巨噬细胞迁移抑制因子在根尖周炎的表达及机制研究

发布时间：2018-07-10 15:39

本文选题：Metformin + MIF　；参考：《武汉大学》2014年博士论文

【摘要】：巨噬细胞迁移抑制因子(Macrophage migration inhibitory factor, MIF)在感染、炎症刺激和应激的条件下迅速被释放到胞外,是调节自身免疫和适应性免疫的一个非常重要的前炎症因子。尽管研究表明T细胞是MIF的主要来源,实际其它细胞也能分泌和产生MIF蛋白,例如巨噬细胞、血管内皮细胞等。核因子κB受体活化因子(receptor activator of nuclear factor kappa B ligand, RANKL)是一个在活化T细胞表面和成骨细胞表面发现的特殊蛋白,在破骨细胞分化和成熟方面起到重要作用。已经证实根尖周大量RANKL的表达与根尖周病理性骨破坏密切相关,与此同时,药物抑制(JRANKL产生在根尖周炎的治疗上开启了新的方向。根尖周炎主要是由细菌感染导致根管内牙髓坏死继而引起根尖周炎症性骨破坏的一种口腔科常见疾病,其病理特征就是根尖周大量炎症细胞表达,多核破骨细胞增多。M1F在其它许多炎症性骨吸收疾病中都有研究并发挥重要作用,然而它在口腔根尖周病骨破坏这一领域未被研究。T细胞在维持口腔对外界抗原和细菌刺激产生免疫的平衡方面起到重要作用,大部分MIF和RANKL蛋白的分泌都来自于T细胞,因此我们猜测MIF可能与RANKL-一起参与了根尖周炎症反应和骨吸收的过程。本课题分为体内和体外两个部分,体内实验拟通过建立大鼠根尖周炎动物模型,观察大鼠根尖周病变过程中各个时期MIF蛋白的表达及变化,并分析其与RANKL,破骨细胞及根尖周骨吸收之间的关系；同时应用药物metformin干预根尖周炎症的发展；体外实验以牙周膜成纤维细胞(human periodontal ligament fibroblasts, hPDLFs)作为靶细胞研究了MIF可能参与根尖周炎的病理过程及作用机制。实验一MIF在实验性大鼠根尖周炎中的表达目的：检测大鼠根尖周病变过程中MIF蛋白的表达与变化,分析其与RANKL 蛋白表达及根尖周骨吸收之间的关系。材料与方法：将36只成年雄性Wistar大鼠下颌第一磨牙开髓,分别于术后0、7、14、21、28和35天取下颌骨,制备组织切片。观察根尖周病变中炎症的变化,HE染色分析骨破坏情况；分别用免疫组织化学和酶组织化学的方法检测MIF、RANKL、MCP-1、CCR2及破骨细胞在根尖周病损中的表达与变化,利用免疫荧光双染定位MIF、RANKL和MCP-1、CCR2。分析MIF表达与RANKL数目及根尖周骨吸收的关系。结果：从0天到28天根尖周骨吸收面积逐渐增大到35天达到稳定。在第7天可以见到少量MIF, RANKL阳性细胞和破骨细胞,14天达到高峰。从21天到35天,MIF, RANKL阳性细胞和破骨细胞表达量减少,MCP-1阳性细胞数从7天到35天持续增加。结论：MIF在大鼠实验性根尖周炎各个时期均有表达,提示它参与了根尖周炎的发病过程。炎症急性期可能通过促进破骨细胞的功能加快牙槽骨吸收,而炎症慢性期,募集大量炎症细胞到根尖区对抗抗原刺激而起到骨保护作用。实验二MIF上调牙周膜细胞RANKL的表达及其机制研究目的：观察MIF和RANKL在人慢性根尖周病损组织中的表达及定位,同时体外实验观察是否rhMIF刺激人牙周膜成纤维细胞产生RANKL蛋白,探讨MIF在慢性根尖周病损发病机制中的作用。方法和材料：从2012年3月至12月在武汉大学口腔医院牙体牙髓科行根尖手术或颌面外科拔牙患者中,收集到人慢性根尖周炎病损组织32例。病损组织用于组织学研究,以观察观察MIF和RANKL在人慢性根尖周病损组织中的表达及定位。同时在体外用不同浓度的rhMIF(0.1、1、5、10ng/ml)刺激hPDLFs,通过免疫荧光,实时定量PCR,ELISA, Westernblot检测RANKL的表达及其相关信号通路的转导。结果：从免疫组织化学染色我们可以看到MIF和RANKL阳性细胞表达在根尖周的病变区,但MIF主要表达在炎症淋巴细胞,许多内皮细胞表达RANKL蛋白。rhMIF上调hPDLFs RANKL在mRNA和蛋白水平的表达,与此同时,rhMIF刺激hPDLFs致使其胞内AKT, P38MAPK, ERK1/2, JNK等信号激酶迅速发生活化。rhMIF启动NF-κBp65核转位,这种核转位可被AKT, P38MAPK抑制剂而不是ERK1/2, JNK抑制剂所逆转。结论：人慢性根尖周炎症组织有大量MIF和RANKL蛋白的表达。rhMIF可通过刺激hPDLFs RANKL蛋白产生参与根尖周炎的免疫反应与骨破坏,rhMIF刺激RANKL的表达依赖Akt-, p38MAPK, NF-κB信号的正向调节以及JNK, ERK1/2的负向调控。实验三Metformin对实验性大鼠根尖周炎的保护作用目的：Metformin是用来降低糖尿病患者血糖浓度的常用药物,近来metformin在骨代谢领域广泛研究。本实验目的主要研究是否metformin对大鼠实验性根尖周炎有保护作用。方法和材料：40只成年雄性Wistar大鼠被随机分为实验组和对照组,实验组从术前一天给予肌注metformin (40mg/kg/day)连续28天,对照组给予生理盐水。两组均在全麻下用球钻打开下颌第一磨牙,于牙髓暴露后2周、4周各处死10只,取出含下颌第一磨牙的下颌骨,拍摄X-ray, Micro CT分析骨破坏情况,常规组织学处理、制作组织切片,再分别做组织学检测、免疫组织化学、免疫荧光和酶组织化学染色。结果：在14天与对照组比较,破骨细胞MIF, RANKL阳性细胞在metformin注射组的数目降低,而OPG阳性细胞在28天增加,同时metformin注射组MIF, RANKL阳性细胞在28天与对照组无明显差异。通过X-ray、Micro CT分析28天metformin注射组的骨吸收吸收面积减少,但是14天组无明显差异。结论：在根尖周炎发展过程中,metformin的保护作用可能体现在调控MIF,RANKL的表达和破骨细胞的分化方面,进而对根尖周骨破坏有一定的抑制作用。
[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 .
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R781.341

【参考文献】