發(fā)布時間：2018-01-07 02:37

  本文關(guān)鍵詞：EphrinB2-EphB4信號通路在炎性骨缺損愈合中的作用　出處：《山東大學》2017年博士論文　論文類型：學位論文

  更多相關(guān)文章： 骨改建 ephrinB2-EphB4信號通路 TNF-α 炎性環(huán)境 siRNA

【摘要】：背景與目的目前,由炎癥、損傷、畸形和腫瘤等原因引起的口腔牙槽骨吸收與頜骨缺損是口腔科許多疾病最常見的臨床表現(xiàn)之一,嚴重影響人們的美觀、咀嚼、發(fā)音以及進一步的義齒修復。因此,如何調(diào)控骨重塑、恢復組織功能成為臨床和基礎(chǔ)研究的重點[1]。我們口腔科的常見病多發(fā)病之一牙周病就是其中的一種以牙槽骨吸收、牙齒松動脫落為主要特征的炎性骨代謝疾病。在牙周病的進程中,骨改建的兩大參與者成骨細胞和破骨細胞均浸浴于慢性炎性環(huán)境中,其功能都會受到不同程度的影響。然而,雖然現(xiàn)有的研究對成骨細胞或者破骨細胞在炎性刺激過程中表現(xiàn)出的功能變化已有了較為清晰的認識,但這些研究大多是將成骨細胞(骨形成)或破骨細胞(骨吸收)各自作為獨立的個體分離開進行研究,因此缺乏在炎性環(huán)境刺激下,成骨細胞和破骨細胞之間在信息交流和互相調(diào)控等方面是否存在失衡的準確信息[2,3]。關(guān)于成骨細胞和破骨細胞之間互相交流的分子信號通路,主要分為以下兩大通路:RANKL-RANK-OPG通路和EphrinB2-EphB4信號通路。以往文獻中報道最多的是RANKL-RANK-OPG信號通路,而對另一通路知之甚少,在口腔領(lǐng)域的研究更少。EphrinB2-EphB4信號通路是2006年由Matsuo等首先報道的骨改建過程中成骨細胞-破骨細胞間信號傳遞的一條新通路,這條信號通路是通過破骨細胞表面表達的ephrinB2和成骨細胞表達的EphB4跨膜蛋白受體間產(chǎn)生雙向信號傳導從而實現(xiàn)信息的傳遞和互相調(diào)控。其作用機理簡言之,破骨細胞表面表達的NFATc1的靶基因ephrinB2和成骨細胞表面表達的EphB4受體相結(jié)合產(chǎn)生雙向的信號傳遞,其中逆向信號由ephrinB2介導將信息傳入破骨細胞,并通過抑制c-Fos-NFATc1的活性從而抑制破骨細胞前體細胞分化為破骨細胞;另一方面,正向信號由EphB4介導通過增強RhoA的活性誘導成骨細胞的分化。Zhao C的研究表明,通過在轉(zhuǎn)基因小鼠的成骨細胞中過度表達EphB4,骨基質(zhì)的沉積量被顯著增加[2]。由EphB4-ephrinB2參與的信號通路在促進骨形成的同時限制了過度的骨吸收,因而對于維持骨改建的平衡起著至關(guān)重要的作用。TNF-α是目前公認的炎性微環(huán)境中最重要的一種內(nèi)源性的炎性介質(zhì),在炎癥過程中起著最基本的作用,也是牙周病發(fā)生發(fā)展過程中重要的介導因子。它的作用原理是通過激活靶細胞內(nèi):NF-kB通路和MAPKS通路如p38,ERK和JNK通路使細胞發(fā)生增殖、分化、炎癥等應激反應[4,5,6]。RNA干擾(RNA interference,RNAi)是廣泛存在于真菌、植物和動物等真核生物體內(nèi)一種序列特異性基因沉默,而小干擾RNA(small interferingRNA,siRNA)是RNA干擾的中間產(chǎn)物,它是通過外源性或內(nèi)源性的雙鏈RNA(dsRNA),特異性結(jié)合了與其序列互補的m RNA,誘導該m RNA的降解,沉默該基因的表達,屬于轉(zhuǎn)錄后水平的基因沉默[7,8]。慢病毒載體是常用的介導RNA干擾的病毒載體之一,其優(yōu)點為既可以感染分裂期細胞,又可以感染非分裂期細胞,且不易誘發(fā)宿主免疫反應,轉(zhuǎn)染效率高,表達的持續(xù)時間較長,因此是基因沉默的有效工具[9,10]。本研究采用慢病毒干擾載體降低小鼠體內(nèi)目的基因ephrinB2和EphB4的表達,研究ephrinB2和EphB4基因沉默后骨缺損處骨形成和骨吸收活動的變化,并進一步探討了 ephrinB2-EphB4信號通路對炎性骨缺損愈合的作用及意義,以期為臨床研發(fā)新的牙周治療方法和牙周病新藥的研發(fā)開辟一條新思路,并為牙周病新藥的研發(fā)提供新的藥物作用靶點,因而具有重要的臨床意義和科研價值。