饥饿诱导环境下成骨细胞自噬与凋亡作用状况的初步研究

发布时间：2018-09-14 14:28

【摘要】：在正畸治疗的过程中,牙齿受到大小合适、方向适宜的正畸力后,压力侧牙槽骨中成骨细胞会大量死亡,破骨细胞增生,导致牙槽骨吸收;张力侧成骨细胞增生,牙槽骨新骨形成,这种发生在牙槽骨的骨组织改建是正畸牙齿移动的基础[1]。目前研究认为,压力侧牙槽骨包括成骨细胞在内的骨细胞大量死亡,是因为正畸力作用下,牙槽骨局部微环境发生了改变,这其中包括压力侧牙周膜压缩变窄、提供营养和氧气的毛细血管在正畸力作用下管腔变窄,循环受阻,血供减少[2],这就导致了压力侧牙槽骨组织细胞处于一个低氧、营养缺乏的饥饿环境[3],那么这种环境是如何造成牙槽骨吸收、压力侧牙槽骨细胞大量死亡的呢?细胞死亡的三种方式:坏死、自噬、凋亡[4],在适宜的正畸力作用下,牙槽骨改建属于牙齿的生理性改建,骨组织不会发生细胞坏死等病理性改变,自噬与凋亡都属于细胞自身主导的程序性死亡方式,他们都分别在压力侧骨组织细胞死亡的过程中扮演了什么样的角色?相互关系如何?本研究针对这个问题,通过建立比格犬正畸牙齿移动模型,在体研究分析了压力侧牙槽骨正畸改建过程中自噬与凋亡表达水平的变化,并进一步通过细胞体外实验观察了成骨细胞在饥饿环境下自噬与凋亡表达水平的变化及相互关系,对压力侧骨组织细胞的消亡关系进行了初步研究。本课题研究内容包括以下两个方面:1.牙齿移动过程中牙槽骨组织细胞自噬与凋亡水平变化的动物实验研究目的:建立比格犬正畸牙齿移动模型,检测在正畸力作用下,压力侧牙槽骨组织形态学变化和骨组织细胞中自噬与凋亡的表达情况。方法:将6只成年比格犬的24颗尖牙随机分为空白对照组、正畸加力组。空白对照组无任何处理,正畸加力组牙齿以种植钉为支抗,以150g正畸力远中移动尖牙,建立比格犬尖牙远中移动模型,按正畸力作用时间分为正畸加力7天组、14天组、28天组。观察正畸力作用下牙齿移动效果,通过he方法分析其压力侧骨组织改建情况,利用免疫组化染色及westernblot方法,分析压力侧牙槽骨组织中自噬与凋亡的作用情况。结果:比格犬尖牙在正畸力作用下,①和空白对照组相比,各实验组尖牙远中移动明显(p0.05),牙齿在正畸力作用下,随作用时间的增加移动距离不断增长(p0.05);②westernblot显示与空白对照组相比,压力侧牙槽骨组织中lc3-Ⅱ/lc3-Ⅰ转化率迅速上升,7天到达顶峰(p0.05),caspase3蛋白表达水平7天后迅速升高,28天到达顶峰(p0.05);③he结果显示与空白对照组相比,压力侧牙槽骨组织随着正畸力作用时间的延长吸收情况不断加重;④免疫组化染色结果显示加力7天组和28天组的lc3染色为阳性,caspase3染色在正畸加力14天组和加力28天组为阳性。结论:适宜的矫治力持续作用下,压力侧牙槽骨组织骨细胞早期出现自噬反应增强的现象,此时骨吸收现象不明显;随着矫治力持续作用,自噬减弱,凋亡开始出现,骨吸收程度也不断加重。这一结果提示,压力侧牙槽骨组织骨细胞早期出现自噬反应增强可能是一种组织保护机制,可以延迟凋亡作用的出现。2.饥饿诱导环境下小鼠成骨细胞自噬与凋亡相互作用的实验研究目的:探讨饥饿条件下自噬与凋亡在小鼠成骨细胞中的发生情况与相互关系。方法:培养初代mc3t3-e1小鼠成骨细胞系,将其分为饥饿诱导组与3-甲基腺素(3-ma)预处理饥饿诱导组。其中,饥饿诱导组采用单纯饥饿诱导法,用厄尔氏平衡盐溶液(earle’sbalancedsaltsolution,ebss)培养1h、2h、3h、4h、5h、6h。3-ma是一种特异性自噬抑制剂,在3-ma预处理饥饿诱导组中,正常培养的小鼠成骨细胞加入3-MA预处理1 h后,再用EBSS平衡盐溶液培养1 h、2h、3 h、4 h、5 h、6 h。应用透射电子显微镜观察饥饿诱导组小鼠成骨细胞自噬与凋亡的形态学变化,应用Western blot免疫印迹法和Annexin V-FITC/PI双标记流式细胞术检测小鼠成骨细胞中自噬相关蛋白LC3,凋亡相关蛋白caspase 3表达水平的变化。结果:①透射电镜结果显示单纯饥饿诱导条件下,细胞在2 h前以自噬为主,2 h后以凋亡为主,并随着时间的延长,凋亡水平逐渐增高;②Western blot结果显示与饥饿诱导组相比,3-MA预处理饥饿诱导组的LC3转化水平在1 h、2 h、3h、4 h、5 h、6 h都出产生了明显的下降趋势(P0.05),caspase 3蛋白表达水平在1 h、2 h、3 h和4 h增高(P0.05),但0 h、5 h和6 h无差异(P0.05);③Annexin V-FITC/PI检测结果证明和单纯饥饿诱导组相比,3-MA预处理组仅在1 h、2 h、3h和4 h促进了细胞凋亡(P0.05),而并未影响到0 h、5 h和6 h的细胞凋亡(P0.05)。结论:在饥饿诱导环境成骨细胞早期发生的自噬作用,可能是细胞避免死亡的一种保护机制,延迟凋亡作用出现的时间。本研究中体内、体外两方面的实验结果初步说明,当正畸力作用于牙齿后,其压力侧牙槽骨组织早期出现的自噬作用加强现象可能是骨组织细胞对抗氧气、营养不足的一种保护机制,可以拮抗、延缓凋亡作用的出现。一旦这种机体保护机制减弱、消失,骨组织细胞就会因凋亡作用的增强而大量消亡、骨组织也随之吸收。
[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.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R783.5

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