【摘要】：在正畸治療的過程中,牙齒受到大小合適、方向適宜的正畸力后,壓力側(cè)牙槽骨中成骨細(xì)胞會(huì)大量死亡,破骨細(xì)胞增生,導(dǎo)致牙槽骨吸收;張力側(cè)成骨細(xì)胞增生,牙槽骨新骨形成,這種發(fā)生在牙槽骨的骨組織改建是正畸牙齒移動(dòng)的基礎(chǔ)[1]。目前研究認(rèn)為,壓力側(cè)牙槽骨包括成骨細(xì)胞在內(nèi)的骨細(xì)胞大量死亡,是因?yàn)檎ψ饔孟?牙槽骨局部微環(huán)境發(fā)生了改變,這其中包括壓力側(cè)牙周膜壓縮變窄、提供營(yíng)養(yǎng)和氧氣的毛細(xì)血管在正畸力作用下管腔變窄,循環(huán)受阻,血供減少[2],這就導(dǎo)致了壓力側(cè)牙槽骨組織細(xì)胞處于一個(gè)低氧、營(yíng)養(yǎng)缺乏的饑餓環(huán)境[3],那么這種環(huán)境是如何造成牙槽骨吸收、壓力側(cè)牙槽骨細(xì)胞大量死亡的呢?細(xì)胞死亡的三種方式:壞死、自噬、凋亡[4],在適宜的正畸力作用下,牙槽骨改建屬于牙齒的生理性改建,骨組織不會(huì)發(fā)生細(xì)胞壞死等病理性改變,自噬與凋亡都屬于細(xì)胞自身主導(dǎo)的程序性死亡方式,他們都分別在壓力側(cè)骨組織細(xì)胞死亡的過程中扮演了什么樣的角色?相互關(guān)系如何?本研究針對(duì)這個(gè)問題,通過建立比格犬正畸牙齒移動(dòng)模型,在體研究分析了壓力側(cè)牙槽骨正畸改建過程中自噬與凋亡表達(dá)水平的變化,并進(jìn)一步通過細(xì)胞體外實(shí)驗(yàn)觀察了成骨細(xì)胞在饑餓環(huán)境下自噬與凋亡表達(dá)水平的變化及相互關(guān)系,對(duì)壓力側(cè)骨組織細(xì)胞的消亡關(guān)系進(jìn)行了初步研究。本課題研究?jī)?nèi)容包括以下兩個(gè)方面:1.牙齒移動(dòng)過程中牙槽骨組織細(xì)胞自噬與凋亡水平變化的動(dòng)物實(shí)驗(yàn)研究目的:建立比格犬正畸牙齒移動(dòng)模型,檢測(cè)在正畸力作用下,壓力側(cè)牙槽骨組織形態(tài)學(xué)變化和骨組織細(xì)胞中自噬與凋亡的表達(dá)情況。方法:將6只成年比格犬的24顆尖牙隨機(jī)分為空白對(duì)照組、正畸加力組�？瞻讓�(duì)照組無任何處理,正畸加力組牙齒以種植釘為支抗,以150g正畸力遠(yuǎn)中移動(dòng)尖牙,建立比格犬尖牙遠(yuǎn)中移動(dòng)模型,按正畸力作用時(shí)間分為正畸加力7天組、14天組、28天組。觀察正畸力作用下牙齒移動(dòng)效果,通過he方法分析其壓力側(cè)骨組織改建情況,利用免疫組化染色及westernblot方法,分析壓力側(cè)牙槽骨組織中自噬與凋亡的作用情況。結(jié)果:比格犬尖牙在正畸力作用下,①和空白對(duì)照組相比,各實(shí)驗(yàn)組尖牙遠(yuǎn)中移動(dòng)明顯(p0.05),牙齒在正畸力作用下,隨作用時(shí)間的增加移動(dòng)距離不斷增長(zhǎng)(p0.05);②westernblot顯示與空白對(duì)照組相比,壓力側(cè)牙槽骨組織中l(wèi)c3-Ⅱ/lc3-Ⅰ轉(zhuǎn)化率迅速上升,7天到達(dá)頂峰(p0.05),caspase3蛋白表達(dá)水平7天后迅速升高,28天到達(dá)頂峰(p0.05);③he結(jié)果顯示與空白對(duì)照組相比,壓力側(cè)牙槽骨組織隨著正畸力作用時(shí)間的延長(zhǎng)吸收情況不斷加重;④免疫組化染色結(jié)果顯示加力7天組和28天組的lc3染色為陽性,caspase3染色在正畸加力14天組和加力28天組為陽性。結(jié)論:適宜的矯治力持續(xù)作用下,壓力側(cè)牙槽骨組織骨細(xì)胞早期出現(xiàn)自噬反應(yīng)增強(qiáng)的現(xiàn)象,此時(shí)骨吸收現(xiàn)象不明顯;隨著矯治力持續(xù)作用,自噬減弱,凋亡開始出現(xiàn),骨吸收程度也不斷加重。這一結(jié)果提示,壓力側(cè)牙槽骨組織骨細(xì)胞早期出現(xiàn)自噬反應(yīng)增強(qiáng)可能是一種組織保護(hù)機(jī)制,可以延遲凋亡作用的出現(xiàn)。2.饑餓誘導(dǎo)環(huán)境下小鼠成骨細(xì)胞自噬與凋亡相互作用的實(shí)驗(yàn)研究目的:探討?zhàn)囸I條件下自噬與凋亡在小鼠成骨細(xì)胞中的發(fā)生情況與相互關(guān)系。方法:培養(yǎng)初代mc3t3-e1小鼠成骨細(xì)胞系,將其分為饑餓誘導(dǎo)組與3-甲基腺素(3-ma)預(yù)處理饑餓誘導(dǎo)組。其中,饑餓誘導(dǎo)組采用單純饑餓誘導(dǎo)法,用厄爾氏平衡鹽溶液(earle’sbalancedsaltsolution,ebss)培養(yǎng)1h、2h、3h、4h、5h、6h。3-ma是一種特異性自噬抑制劑,在3-ma預(yù)處理饑餓誘導(dǎo)組中,正常培養(yǎng)的小鼠成骨細(xì)胞加入3-MA預(yù)處理1 h后,再用EBSS平衡鹽溶液培養(yǎng)1 h、2h、3 h、4 h、5 h、6 h。應(yīng)用透射電子顯微鏡觀察饑餓誘導(dǎo)組小鼠成骨細(xì)胞自噬與凋亡的形態(tài)學(xué)變化,應(yīng)用Western blot免疫印跡法和Annexin V-FITC/PI雙標(biāo)記流式細(xì)胞術(shù)檢測(cè)小鼠成骨細(xì)胞中自噬相關(guān)蛋白LC3,凋亡相關(guān)蛋白caspase 3表達(dá)水平的變化。結(jié)果:①透射電鏡結(jié)果顯示單純饑餓誘導(dǎo)條件下,細(xì)胞在2 h前以自噬為主,2 h后以凋亡為主,并隨著時(shí)間的延長(zhǎng),凋亡水平逐漸增高;②Western blot結(jié)果顯示與饑餓誘導(dǎo)組相比,3-MA預(yù)處理饑餓誘導(dǎo)組的LC3轉(zhuǎn)化水平在1 h、2 h、3h、4 h、5 h、6 h都出產(chǎn)生了明顯的下降趨勢(shì)(P0.05),caspase 3蛋白表達(dá)水平在1 h、2 h、3 h和4 h增高(P0.05),但0 h、5 h和6 h無差異(P0.05);③Annexin V-FITC/PI檢測(cè)結(jié)果證明和單純饑餓誘導(dǎo)組相比,3-MA預(yù)處理組僅在1 h、2 h、3h和4 h促進(jìn)了細(xì)胞凋亡(P0.05),而并未影響到0 h、5 h和6 h的細(xì)胞凋亡(P0.05)。結(jié)論:在饑餓誘導(dǎo)環(huán)境成骨細(xì)胞早期發(fā)生的自噬作用,可能是細(xì)胞避免死亡的一種保護(hù)機(jī)制,延遲凋亡作用出現(xiàn)的時(shí)間。本研究中體內(nèi)、體外兩方面的實(shí)驗(yàn)結(jié)果初步說明,當(dāng)正畸力作用于牙齒后,其壓力側(cè)牙槽骨組織早期出現(xiàn)的自噬作用加強(qiáng)現(xiàn)象可能是骨組織細(xì)胞對(duì)抗氧氣、營(yíng)養(yǎng)不足的一種保護(hù)機(jī)制,可以拮抗、延緩凋亡作用的出現(xiàn)。一旦這種機(jī)體保護(hù)機(jī)制減弱、消失,骨組織細(xì)胞就會(huì)因凋亡作用的增強(qiáng)而大量消亡、骨組織也隨之吸收。