材料與方法第一部分小鼠炎性微環(huán)境模型的構(gòu)建及對骨缺損愈合的影響方法雄性C57BL/6小鼠30只,體重18-20g,隨機分為3組:0.5μg/kg組(下頜骨缺損,隔日腹腔注射TNF-α 0.5μg/kg)、3μg/kg組(下頜骨缺損,隔日腹腔注射TNF-α3μg/kg)和5μg/kg組(下頜骨缺損,隔日腹腔注射TNF-α5μg/kg)。于術(shù)后當天、3天、7天、10天、14天通過小鼠的內(nèi)眥靜脈采血,酶聯(lián)免疫吸附實驗檢測血液中TNF-α的水平;36只雄性C57BL/6小鼠體內(nèi)建立下頜骨實驗性骨缺損,手術(shù)當天開始腹腔注射0、0.5、3或5μg/kg的TNF-α,隔天注射一次。于術(shù)后3天、7天、14天取缺損側(cè)的下頜骨組織,HE染色觀察骨缺損處新骨形成的情況。結(jié)果Elisa(酶聯(lián)免疫吸附實驗)結(jié)果顯示:0.5μg/kg腹腔注射組3天時血清中TNF-α的含量上升至1.50±0.25 ng/ml,14天時又降低至0.31±0.35 ng/ml,呈先上升后下降的趨勢;而3μg/kg組呈先上升后下降的趨勢,10天時小鼠血清中TNF-α的含量最高,但14天時又有所下降;5μg/kg組血清中TNF-α的含量隨時間呈現(xiàn)升高的趨勢,7天后達到穩(wěn)定的血藥濃度。因此,為了在小鼠體內(nèi)形成穩(wěn)定的炎性環(huán)境,我們選擇了 5μg/kgTNF-α腹腔注射,隔天注射一次的方法用于后續(xù)的實驗。HE染色結(jié)果發(fā)現(xiàn),術(shù)后3天,0.5μg/kg組、3μg/kg組和5μg/kg組新生骨量低于對照組(0μg/kg組),但四者無統(tǒng)計學差異(p0.05);術(shù)后7天和14天時,3μg/kg組和5μg/kg組缺損處的新生骨量顯著低于對照組(p0.05),但0.5μg/kg組與對照組相比,新生骨量無統(tǒng)計學差異(p0.05)。第二部分EphrinB2和EphB4小干擾RNA慢病毒載體的構(gòu)建和包裝方法1.包含目的基因的siRNA干擾載體的構(gòu)建委托ThermoFisher Scientific公司合成含設(shè)計好的針對ephrinB2和EphB4干擾序列的單鏈DNA(single strand DNA,ssDNA)oligo,共8對,然后退火延伸形成互補雙鏈。用特異限制性內(nèi)切酶酶切干擾序列兩端所含酶切位點后,使用DNA連接酶接入酶切后的pcDNA6.2TM-GW/EmGFP-miR載體上,將產(chǎn)物轉(zhuǎn)染進入細菌感受態(tài)細胞,測序驗證。PCR反應檢測ephrinB2和EphB4的mRNA水平,篩選干擾效率最高的兩組干擾載體和慢病毒目的載體pLenti6.3/V5-DEST重組,以獲得含干擾序列的慢病毒表達載體。2.慢病毒載體的包裝和滴度測定擴增構(gòu)建完成的含有目的基因干擾序列的慢病毒表達載體,將其與三種包裝載體質(zhì)粒pLP1,pLP2和pLP/VSVG共轉(zhuǎn)染HEK293T細胞,包裝慢病毒并收集病毒原液,超速離心濃縮后,梯度稀釋法測定病毒的滴度,分裝保存?zhèn)溆�。結(jié)果1.包含目的基因的siRNA干擾載體的構(gòu)建DNA序列測定結(jié)果證明所插入的片段序列與合成序列完全一致,表明已成功構(gòu)建了針對ephrinB2和EphB4基因的干擾載體。轉(zhuǎn)染48小時后,PCR篩選結(jié)果顯示,針對目的基因ephrinB2,1#干擾載體的基因沉默效果最佳(87%),針對目的基因EphB4,2#干擾載體的基因沉默效果最佳(77%)。所以我們選擇了ephrinB2 siRNA1#和 EphB4 siRNA 2#載體和慢病毒目的載體 pLenti6.3/V5-DEST進行重組,獲得含靶基因干擾序列的慢病毒表達載體。2.慢病毒載體的包裝和滴度測定將Lenti-E2 1#和Lenti-E4 2#慢病毒感染HEK293T細胞,96小時后梯度稀釋法測定病毒的滴度,經(jīng)計算得知,Lenti-E2 1#病毒的滴度為3.5*108TU/ml,Lenti-E42#病毒的滴度為2.5*108TU/ml,分別命名為pLenti6.3-efnb2siRNA和pLenti6.3-ephb4siRNA,將慢病毒液稀釋至1*108TU/ml,分裝保存于-80度備用。