本文關(guān)鍵詞：牙齦卟啉單胞菌脂多糖對破骨細(xì)胞EphA2表達(dá)的調(diào)控　出處：《吉林大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Pg-LPS 破骨細(xì)胞 EphA2

【摘要】：背景 牙周炎是以牙周結(jié)締組織破壞和牙槽骨吸收為特點的慢性炎癥性疾病，牙菌斑生物膜中的牙齦卟啉單胞菌（Porphyromonasgingivalis，Pg）是牙周病變區(qū)主要的優(yōu)勢菌之一，該菌能夠產(chǎn)生大量的毒力因子，如內(nèi)毒素、胰酶樣蛋白酶等，這些毒力因子是引起牙周結(jié)締組織的破壞和牙槽骨的吸收的重要因素，其中內(nèi)毒素也稱脂多糖（lipopolysaccharide，LPS）是革蘭氏陰性細(xì)菌細(xì)胞壁成分,是引發(fā)牙槽骨吸收的關(guān)鍵因子，它的作用是雙方面的，一方面它可以通過促進破骨細(xì)胞的分化促進骨吸收，同時還可以抑制成骨細(xì)胞的分化而抑制骨形成。骨重建（bone remodeling）是一種有序、耦聯(lián)（coupling）的骨吸收和骨形成過程，兩者間處于動態(tài)平衡以維持骨組織內(nèi)環(huán)境的穩(wěn)態(tài)（homeostasis），且伴隨終生。近來研究表明，ephrin/Eph雙向信號轉(zhuǎn)導(dǎo)通路參與調(diào)節(jié)骨代謝平衡，對維持骨穩(wěn)態(tài)起重要作用，而其中的ephrinA2/EphA2信號在骨重建的骨吸收階段發(fā)揮顯著作用，而EphA2信號分子是否參與Pg-LPS介導(dǎo)的炎癥性骨吸收，目前國內(nèi)外尚未見研究報道�；谝陨媳尘把芯�，我們提出了假設(shè)，，Pg-LPS是否通過EphA2/ephrinA2雙向信號通路參與牙槽骨吸收呢？因此本實驗通過用Pg-LPS與小鼠單核巨噬細(xì)胞RAW264.7共培養(yǎng)，觀察EphA2表達(dá)的變化，以探討EphA2信號分子在Pg-LPS介導(dǎo)的炎癥性吸收過程中的作用。 方法： 使用濃度為10g/ml的Pg-LPS與RAW264.7細(xì)胞共培養(yǎng)，分別在1、3、5d三個時間點，采用RT-PCR和Weston blotting技術(shù)分別檢測EphA2基因和破骨相關(guān)基因的表達(dá)及EphA2蛋白質(zhì)的表達(dá)，并通過TRAP染色檢測破骨細(xì)胞的分化成熟情況。 結(jié)果： 1. RT-PCR結(jié)果顯示：在Pg-LPS與RAW264.7細(xì)胞共培養(yǎng)0.5h、1h、2h三個時間點，實驗組EphA2基因的表達(dá)比對照組明顯增高。 2. RT-PCR結(jié)果顯示：在Pg-LPS與RAW264.7細(xì)胞共培養(yǎng)后，EphA2基因的表達(dá)在3d和5d時，實驗組較對照組均有明顯增加（P0.01），其中3d組較5d組，EphA2基因的表達(dá)更為明顯。但在1d時兩組間EphA2基因表達(dá)無顯著性差異。伴隨著EphA2基因上調(diào)的同時，同時破骨相關(guān)基因c-fos、NFATc1、CtsK、ACP5、MMP9的表達(dá)都明顯升高（P 0.05），但是CtsK在1d時和c-fos在5d時的表達(dá)無明顯差異。 3. Weston blotting結(jié)果顯示：在Pg-LPS與RAW264.7細(xì)胞共培養(yǎng)后，在1d時實驗組較對照組EphA2的表達(dá)明顯增加，但是在3、5d兩個時間點EphA2的表達(dá)無明顯差異。 4. TRAP染色結(jié)果顯示：在Pg-LPS與RAW264.7細(xì)胞共培養(yǎng)5d后,實驗組較對照組的TRAP陽性多核細(xì)胞（細(xì)胞核≥3）數(shù)目明顯增多。 結(jié)論： 1.在無破骨誘導(dǎo)的條件下，Pg-LPS能夠促進RAW264.7細(xì)胞表達(dá)EphA2基因。 2.在破骨誘導(dǎo)條件下，Pg-LPS在中期和晚期能夠促進RAW264.7細(xì)胞向破骨細(xì)胞分化，但是在早期促進作用不明顯。 3. Pg-LPS可能通過EphA2/ephrinA2信號通路參與調(diào)節(jié)RAW264.7細(xì)胞向破骨細(xì)胞分化的過程。
[Abstract]:background
Periodontitis is a chronic inflammatory disease of the periodontal connective tissue destruction and alveolar bone absorption characteristics, in plaque of Porphyromonas gingivalis (Porphyromonasgingivalis, Pg) is one of the dominant bacteria of periodontal disease in the region, the bacteria can produce a large number of virulence factors, such as endotoxin, trypsin like protease. These virulence factors are the important factors causing the absorption of periodontal connective tissue destruction and alveolar bone, which is also called endotoxin lipopolysaccharide (lipopolysaccharide, LPS) is a gram-negative bacterial cell wall components, is the key factor causing the absorption of alveolar bone, its role is twofold, on the one hand it can promote the differentiation of osteoclasts to promote bone absorption, but also can inhibit osteoblast differentiation and inhibit bone formation. Bone reconstruction (bone remodeling) is a kind of orderly, coupling (coupling) of bone resorption and bone formation. Between the two, in a dynamic balance to maintain a steady state in the bone tissue environment (homeostasis), and lifelong. Recent studies showed that ephrin/Eph bidirectional signal transduction pathway involved in the regulation of bone metabolism, plays an important role in maintaining bone homeostasis, and one of the ephrinA2/EphA2 signal in bone resorption phase and play a significant role. EphA2 signaling is involved in Pg-LPS mediated inflammatory bone resorption, it has not yet been reported. Based on the above background, we put forward the hypothesis, whether the Pg-LPS through the EphA2/ephrinA2 bidirectional signaling pathways involved in alveolar bone resorption? So Pg-LPS and RAW264.7 in mouse macrophage co culture through this experiment, to observe the changes of the expression of EphA2 the inflammatory process in order to investigate the absorption of EphA2 signal molecules in Pg-LPS mediated in vitro.
Method錛

本文編號：1384529