本文選題：Metformin + MIF　； 參考：《武漢大學(xué)》2014年博士論文

【摘要】：巨噬細(xì)胞遷移抑制因子(Macrophage migration inhibitory factor, MIF)在感染、炎癥刺激和應(yīng)激的條件下迅速被釋放到胞外,是調(diào)節(jié)自身免疫和適應(yīng)性免疫的一個(gè)非常重要的前炎癥因子。盡管研究表明T細(xì)胞是MIF的主要來(lái)源,實(shí)際其它細(xì)胞也能分泌和產(chǎn)生MIF蛋白,例如巨噬細(xì)胞、血管內(nèi)皮細(xì)胞等。核因子κB受體活化因子(receptor activator of nuclear factor kappa B ligand, RANKL)是一個(gè)在活化T細(xì)胞表面和成骨細(xì)胞表面發(fā)現(xiàn)的特殊蛋白,在破骨細(xì)胞分化和成熟方面起到重要作用。已經(jīng)證實(shí)根尖周大量RANKL的表達(dá)與根尖周病理性骨破壞密切相關(guān),與此同時(shí),藥物抑制(JRANKL產(chǎn)生在根尖周炎的治療上開(kāi)啟了新的方向。 根尖周炎主要是由細(xì)菌感染導(dǎo)致根管內(nèi)牙髓壞死繼而引起根尖周炎癥性骨破壞的一種口腔科常見(jiàn)疾病,其病理特征就是根尖周大量炎癥細(xì)胞表達(dá),多核破骨細(xì)胞增多。M1F在其它許多炎癥性骨吸收疾病中都有研究并發(fā)揮重要作用,然而它在口腔根尖周病骨破壞這一領(lǐng)域未被研究。T細(xì)胞在維持口腔對(duì)外界抗原和細(xì)菌刺激產(chǎn)生免疫的平衡方面起到重要作用,大部分MIF和RANKL蛋白的分泌都來(lái)自于T細(xì)胞,因此我們猜測(cè)MIF可能與RANKL-一起參與了根尖周炎癥反應(yīng)和骨吸收的過(guò)程。本課題分為體內(nèi)和體外兩個(gè)部分,體內(nèi)實(shí)驗(yàn)擬通過(guò)建立大鼠根尖周炎動(dòng)物模型,觀察大鼠根尖周病變過(guò)程中各個(gè)時(shí)期MIF蛋白的表達(dá)及變化,并分析其與RANKL,破骨細(xì)胞及根尖周骨吸收之間的關(guān)系；同時(shí)應(yīng)用藥物metformin干預(yù)根尖周炎癥的發(fā)展；體外實(shí)驗(yàn)以牙周膜成纖維細(xì)胞(human periodontal ligament fibroblasts, hPDLFs)作為靶細(xì)胞研究了MIF可能參與根尖周炎的病理過(guò)程及作用機(jī)制。 實(shí)驗(yàn)一MIF在實(shí)驗(yàn)性大鼠根尖周炎中的表達(dá) 目的：檢測(cè)大鼠根尖周病變過(guò)程中MIF蛋白的表達(dá)與變化,分析其與RANKL 蛋白表達(dá)及根尖周骨吸收之間的關(guān)系。 材料與方法：將36只成年雄性Wistar大鼠下頜第一磨牙開(kāi)髓,分別于術(shù)后0、7、14、21、28和35天取下頜骨,制備組織切片。觀察根尖周病變中炎癥的變化,HE染色分析骨破壞情況；分別用免疫組織化學(xué)和酶組織化學(xué)的方法檢測(cè)MIF、RANKL、MCP-1、CCR2及破骨細(xì)胞在根尖周病損中的表達(dá)與變化,利用免疫熒光雙染定位MIF、RANKL和MCP-1、CCR2。分析MIF表達(dá)與RANKL數(shù)目及根尖周骨吸收的關(guān)系。 結(jié)果：從0天到28天根尖周骨吸收面積逐漸增大到35天達(dá)到穩(wěn)定。在第7天可以見(jiàn)到少量MIF, RANKL陽(yáng)性細(xì)胞和破骨細(xì)胞,14天達(dá)到高峰。從21天到35天,MIF, RANKL陽(yáng)性細(xì)胞和破骨細(xì)胞表達(dá)量減少,MCP-1陽(yáng)性細(xì)胞數(shù)從7天到35天持續(xù)增加。 結(jié)論：MIF在大鼠實(shí)驗(yàn)性根尖周炎各個(gè)時(shí)期均有表達(dá),提示它參與了根尖周炎的發(fā)病過(guò)程。炎癥急性期可能通過(guò)促進(jìn)破骨細(xì)胞的功能加快牙槽骨吸收,而炎癥慢性期,募集大量炎癥細(xì)胞到根尖區(qū)對(duì)抗抗原刺激而起到骨保護(hù)作用。 實(shí)驗(yàn)二MIF上調(diào)牙周膜細(xì)胞RANKL的表達(dá)及其機(jī)制研究 目的：觀察MIF和RANKL在人慢性根尖周病損組織中的表達(dá)及定位,同時(shí)體外實(shí)驗(yàn)觀察是否rhMIF刺激人牙周膜成纖維細(xì)胞產(chǎn)生RANKL蛋白,探討MIF在慢性根尖周病損發(fā)病機(jī)制中的作用。 