本文選題：BMP9 + TNF-α��； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：年輕恒牙根尖周炎是臨床上一種常見病、多發(fā)病。由于患牙的牙根尚未發(fā)育成熟,不及時治療將導(dǎo)致患牙的松動,甚至提前脫落�，F(xiàn)階段治療手段往往僅能形成根尖屏障,并不能解決牙根發(fā)育不足,根管粗大,冠根比例不協(xié)調(diào)等問題。所以,臨床上急需探索新的治療方法。近年來,利用組織工程技術(shù)進(jìn)行組織修復(fù)越來越引起研究者興趣,也為我們探索新的治療根尖周組織缺損提供新的思路。根尖牙乳頭干細(xì)胞(Stem Cells from the Apical Papilla,SCAP)是5種牙源性相關(guān)干細(xì)胞其中之一,對于牙本質(zhì)及牙根發(fā)育具有重要作用,是修復(fù)根尖組織缺損理想細(xì)胞。本課題前期研究構(gòu)建了永生化的根尖乳頭干細(xì)胞(immortalized Stem Cells from the Apical Papilla,iSCAP),永生化后的細(xì)胞保持了原有細(xì)胞的多向分化的特性,并且解決了細(xì)胞數(shù)量不足的難題。骨形態(tài)發(fā)生蛋白9(bone morphogenetic protein,BMP9)是BMP家族迄今發(fā)現(xiàn)的能夠誘導(dǎo)干細(xì)胞成骨分化最強因子之一,本課題前期研究證實BMP9能夠有效誘導(dǎo)iSCAP細(xì)胞成骨分化。近年來,隨著組織工程技術(shù)的發(fā)展,微環(huán)境對于干細(xì)胞定向分化的作用的影響越來越受到研究者的重視。在根尖周炎炎性微環(huán)境下,會有大量炎癥因子釋放,其中腫瘤壞死因子α(tumournecrosisfactor-α,tnf-α)作為根尖周炎最重要的致炎因子之一,其在炎癥期間大量釋放對bmp9效誘導(dǎo)iscap細(xì)胞成骨分化的影響尚未可知。因此,本研究意在解決以下問題:(1)檢測根尖周炎進(jìn)展過程中tnf-α表達(dá)及其與根尖骨組織缺損的關(guān)系;(2)探討過表達(dá)tnf-α對bmp9效誘導(dǎo)iscap細(xì)胞成骨分化的影響;(3)探討在高濃度tnf-α(10ng/ml)的環(huán)境下,bmp9是否依然能夠誘導(dǎo)根尖牙乳頭干細(xì)胞成骨分化。為了探討以上問題,本研究第一部分建立了牙根未發(fā)育完全的sd大鼠根尖周炎模型,分別在不同時間點micro-ct掃描并計算根尖陰影面積。我們發(fā)現(xiàn)牙根發(fā)育停止,根尖暗影開始于牙齒開髓后第3天,14天后至28天根尖暗影面積急劇增大,28天后暗影面積趨于穩(wěn)定。采用了he染色方法觀察根尖缺損區(qū)組織形態(tài),發(fā)現(xiàn)骨質(zhì)缺損區(qū)充滿了結(jié)締組織,并有大量炎性細(xì)胞浸潤。免疫組織化學(xué)檢測不同時間點tnf-α表達(dá),發(fā)現(xiàn)根尖tnf-α陽性表達(dá)與根尖暗影面積呈正相關(guān)。第二部分探討不同濃度的重組tnf-α蛋白(0.1ng/ml、1.0ng/ml、10ng/ml、100ng/ml)對bmp9誘導(dǎo)iscap細(xì)胞成骨分化的影響。發(fā)現(xiàn)alp表達(dá)在tnf-α(10ng/ml、100ng/ml)組顯著降低,qpcr檢測相關(guān)目的基因ocn、opn表達(dá)明顯降低,鈣鹽沉積受到抑制,說明tnf-α(10ng/ml、100ng/ml)抑制bmp9誘導(dǎo)iscap細(xì)胞成骨分化。裸鼠體內(nèi)的異位成骨實驗發(fā)現(xiàn),加入TNF-α(10 ng/ml)組較陽性對照組BMP9組異位骨質(zhì)塊體積明顯減小,并且骨質(zhì)內(nèi)骨小梁密度低,數(shù)目少,Masson染色亦發(fā)現(xiàn)TNF-α(10 ng/ml)組骨質(zhì)礦化程度低,骨質(zhì)不成熟。為了探討過表達(dá)BMP9是否能夠在高濃度TNF-α(10 ng/ml)存在的情況下仍然能夠促進(jìn)iSCAP細(xì)胞成骨分化,第三部分我們逐步提高了Ad-BMP9表達(dá),發(fā)現(xiàn)ALP表達(dá)逐漸上升,qPCR檢測到OCN、OPN表達(dá)逐步提高,茜素紅著色的晚期鈣鹽沉積結(jié)節(jié)增多,說明BMP9在高濃度TNF-α(10 ng/ml)存在的情況下仍然能夠促進(jìn)iSCAP細(xì)胞成骨分化。綜上所述,TNF-α在根尖周炎炎癥進(jìn)展過程中大量釋放,并且與根尖骨質(zhì)缺損正相關(guān)。高濃度的TNF-α(10 ng/ml)能夠顯著抑制BMP9誘導(dǎo)iSCAP細(xì)胞成骨分化。BMP9仍然能在高濃度的TNF-α(10 ng/ml)環(huán)境下促進(jìn)iSCAP細(xì)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).

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R781.341

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