發(fā)布時間：2018-07-10 15:39

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

【摘要】：巨噬細胞遷移抑制因子(Macrophage migration inhibitory factor, MIF)在感染、炎癥刺激和應激的條件下迅速被釋放到胞外,是調節(jié)自身免疫和適應性免疫的一個非常重要的前炎癥因子。盡管研究表明T細胞是MIF的主要來源,實際其它細胞也能分泌和產生MIF蛋白,例如巨噬細胞、血管內皮細胞等。核因子κB受體活化因子(receptor activator of nuclear factor kappa B ligand, RANKL)是一個在活化T細胞表面和成骨細胞表面發(fā)現的特殊蛋白,在破骨細胞分化和成熟方面起到重要作用。已經證實根尖周大量RANKL的表達與根尖周病理性骨破壞密切相關,與此同時,藥物抑制(JRANKL產生在根尖周炎的治療上開啟了新的方向。 根尖周炎主要是由細菌感染導致根管內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疾病,其病理特征就是根尖周大量炎癥細胞表達,多核破骨細胞增多。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域未被研究。T細胞在維持口腔對外界抗原和細菌刺激產生免疫的平衡方面起到重要作用,大部分MIF和RANKL蛋白的分泌都來自于T細胞,因此我們猜測MIF可能與RANKL-一起參與了根尖周炎癥反應和骨吸收的過程。本課題分為體內和體外兩個部分,體內實驗擬通過建立大鼠根尖周炎動物模型,觀察大鼠根尖周病變過程中各個時期MIF蛋白的表達及變化,并分析其與RANKL,破骨細胞及根尖周骨吸收之間的關系；同時應用藥物metformin干預根尖周炎癥的發(fā)展；體外實驗以牙周膜成纖維細胞(human periodontal ligament fibroblasts, hPDLFs)作為靶細胞研究了MIF可能參與根尖周炎的病理過程及作用機制。 實驗一MIF在實驗性大鼠根尖周炎中的表達 目的：檢測大鼠根尖周病變過程中MIF蛋白的表達與變化,分析其與RANKL 蛋白表達及根尖周骨吸收之間的關系。 材料與方法：將36只成年雄性Wistar大鼠下頜第一磨牙開髓,分別于術后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和酶組織化學的方法檢測MIF、RANKL、MCP-1、CCR2及破骨細胞在根尖周病損中的表達與變化,利用免疫熒光雙染定位MIF、RANKL和MCP-1、CCR2。分析MIF表達與RANKL數目及根尖周骨吸收的關系。 結果：從0天到28天根尖周骨吸收面積逐漸增大到35天達到穩(wěn)定。在第7天可以見到少量MIF, RANKL陽性細胞和破骨細胞,14天達到高峰。從21天到35天,MIF, RANKL陽性細胞和破骨細胞表達量減少,MCP-1陽性細胞數從7天到35天持續(xù)增加。 結論：MIF在大鼠實驗性根尖周炎各個時期均有表達,提示它參與了根尖周炎的發(fā)病過程。炎癥急性期可能通過促進破骨細胞的功能加快牙槽骨吸收,而炎癥慢性期,募集大量炎癥細胞到根尖區(qū)對抗抗原刺激而起到骨保護作用。 實驗二MIF上調牙周膜細胞RANKL的表達及其機制研究 目的：觀察MIF和RANKL在人慢性根尖周病損組織中的表達及定位,同時體外實驗觀察是否rhMIF刺激人牙周膜成纖維細胞產生RANKL蛋白,探討MIF在慢性根尖周病損發(fā)病機制中的作用。 方法和材料：從2012年3月至12月在武漢大學口腔醫(yī)院牙體牙髓科行根尖手術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及定位。同時在體外用不同濃度的rhMIF(0.1、1、5、10ng/ml)刺激hPDLFs,通過免疫熒光,實時定量PCR,ELISA, Westernblot檢測RANKL的表達及其相關信號通路的轉導。 結果：從免疫組織化學染色我們可以看到MIF和RANKL陽性細胞表達在根尖周的病變區(qū),但MIF主要表達在炎癥淋巴細胞,許多內皮細胞表達RANKL蛋白。rhMIF上調hPDLFs RANKL在mRNA和蛋白水平的表達,與此同時,rhMIF刺激hPDLFs致使其胞內AKT, P38MAPK, ERK1/2, JNK等信號激酶迅速發(fā)生活化。rhMIF啟動NF-κBp65核轉位,這種核轉位可被AKT, P38MAPK抑制劑而不是ERK1/2, JNK抑制劑所逆轉。 結論：人慢性根尖周炎癥組織有大量MIF和RANKL蛋白的表達。rhMIF可通過刺激hPDLFs RANKL蛋白產生參與根尖周炎的免疫反應與骨破壞,rhMIF刺激RANKL的表達依賴Akt-, p38MAPK, NF-κB信號的正向調節(jié)以及JNK, ERK1/2的負向調控。 實驗三Metformin對實驗性大鼠根尖周炎的保護作用 目的：Metformin是用來降低糖尿病患者血糖濃度的常用藥物,近來metformin在骨代謝領域廣泛研究。本實驗目的主要研究是否metformin對大鼠實驗性根尖周炎有保護作用。 方法和材料：40只成年雄性Wistar大鼠被隨機分為實驗組和對照組,實驗組從術前一天給予肌注metformin (40mg/kg/day)連續(xù)28天,對照組給予生理鹽水。兩組均在全麻下用球鉆打開下頜第一磨牙,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)組織學處理、制作組織切片,再分別做組織學檢測、免疫組織化學、免疫熒光和酶組織化學染色。 結果：在14天與對照組比較,破骨細胞MIF, RANKL陽性細胞在metformin注射組的數目降低,而OPG陽性細胞在28天增加,同時metformin注射組MIF, RANKL陽性細胞在28天與對照組無明顯差異。通過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天組無明顯差異。 結論：在根尖周炎發(fā)展過程中,metformin的保護作用可能體現在調控MIF,RANKL的表達和破骨細胞的分化方面,進而對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 .
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R781.341

【參考文獻】

相關期刊論文 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

，

本文編號：2113831