方法和材料：從2012年3月至12月在武漢大學(xué)口腔醫(yī)院牙體牙髓科行根尖手術(shù)或頜面外科拔牙患者中,收集到人慢性根尖周炎病損組織32例。病損組織用于組織學(xué)研究,以觀察觀察MIF和RANKL在人慢性根尖周病損組織中的表達(dá)及定位。同時(shí)在體外用不同濃度的rhMIF(0.1、1、5、10ng/ml)刺激hPDLFs,通過(guò)免疫熒光,實(shí)時(shí)定量PCR,ELISA, Westernblot檢測(cè)RANKL的表達(dá)及其相關(guān)信號(hào)通路的轉(zhuǎn)導(dǎo)。 結(jié)果：從免疫組織化學(xué)染色我們可以看到MIF和RANKL陽(yáng)性細(xì)胞表達(dá)在根尖周的病變區(qū),但MIF主要表達(dá)在炎癥淋巴細(xì)胞,許多內(nèi)皮細(xì)胞表達(dá)RANKL蛋白。rhMIF上調(diào)hPDLFs RANKL在mRNA和蛋白水平的表達(dá),與此同時(shí),rhMIF刺激hPDLFs致使其胞內(nèi)AKT, P38MAPK, ERK1/2, JNK等信號(hào)激酶迅速發(fā)生活化。rhMIF啟動(dòng)NF-κBp65核轉(zhuǎn)位,這種核轉(zhuǎn)位可被AKT, P38MAPK抑制劑而不是ERK1/2, JNK抑制劑所逆轉(zhuǎn)。 結(jié)論：人慢性根尖周炎癥組織有大量MIF和RANKL蛋白的表達(dá)。rhMIF可通過(guò)刺激hPDLFs RANKL蛋白產(chǎn)生參與根尖周炎的免疫反應(yīng)與骨破壞,rhMIF刺激RANKL的表達(dá)依賴(lài)Akt-, p38MAPK, NF-κB信號(hào)的正向調(diào)節(jié)以及JNK, ERK1/2的負(fù)向調(diào)控。 實(shí)驗(yàn)三Metformin對(duì)實(shí)驗(yàn)性大鼠根尖周炎的保護(hù)作用 目的：Metformin是用來(lái)降低糖尿病患者血糖濃度的常用藥物,近來(lái)metformin在骨代謝領(lǐng)域廣泛研究。本實(shí)驗(yàn)?zāi)康闹饕芯渴欠駇etformin對(duì)大鼠實(shí)驗(yàn)性根尖周炎有保護(hù)作用。 方法和材料：40只成年雄性Wistar大鼠被隨機(jī)分為實(shí)驗(yàn)組和對(duì)照組,實(shí)驗(yàn)組從術(shù)前一天給予肌注metformin (40mg/kg/day)連續(xù)28天,對(duì)照組給予生理鹽水。兩組均在全麻下用球鉆打開(kāi)下頜第一磨牙,于牙髓暴露后2周、4周各處死10只,取出含下頜第一磨牙的下頜骨,拍攝X-ray, Micro CT分析骨破壞情況,常規(guī)組織學(xué)處理、制作組織切片,再分別做組織學(xué)檢測(cè)、免疫組織化學(xué)、免疫熒光和酶組織化學(xué)染色。 結(jié)果：在14天與對(duì)照組比較,破骨細(xì)胞MIF, RANKL陽(yáng)性細(xì)胞在metformin注射組的數(shù)目降低,而OPG陽(yáng)性細(xì)胞在28天增加,同時(shí)metformin注射組MIF, RANKL陽(yáng)性細(xì)胞在28天與對(duì)照組無(wú)明顯差異。通過(guò)X-ray、Micro CT分析28天metformin注射組的骨吸收吸收面積減少,但是14天組無(wú)明顯差異。 結(jié)論：在根尖周炎發(fā)展過(guò)程中,metformin的保護(hù)作用可能體現(xiàn)在調(diào)控MIF,RANKL的表達(dá)和破骨細(xì)胞的分化方面,進(jìn)而對(duì)根尖周骨破壞有一定的抑制作用。
[Abstract]:Nuclear factor kappa B ligand ( RANKL ) is a special protein found on the surface of activated T cell and the surface of osteoblasts , which is a special protein found on the surface of activated T cell and the surface of osteoblasts .

