TNF-α过表达抑制BMP9诱导根尖牙乳头干细胞成骨分化的相关研究

发布时间：2018-05-01 13:36

本文选题：BMP9 + TNF-α　；参考：《重庆医科大学》2017年硕士论文

【摘要】：年轻恒牙根尖周炎是临床上一种常见病、多发病。由于患牙的牙根尚未发育成熟,不及时治疗将导致患牙的松动,甚至提前脱落。现阶段治疗手段往往仅能形成根尖屏障,并不能解决牙根发育不足,根管粗大,冠根比例不协调等问题。所以,临床上急需探索新的治疗方法。近年来,利用组织工程技术进行组织修复越来越引起研究者兴趣,也为我们探索新的治疗根尖周组织缺损提供新的思路。根尖牙乳头干细胞(Stem Cells from the Apical Papilla,SCAP)是5种牙源性相关干细胞其中之一,对于牙本质及牙根发育具有重要作用,是修复根尖组织缺损理想细胞。本课题前期研究构建了永生化的根尖乳头干细胞(immortalized Stem Cells from the Apical Papilla,iSCAP),永生化后的细胞保持了原有细胞的多向分化的特性,并且解决了细胞数量不足的难题。骨形态发生蛋白9(bone morphogenetic protein,BMP9)是BMP家族迄今发现的能够诱导干细胞成骨分化最强因子之一,本课题前期研究证实BMP9能够有效诱导iSCAP细胞成骨分化。近年来,随着组织工程技术的发展,微环境对于干细胞定向分化的作用的影响越来越受到研究者的重视。在根尖周炎炎性微环境下,会有大量炎症因子释放,其中肿瘤坏死因子α(tumournecrosisfactor-α,tnf-α)作为根尖周炎最重要的致炎因子之一,其在炎症期间大量释放对bmp9效诱导iscap细胞成骨分化的影响尚未可知。因此,本研究意在解决以下问题:(1)检测根尖周炎进展过程中tnf-α表达及其与根尖骨组织缺损的关系;(2)探讨过表达tnf-α对bmp9效诱导iscap细胞成骨分化的影响;(3)探讨在高浓度tnf-α(10ng/ml)的环境下,bmp9是否依然能够诱导根尖牙乳头干细胞成骨分化。为了探讨以上问题,本研究第一部分建立了牙根未发育完全的sd大鼠根尖周炎模型,分别在不同时间点micro-ct扫描并计算根尖阴影面积。我们发现牙根发育停止,根尖暗影开始于牙齿开髓后第3天,14天后至28天根尖暗影面积急剧增大,28天后暗影面积趋于稳定。采用了he染色方法观察根尖缺损区组织形态,发现骨质缺损区充满了结缔组织,并有大量炎性细胞浸润。免疫组织化学检测不同时间点tnf-α表达,发现根尖tnf-α阳性表达与根尖暗影面积呈正相关。第二部分探讨不同浓度的重组tnf-α蛋白(0.1ng/ml、1.0ng/ml、10ng/ml、100ng/ml)对bmp9诱导iscap细胞成骨分化的影响。发现alp表达在tnf-α(10ng/ml、100ng/ml)组显著降低,qpcr检测相关目的基因ocn、opn表达明显降低,钙盐沉积受到抑制,说明tnf-α(10ng/ml、100ng/ml)抑制bmp9诱导iscap细胞成骨分化。裸鼠体内的异位成骨实验发现,加入TNF-α(10 ng/ml)组较阳性对照组BMP9组异位骨质块体积明显减小,并且骨质内骨小梁密度低,数目少,Masson染色亦发现TNF-α(10 ng/ml)组骨质矿化程度低,骨质不成熟。为了探讨过表达BMP9是否能够在高浓度TNF-α(10 ng/ml)存在的情况下仍然能够促进iSCAP细胞成骨分化,第三部分我们逐步提高了Ad-BMP9表达,发现ALP表达逐渐上升,qPCR检测到OCN、OPN表达逐步提高,茜素红着色的晚期钙盐沉积结节增多,说明BMP9在高浓度TNF-α(10 ng/ml)存在的情况下仍然能够促进iSCAP细胞成骨分化。综上所述,TNF-α在根尖周炎炎症进展过程中大量释放,并且与根尖骨质缺损正相关。高浓度的TNF-α(10 ng/ml)能够显著抑制BMP9诱导iSCAP细胞成骨分化。BMP9仍然能在高浓度的TNF-α(10 ng/ml)环境下促进iSCAP细胞成骨分化。
[Abstract]:The root apical periodontitis of young permanent teeth is a common disease in the clinic. Because the root of the tooth is not mature, the treatment will lead to the loosening of the tooth, even in advance. At this stage, the treatment means can only form the root tip barrier, and can not solve the problems of the root development, the root canal and the proportion of the crown and root. In recent years, new therapies are urgently needed. In recent years, tissue engineering has attracted more and more interest in tissue repair. It also provides a new way for us to explore new treatment of periapical tissue defects. Stem Cells from the Apical Papilla (SCAP) is 5 kinds of odontogenic stem cells. 1. It is an ideal cell for dentin and root development, which is an ideal cell for the repair of the apical tissue defects. The early study of this topic constructed immortalized Stem Cells from the Apical Papilla, iSCAP. The immortalized cells kept the characteristics of the multidirectional differentiation of the original cells, and solved the details. Bone morphogenetic protein 9 (bone morphogenetic protein, BMP9) is one of the strongest factors that the BMP family has found to induce osteogenic differentiation of stem cells so far. This project has proved that BMP9 can effectively induce the osteogenic differentiation of iSCAP cells. In recent years, with the development of tissue engineering technology, microenvironment has been applied to dry fine. More and more attention has been paid to the effect of cell orientation differentiation. A large number of inflammatory factors are released in the hot periapical microenvironment, in which tumournecrosisfactor- alpha (tnf- alpha) is one of the most