrinB1或其他因子对其提供了代偿作用。因此,我们推断EphB4在炎性骨缺损愈合的过程中起着至关重要的作用。
[Abstract]:Background and objective: at present, the inflammation, injury, oral alveolar bone deformity and other reasons caused by tumor uptake and jaw defect is one of the most common clinical manifestation of many diseases in Department of Stomatology, seriously affects people's appearance, chewing, pronunciation and further denture. Therefore, how to regulate bone remodeling, restore tissue function has become the focus of clinical [1]. on the basis of the Department of Stomatology and our common disease of a periodontal disease is one of the absorption of alveolar bone, teeth loose for inflammatory disease of bone metabolism mainly features. In the process of periodontal disease, bone remodeling in two participants of osteoblasts and osteoclasts in chronic bath the inflammatory environment, its function will be affected in different degrees. However, although the existing research on osteoblasts or osteoclasts showed in inflammatory stimulation in the process of change has a function For a clear understanding, but most of these studies is the osteoblasts (bone formation) and osteoclasts (bone resorption) respectively as independent individuals from research, thus lacking in inflammatory stimuli, osteoblasts and osteoclasts are molecular pathways to communicate accurate information [2,3]. the imbalance on osteoblasts and osteoclasts in the exchange of information and mutual control etc., mainly divided into the following two major pathways: RANKL-RANK-OPG pathway and EphrinB2-EphB4 pathway. Reported in the literature is the most RANKL-RANK-OPG signaling pathway, another pathway is poorly understood, less research of.EphrinB2-EphB4 signal pathway in stomatology is the first reported by Matsuo in 2006 reconstruction of the bone during osteoblast break a new pathway of signal transfer between bone cells, this pathway is by osteoclasts The surface expression of ephrinB2 and the expression of osteogenic EphB4 transmembrane protein receptor is generated between the bidirectional signaling transfer so as to realize the information and mutual regulation. Its mechanism of action in short, target gene ephrinB2 osteoclast surface NFATc1 expression and osteogenic cell surface expression of EphB4 receptors produced by combination of two-way signal transmission. The reverse signal mediated by ephrinB2 transmits information to the osteoclast, and by inhibiting c-Fos-NFATc1 activity and inhibit osteoclast precursor cells differentiate into osteoclasts; on the other hand, the positive signal mediated by EphB4 through enhancing the activity of RhoA induced differentiation of bone cells of.Zhao C showed that in transgenic over expression of EphB4 mice into bone cells, bone matrix deposition was significantly increased by EphB4-ephrinB2 in the [2]. signaling pathway in promoting bone formation while limiting excessive The bone resorption, and to maintain the balance of bone remodeling plays a crucial role in.TNF- alpha is an endogenous inflammatory mediators most important inflammatory microenvironment in the currently recognized, plays the most basic role in the inflammatory process, and periodontal disease important mediators in the development process of action. It is the principle of the target cells through activation of NF-kB pathway and MAPKS pathway such as p38, ERK and JNK pathways make the cell proliferation, differentiation, inflammation and stress response of [4,5,6].RNA interference (RNA interference, RNAi) is widespread in fungi, a sequence specific gene silencing in plants and animal and eukaryotic organisms. Small interfering RNA (small interferingRNA siRNA) is the intermediate product of RNA interference, it is through the double stranded RNA exogenous or endogenous (dsRNA), specific binding with m RNA complementary sequence, the M degradation induced by RNA, the gene silencing The expression of [7,8]. gene silencing lentivirus vector belongs to the post transcriptional level is one of the widely used viral vector mediated RNA interference, the utility model has the advantages of mitotic cells can be infected, and can infect non dividing cells, and induce the host immune response, high transfection efficiency and expression of long duration, so it is the expression of gene silencing of [9,10]. is an effective tool for this study used lentivirus vector to reduce mouse gene ephrinB2 and EphB4, and the changes of bone resorption activity of bone defect and bone formation of ephrinB2 after EphB4 gene silencing, and further discusses the role and significance of ephrinB2-EphB4 signal pathway on the healing of inflammatory bone defects, in order to research for the clinical development of new periodontal treatment and periodontal disease drugs opens up a new idea, and to provide new drug targets for drug development of periodontal disease, and for The clinical significance and important scientific value. Materials and methods the first part in inflammatory microenvironment model and effects on bone defect healing method of 30 male C57BL/6 mice, weighing 18-20g, were randomly divided into 3 groups: 0.5 g/kg group (mandibular defect, intraperitoneal injected with TNF- alpha 0.5 g/kg), 3 g/kg group (mandibular defect, intraperitoneal injected with TNF- alpha 3 g/kg) and 5 g/kg (group of mandibular defect, intraperitoneal injected with TNF- alpha 5 g/kg). On the day after the surgery, 3 days, 7 days, 10 days, 14 days through the inner canthus vein blood of mice, ELISA. Checking the blood level of TNF- alpha; 36 male C57BL/6 mice to establish experimental mandibular bone defect surgery were intraperitoneally injected with 0,0.5,3 or 5 g/kg TNF- alpha, the injection time. After 3 days, 7 days, 14 days from the side of the mandibular defect tissue, HE staining of bone defect the new bone formation condition .缁撴灉Elisa(閰惰仈鍏嶇柅鍚搁檮瀹為獙)缁撴灉鏄剧ず:0.5渭g/kg鑵硅厰娉ㄥ皠缁，

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