In vivo and in vitro , we hypothesized that MIF might be involved in the pathogenesis of periapical periodontitis and bone resorption . In vivo and in vitro , we hypothesized that MIF might be involved in the pathogenesis of apical periodontitis and bone resorption in many other inflammatory bone resorption diseases .
At the same time , drug metformin was used to interfere with the development of apical periodontitis .
In vitro , human periodontal ligament fibroblasts ( hPDLFs ) were used as target cells to study the pathological process and mechanism of MIF in apical periodontitis .

Expression of experimental one MIF in experimental rat periapical periodontitis

Objective : To detect the expression and change of MIF protein in periapical lesion of rats and to analyze the expression of MIF protein and RANKL .

Protein expression and periapical bone resorption .

Materials and Methods : Thirty - six adult male Wistar rats were divided into the first molar and the first molar , and the mandible was taken from 0 , 7 , 14 , 21 , 28 and 35 days after operation , and the tissue sections were prepared . The changes of inflammation in periapical lesions were observed and the bone destruction was analyzed by HE staining .
The expression and change of MIF , RANKL , MCP - 1 , CCR2 and broken cell in periapical lesions were detected by immunohistochemistry and immunohistochemistry respectively . MIF , RANKL and MCP - 1 , CCR2 were detected by immunofluorescence double staining . The relationship between MIF expression and RANKL and periapical bone resorption was analyzed .

Results : The area of bone resorption gradually increased to 35 days from 0 to 28 days . On Day 7 , a small amount of MIF , RANKL - positive cells and osteocytes were seen to peak . From 21 to 35 days , the expression of MIF , RANKL - positive cells and osteocytes decreased , and the number of MCP - 1 - positive cells increased from 7 days to 35 days .

Conclusion : MIF may be involved in the pathogenesis of periapical periodontitis in rats with periapical periodontitis . The acute stage of inflammation may accelerate alveolar bone resorption by promoting the function of Osteoclasts , and inflammatory chronic phase , raise a large amount of inflammatory cells to the apical area to resist the antigen stimulation to play a role of bone protection .

Study on the up - regulation of RANKL expression in periodontal ligament cells by experiment two MIF and its mechanism

Objective : To observe the expression and localization of MIF and RANKL in chronic periapical diseases of human chronic apical diseases .

Methods : From March to December 2012 , 32 patients with chronic periapical periodontitis were collected from the apical or maxillofacial surgery of the dental pulp of the dental pulp department of Wuhan University . The lesions were used in histological study to observe the expression and localization of MIF and RANKL in human chronic apical periapical diseases .

Results : We can see MIF and RANKL - positive cells expressed in periapical lesions from immunohistochemical staining , but MIF is mainly expressed in inflammatory lymphocytes and many endothelial cells express RANKL protein . rhMIF stimulates the expression of hPDLFs RANKL at mRNA and protein levels . At the same time , rhMIF stimulates hPDLFs to rapidly activate the signal kinase of hPDLFs .

Conclusion : The expression of MIF and RANKL protein in human chronic periapical periodontitis tissue can be induced by stimulating hPDLFs RANKL protein . rhMIF can stimulate the expression of RANKL in the positive regulation of signal transduction , as well as the negative regulation of MAPK , NF - 魏B signal in human chronic apical periodontitis .

Protective effect of experimental three - Metformin on periapical periodontitis in experimental rats

Objective : Metformin is a common drug used to reduce blood glucose concentration in diabetic patients . Recently , metformin has been widely used in bone metabolism .

Methods and Materials : Forty adult male Wistar rats were randomly divided into experimental group and control group . The experimental group received intramuscular injection of metformin ( 40mg / kg / day ) for 28 days from the previous day , and the control group received normal saline . All the 10 rats were sacrificed 2 weeks after the exposure of the pulp and 10 rats were sacrificed at 4 weeks .

Results : Compared with the control group , the number of MIF and RANKL - positive cells decreased in the metformin injection group , while the OPG - positive cells increased in 28 days , while in the metformin injection group MIF and RANKL - positive cells were not significantly different from the control group on 28 days .

Conclusion : In the development of periapical periodontitis , the protective effect of metformin may be reflected in the regulation of MIF , RANKL expression and differentiation , which can inhibit the damage of periapical bone .
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R781.341

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;CD74 and macrophage migration inhibitory factor as therapeutic targets in gastric cancer[J];World Journal of Gastroenterology;2012年18期

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本文編號(hào)：2113831