本文選題：Pg-LPS + 成骨細(xì)胞　； 參考：《吉林大學(xué)》2014年碩士論文

【摘要】：背景 牙齦卟啉單胞菌（Porphyromonas gingivalis，Pg）是牙周病重要的可疑致病菌，該菌可產(chǎn)生大量的毒力因子，如內(nèi)毒素、膠原酶、胰酶樣蛋白酶等，將引起牙周結(jié)締組織的破壞和牙槽骨的吸收，其中內(nèi)毒素也稱脂多糖（lipopolysaccharide，LPS）是引發(fā)牙槽骨吸收的關(guān)鍵因子，一方面它可以通過促進(jìn)破骨細(xì)胞的分化促進(jìn)骨吸收，另一方面還可以抑制成骨細(xì)胞的分化而抑制骨形成。雖然目前關(guān)于LPS引起的骨穩(wěn)態(tài)失衡的機(jī)制研究已經(jīng)在廣泛進(jìn)行，但是對于LPS如何調(diào)控成骨細(xì)胞和破骨細(xì)胞的具體分子機(jī)制尚不十分清楚。近年來Eph/ephrin信號分子在骨重建過程的作用備受學(xué)者們關(guān)注。新近研究表明，破骨細(xì)胞和成骨細(xì)胞間的EphA2/ephrinA2信號分子在骨重建過程中，是啟動骨吸收的重要信號分子。當(dāng)成骨細(xì)胞和破骨細(xì)胞表面EphA2/ephrinA2發(fā)生接觸后，破骨細(xì)胞接收的正向信號和逆向信號都能促進(jìn)破骨細(xì)胞分化，加速骨吸收過程。經(jīng)成骨細(xì)胞表面EphA2受體傳導(dǎo)的正向信號能夠抑制成骨細(xì)胞分化，抑制骨形成過程。 隨著骨組織局部微環(huán)境的變化而引起的成骨細(xì)胞和破骨細(xì)胞之間功能狀態(tài)改變，最終實(shí)現(xiàn)由骨穩(wěn)態(tài)向骨吸收的轉(zhuǎn)變過程。但目前關(guān)于Pg-LPS是否通過EphA2/ephrinA2信號通路參與啟動牙槽骨吸收過程國內(nèi)外未見相關(guān)報道。本實(shí)驗(yàn)嘗試在體外環(huán)境下通過使用Pg-LPS與MC3T3-E1細(xì)胞共培養(yǎng)，檢測細(xì)胞表達(dá)EphA2的變化，初步了解Pg-LPS對EphA2/ephrinA2信號通路調(diào)控的影響。 方法： 使用濃度為1g/ml的Pg-LPS與MC3T3-E1共培養(yǎng)，分別在3、7、14d三個時間點(diǎn)，，采用RT-PCR和Western blotting技術(shù)分別檢測EphA2和成骨相關(guān)基因的表達(dá)及EphA2蛋白質(zhì)的表達(dá)，并通過PNPP法測定堿性磷酸酶活性。 結(jié)果： 1. RT-PCR結(jié)果顯示：在Pg-LPS與MC3T3-E1細(xì)胞共培養(yǎng)后，實(shí)驗(yàn)組EphA2基因的表達(dá)比對照組明顯增高。在Pg-LPS作用10min后，實(shí)驗(yàn)組EphA2基因的表達(dá)無明顯差異。作用1h后，EphA2基因的表達(dá)最為明顯。 2. RT-PCR結(jié)果顯示：在Pg-LPS與MC3T3-E1細(xì)胞共培養(yǎng)后，EphA2基因的表達(dá)在3d和7d時，實(shí)驗(yàn)組較對照組均有明顯增加（P0.01），其中3d組較7d組，EphA2基因的表達(dá)更為明顯。但在14d時兩組間EphA2基因表達(dá)無顯著性差異。在3d和7d時，伴隨著EphA2基因上調(diào)的同時，ALP和Sp7基因出現(xiàn)了顯著下降（P 0.01），ALP基因在7d時下降最明顯，在14d時ALP和Sp7的表達(dá)則無顯著性差異。但是Pg-LPS對Runx2和ColⅠ基因的表達(dá)在3、7和14d時均無明顯影響。 3. Western blotting結(jié)果顯示：在Pg-LPS與MC3T3-E1細(xì)胞共培養(yǎng)后，在7d時實(shí)驗(yàn)組較對照組EphA2的表達(dá)明顯增加，但是在3、14d兩個時間點(diǎn)EphA2的表達(dá)無明顯差異。 4. ALP活性結(jié)果顯示：在3、7、14d三個時間點(diǎn)，Pg-LPS都能夠抑制堿性磷酸酶活性，其中3d時，對堿性磷酸酶活性抑制最明顯，實(shí)驗(yàn)組比對照組降低35%（P 0.01）；在7d時，Pg-LPS的抑制作用最弱，實(shí)驗(yàn)組比對照組降低17%（P 0.05）。 結(jié)論： 1.在無成骨誘導(dǎo)的條件下，Pg-LPS能夠促進(jìn)MC3T3-E1細(xì)胞表達(dá)EphA2基因。 2.在成骨誘導(dǎo)條件下，Pg-LPS在早期和中期能夠抑制MC3T3-E1細(xì)胞向成骨細(xì)胞分化，但是在晚期抑制作用不明顯。 3. Pg-LPS可能通過EphA2/ephrinA2信號通路參與調(diào)節(jié)MC3T3-E1細(xì)胞向成骨細(xì)胞分化的過程。
[Abstract]:background
Porphyromonas gingivalis (Pg) is an important suspicious pathogen of periodontitis. This bacterium can produce a large number of virulence factors, such as endotoxin, collagenase, trypsin like protease and so on, which will cause the destruction of periodontal connective tissue and the absorption of alveolar bone, and the endotoxin, also known as lipopolysaccharide (LPS), is the cause of alveolar bone. The key factor in absorption is that it can promote bone absorption by promoting osteoclast differentiation, on the other hand it can inhibit osteoblast differentiation and inhibit bone formation. Although the mechanism of LPS induced bone homeostasis has been extensively studied, how LPS regulates osteoblasts and osteoclasts. The molecular mechanism of body molecules is not yet very clear. In recent years, the role of Eph/ephrin signal molecules in bone remodeling has attracted much attention. Recent studies have shown that the EphA2/ephrinA2 signal molecules between osteoclasts and osteoblasts are important signals to start bone resorption during bone reconstruction. As the surface of bone and osteoclast, EphA2/eph After rinA2 exposure, both positive and reverse signals received by osteoclast can promote osteoclast differentiation and accelerate bone absorption. The positive signals transmitted by EphA2 receptors on the surface of osteoblasts can inhibit osteoblast differentiation and inhibit the formation of bone.
The changes in the functional state of osteoblasts and osteoclasts caused by the changes in the local microenvironment of the bone tissue, finally realized the process of the transition from bone homeostasis to bone absorption. However, there is no report about whether Pg-LPS is involved in the initiation of alveolar bone absorption through the EphA2/ephrinA2 signaling pathway. In the environment, the expression of EphA2 was detected by co culture of Pg-LPS and MC3T3-E1 cells, and the effect of Pg-LPS on the regulation of EphA2/ephrinA2 signaling pathway was preliminarily understood.
Method錛

本文編號：1893150