第三部分EphrinB2-EphB4信號通路對炎性環(huán)境下骨缺損愈合的影響方法選用6-8周齡健康雄性C57BL/6小鼠108只,隨機分成pLenti6.3-ctrl、pLenti6.3-efnb2siRNA 和 pLenti6.3-ephb4siRNA 3 組,建立下頜骨骨缺損,各組缺損內(nèi)分別注射 5μl pLenti6.3-ctrl,pLenti6.3-efnb2siRNA 和pLenti6.3-ephb4siRNA,隔日腹腔注射 TNF-α 5μg/kg,分別于術(shù)后 7、14 和 21天取缺損側(cè)的小鼠下頜骨標本,Real-time PCR檢測骨缺損處ephrinB2和EphB4兩種因子以及成骨因子Runx2,Osterix,OC,ALP,BSP和骨吸收因子Nfatc1 mRNA的表達;Western blot檢測Runx2和BSP蛋白的表達;HE染色觀察骨缺損處新骨形成的情況;免疫組化觀察Runx2和OC在骨缺損處的定位和蛋白表達情況,并進行光密度值分析;TRAP染色觀察缺損處骨吸收情況并進行破骨細胞計數(shù)。結(jié)果Realtime-PCR 結(jié)果顯示,實驗組(pLenti6.3-efnb2siRNA 組和pLenti6.3-ephb4siRNA組)目的基因ephrinB2和EphB4的表達水平相對于對照組pLenti6.3-ctrl組在三個時間點(7天、14天和21天)均是顯著降低的(p0.05);7天和14天時,pLenti6.3-ephb4組Runx2和Osterix mRNA表達顯著低于對照組(分別p0.01,p0.05),而ALPmRNA的表達量在三個時間點均顯著低于對照組,晚期成骨因子OC和BSP mRNA的表達量在14天和21天時亦顯著低于對照組(分別p0.01,p0.05),而Nfatc1(7天和14天)mRNA的表達顯著高于對照組(分別p0.01,p0.05);Western blot結(jié)果顯示,Runx2和BSP蛋白表達的結(jié)果與PCR分析相一致;HE染色發(fā)現(xiàn),14天時,pLenti6.3-ephb4組新生骨量顯著低于對照組(p0.01);免疫組化結(jié)果顯示:Runx2和OC在小鼠的成骨細胞、骨細胞和成纖維細胞中均有廣泛的表達,光密度分析顯示Runx2和OC蛋白表達的結(jié)果與PCR分析相一致;TRAP染色結(jié)果表明:7天和14天時,pLenti6.3-ephb4siRNA組TRAP(+)破骨細胞數(shù)顯著高于對照組(分別p0.01,p0.05),而pLenti6.3-efnb2siRNA組骨形成因子和骨吸收因子、缺損處的新生骨量以及破骨細胞計數(shù)與對照組相比均無統(tǒng)計學差異(p0.05)。結(jié)論1.采用5μg/kgTNF-α腹腔注射,隔天注射一次的方式,成功構(gòu)建了小鼠體內(nèi)穩(wěn)定的炎性環(huán)境模型。2.成功構(gòu)建了針對目的基因ephrinB2和EphB4的穩(wěn)定高效的慢病毒載體:pLenti6.3-efnb2siRNA 和 pLenti6.3-ephb4siRNA,基因沉默效果分別為 87%和87%,可用于后續(xù)的體內(nèi)實驗。3.通過下調(diào)EphrinB2和EphB4的表達發(fā)現(xiàn):EphB4表達降低,骨形成抑制,同時伴有成骨相關(guān)標志物mRNA和蛋白表達的下降,破骨細胞數(shù)及破骨標志物表達的升高;而ephrinB2表達降低,上述各指標無明顯影響,推測可能由于體內(nèi)EphrinB1或其他因子對其提供了代償作用。因此,我們推斷EphB4在炎性骨缺損愈合的過程中起著至關(guān)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 .緇撴灉Elisa(閰惰仈鍏嶇柅鍚擱檮瀹為獙)緇撴灉鏄劇ず:0.5渭g/kg鑵硅厰娉ㄥ皠緇，

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