[Abstract]:In the process of orthodontic treatment, a large number of osteoblasts in alveolar bone on the pressure side will die and osteoclasts will proliferate, leading to alveolar bone resorption; osteoblasts on the tension side will proliferate, alveolar bone new bone formation, which occurs in the alveolar bone tissue remodeling is the basis of orthodontic tooth movement [1]. Current studies suggest that a large number of osteoblasts, including osteoblasts, in the pressure-side alveolar bone die because of changes in the local microenvironment of the alveolar bone under orthodontic forces, including narrowing of the pressure-side periodontal ligament, narrowing of the capillaries that provide nutrients and oxygen under orthodontic forces, narrowing of the lumen, blocking of circulation and decreasing of blood supply [2]. This leads to a hypoxic, nutrient-deficient starvation environment for alveolar bone cells on the pressure side [3]. How does this environment result in alveolar bone resorption and a large number of alveolar bone cells on the pressure side die? Three ways of cell death: necrosis, autophagy, apoptosis [4], alveolar bone remodeling belongs to teeth under the appropriate orthodontic force. Physiological remodeling, bone tissue will not occur cell necrosis and other pathological changes, autophagy and apoptosis belong to the cell-dominated mode of programmed death, they are respectively in the pressure side of bone tissue cell death in the process of what role? How is the relationship? This study aimed at this problem, through the establishment of beagle dogs correct The expression of autophagy and apoptosis in pressure-side alveolar bone during orthodontic remodeling was studied in vivo. The expression of autophagy and apoptosis in osteoblasts under starvation was observed in vitro, and the relationship between autophagy and apoptosis was studied. The main contents of this study include the following two aspects: 1. Animal experimental study on the changes of autophagy and apoptosis of alveolar bone cells during tooth movement Objective: To establish a model of orthodontic tooth movement in beagle dogs and detect the morphological changes of alveolar bone on the pressure side and autophagy and apoptosis in bone tissue cells under orthodontic force. Methods: Twenty-four canines of six adult beagles were randomly divided into two groups: the control group and the orthodontic afterburner group. The effect of orthodontic force on tooth movement was observed. The remodeling of alveolar bone tissue on the pressure side was analyzed by he method. The effects of autophagy and apoptosis in alveolar bone tissue on the pressure side were analyzed by immunohistochemical staining and Western blot. Compared with the control group, the distal movement of canines in each experimental group was obvious (p0.05), and the distal movement of teeth increased with the increase of orthodontic force (p0.05). 2) Western blot showed that compared with the blank control group, the transformation rate of lc3-II/lc3-I in alveolar bone tissue on the pressure side increased rapidly, reached the peak on 7 days (p0.05), and caspase-3 protein expression level reached the peak on 7 days. The results of he showed that the absorption of alveolar bone tissue on the pressure side was aggravated with the extension of orthodontic treatment time compared with the blank control group; the results of immunohistochemical staining showed that LC3 staining