important inflammatory factors of periapical periodontitis, which releases a large number of BMP9 induced iscap cells in the period of inflammation. The effect of bone differentiation has not been known. Therefore, this study aims to solve the following problems: (1) to detect the expression of tnf- alpha and its relationship with the apical bone tissue defect during the progression of periapical periodontitis; (2) to explore the effect of overexpressing tnf- alpha on the osteogenic differentiation of BMP9 induced iscap cells; (3) whether BMP9 still remains in the environment of high concentration tnf- alpha (10ng/ml). In order to explore the above problems, the first part of this study was to establish a SD rat model of root apex periapical periodontitis without complete root development. Micro-CT scanning at different time points and calculating the area of the root apex shadow. We found that the root development stopped and the root tip shadow began third days after the tooth opening, 14 The shadow area of the root tip increased sharply from day to day to 28 days, and the shadow area tended to be stable after 28 days. The tissue morphology of the apex defect area was observed by HE staining. The bone defect area was found to be full of connective tissue and a large number of inflammatory cells were infiltrated. The expression of tnf- alpha at different time points was detected by immunohistochemistry, and the positive expression of tnf- alpha and root apex was found. The effect of the recombinant tnf- alpha protein (0.1ng/ml, 1.0ng/ml, 10ng/ml, 100ng/ml) on the osteogenesis of iscap cells induced by BMP9 was investigated in the second part. It was found that the expression of ALP in tnf- alpha (10ng/ml, 100ng/ml) group was significantly reduced, the qPCR detection related target genes were significantly reduced, and the calcium salt deposition was inhibited. The results showed that tnf- alpha (10ng/ml, 100ng/ml) inhibited BMP9 induced osteogenic differentiation of iscap cells. The ectopic osteogenesis in nude mice showed that the volume of ectopic bone mass decreased significantly in the TNF- alpha (10 ng/ml) group as compared with that of the positive control group, and the density of bone trabecula in the bone was low and the number was small. Masson staining also found the low degree of bone mineralization in TNF- a (10 ng/ml) group. Osteogenesis immaturity. In order to investigate whether the expression of BMP9 can still promote the osteogenesis of iSCAP cells in the presence of high concentration of TNF- alpha (10 ng/ml), the third part gradually increased the expression of Ad-BMP9, found that the expression of ALP increased gradually, qPCR detected OCN, the OPN table increased gradually, and the late calcium salt nodules with alizarin red color In addition, it is suggested that BMP9 can still promote osteogenic differentiation of iSCAP cells in the presence of high concentration of TNF- alpha (10 ng/ml). To sum up, TNF- alpha is released during the progress of periapical periodontitis, and is positively related to the apical bone defect. High concentration of TNF- alpha (10 ng/ml) can significantly inhibit BMP9 induced iSCAP cell osteogenesis differentiation.BMP9 still. It can promote osteogenic differentiation of iSCAP cells in a high concentration of TNF- alpha (10 ng/ml).

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.341

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