was positive in the 7-day group and 28-day group, and caspase 3 staining was positive in the 14-day group and 28-day group after orthodontic treatment. CONCLUSION: The autophagy of alveolar bone cells on the pressure side was enhanced at the early stage under the sustained effect of appropriate orthodontic force, but the bone resorption was not obvious at this time. With the sustained effect of orthodontic force, the autophagy was weakened, the apoptosis began to appear, and the bone resorption was aggravated. Early enhancement of autophagy may be a tissue protective mechanism that delays apoptosis. 2. Experimental study on the interaction between autophagy and apoptosis of mouse osteoblasts in starvation-induced environment Objective: To investigate the occurrence and correlation of autophagy and apoptosis in mouse osteoblasts under starvation. C3t3-e1 mouse osteoblasts were divided into starvation-inducing group and starvation-inducing group pretreated with 3-methyladenine (3-ma). starvation-inducing group was treated with simple starvation-inducing method and cultured with earle's balanced salt solution (ebss) for 1 h, 2 h, 3 h, 4 h, 5 h, 6 h.3-ma, which was a specific autophagy inhibitor. In the starvation-induced group, the normal cultured osteoblasts were pretreated with 3-MA for 1 h, then cultured in EBSS equilibrium salt solution for 1 h, 2 h, 3 h, 4 h, 5 h and 6 h. The morphological changes of autophagy and apoptosis of osteoblasts in the starvation-induced group were observed by transmission electron microscopy. Western blot immunoblot and Annexin V-FITC/PI flow cytometry were used. The expression of autophagy-related protein LC3 and apoptosis-related protein caspase-3 in mouse osteoblasts were detected by cytometry. Results: The results of transmission electron microscopy showed that autophagy was predominant before 2 hours and apoptosis was predominant after 2 hours under starvation-induced condition, and the level of apoptosis gradually increased with the prolongation of time. Compared with starvation induction group, the level of LC3 transformation in 3-MA pretreatment starvation induction group decreased significantly at 1 h, 2 h, 3 h, 4 h, 5 h and 6 h (P 0.05). The expression of caspase-3 protein increased at 1 h, 2 h, 3 h and 4 h (P 0.05), but there was no difference between 0 h, 5 h and 6 h (P 0.05). The results of Annexin V-FITC/PI showed that the expression level of caspase-3 protein in starvation induction group was significantly lower than that in starvation induction group. Compared with 3-MA pretreatment, 3-MA pretreatment only promoted apoptosis at 1 h, 2 h, 3 h and 4 h (P 0.05), but did not affect apoptosis at 0 h, 5 h and 6 h (P 0.05). Conclusion: Autophagy in the early stage of osteoblasts in starvation-induced environment may be a protective mechanism to prevent cell death and delay the time of apoptosis. External experimental results preliminarily show that the autophagy enhancement of alveolar bone tissue on the pressure side may be a protective mechanism of bone tissue cells against oxygen and nutritional deficiency, which can antagonize and delay the occurrence of apoptosis. The cells will be destroyed by the enhancement of apoptosis and the bone tissue will also be absorbed.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R783.5

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