骨膜蛋白在正畸牙周膜改建中的表达及调控机制

发布时间：2018-06-29 10:20

本文选题：骨膜蛋白 + 正畸　；参考：《南方医科大学》2014年博士论文

【摘要】：研究背景和目的：长期以来,正畸治疗错合畸形的一个突出问题就是临床治疗疗程长,也是众多牙颌畸形患者特别是成年患者决定是否矫治的关键因素之一。研究如何在对牙周组织、牙根和牙髓无可逆性损伤的条件下加快正畸牙齿移动,缩短正畸治疗疗程,成为本学科的研究热点和难点。现在人们的研究热点逐渐集中到机体对正畸力的生物学反应上。在正畸临床中发现没有牙周膜的粘连牙或者种植牙都不能发生移动,这就提示我们：如果只有力的作用,而没有牙周膜的改建,牙齿还不能移向所希望的位置或维持在必要的位置上,因此正畸牙齿移动有赖于牙周组织的相应反应,正畸牙齿的移动必须以牙周膜和牙槽骨的改建为前提。在正畸治疗过程中,机械力刺激通过牙齿传递到牙周膜,引发和激活其内部各种效应细胞的生物代谢活性,从而影响牙周组织的塑形和改建,最终使牙齿移动到位。也就是说牙周膜是正畸治疗的生理介质,它是一种变异的骨膜,具有明显的骨沉积和骨吸收能力；位于牙骨质与牙槽骨两种硬组织之间,由致密结缔组织所构成,多数纤维排列成束,其两端分别埋于牙骨质内牙槽窝的骨壁里,使牙齿固位于牙槽窝内。胶原纤维成束状排列,它可充分缓冲和吸收作用力,在牙周组织、牙周膜损伤修复中起着至关重要的作用。牙周膜在接受机械力刺激后,通过信号转导系统将力学刺激转化为生物学信号,引起牙周组织代谢的改变和牙槽骨改建等一系列生物学反应,这是一个十分复杂的过程,涉及到多种信号转导分子和通道以及各种基质蛋白、细胞因子。骨膜蛋白(Periostin,PN)是一种新发现的多功能细胞外基质蛋白,分子量为90kD,在骨膜和牙周韧带定位表达。骨膜蛋白可被转化生长因子β(TGFβ)诱导表达,其支持细胞粘附、胶原蛋白的交联和调节纤维形成,对牙周组织的完整性、牙齿发育、萌出以及机械应激导致的组织修复和再生发挥着重要的作用；被认为是对牙周膜稳态调节有关键作用的细胞外基质蛋白,其可能对牙周病及正畸牙移动中牙周组织改建也起着关键的作用。 PN与TGF-β1的关系越来越受各界的关注,多项研究表明PN参与多个系统疾病的发生发展,且其表达与TGF-β1有密切关系。气道上皮细胞分泌PN可激活TGF-β1,进而促进气道纤维化并降低气道弹性导致哮喘发生；在心脏方面研究发现PN参与心肌胶原的合成和成熟,培养鼠胚胎时期的成纤维细胞,经TGF-β1刺激后,PN蛋白基因敲除后胶原的合成量仅为野生型的胚胎鼠胶原合成量的32%。由此可见PN蛋白是成纤维细胞合成分泌胶原的主要信号通道。Norris研究发现在PN基因敲除小鼠中心肌表现出较低的胶原蛋白变性温度,说明交联的胶原蛋白水平降低,PN可调节Ⅰ型胶原纤维形成,而且能够与Ⅰ型胶原直接相互作用,从而成为纤维结缔组织的生物力学特性的重要作用介质。Rios利用X线及Micro-ct观察敲除骨膜蛋白基因的小鼠牙齿牙周膜发育状况,牙齿发育萌出到建立正常咬合前,PN-null小鼠牙周膜于正常对照组相比无差别；牙齿萌出承受咀嚼力后3月龄实验组小鼠牙周组织出现明显破坏、牙周附着降低、牙槽骨吸收,表明在咬合力作用下骨膜蛋白在维持牙周膜的完整性和功能方面起着不可或缺的作用,在功能减退组的表达水平的改变可能会被认为是一种对环境变化的适应形式。 Choi降低Wistar大鼠的右上第一磨牙咬合后的研究结果显示：最初牙周膜纤维数量减少、纤维变薄、随后牙周膜纤维和它们在牙槽骨的附着排列变得混乱,最终牙周膜纤维失去其网状结构。PNmRNA表达在24h大幅下调,这一变化持续在整个实验过程中。证实在缺乏机械应力的情况下,伴随着牙周膜中PN水平的减少,牙周膜纤维系统开始出现退化。Wen对TGF-β1和FAK调节PN在牙周成纤维细胞中的表达的作用进行了研究,用TGF-β1处理显著增加PNmRNA水平,粘着斑激酶(Focal Adhesion Kinase FAK)抑制剂有衰减作用。即使用循环应力刺激后,FAK敲除的成纤维细胞中也没有检测至PNmRNA的表达。这些结果提示,PN蛋白在人牙周膜中强烈表达,可能会涉及FAK依赖的通路的调节。最新研究证实炎症介质(TNF-α)和细菌毒素因子(LPS)能显著降低牙周膜成纤维细胞表达骨膜蛋白,表明PN在牙周病发生发展过程中是一个重要因子。这也提示我们在牙周患者进行正畸治疗时,口腔卫生的清洁护理、炎症的治疗控制是多么重要的前提；否则牙周组织遭到破坏、正畸牙周组织的改建、骨组织改建和正畸牙齿的移动根本无从谈起。 PN作为一种重要的细胞外基质蛋白已引起广泛的关注,在全身多个系统都具有重要的作用,现对其研究也日渐深入。PN作为应力作用下维持牙周膜的完整性和功能方面起着必不可少作用的关键因子,对其体外方面研究多集中于牙周膜细胞加力机械刺激,涉及多因素体内研究却相对较少。通过建立小鼠正畸牙齿移动模型来研究PN在正畸牙齿移动过程中牙周组织改建作用还未见相关报道,PN在各疾病中特别是牙周组织的作用及其机制的研究尚不清楚。本研究将首次建立小鼠正畸牙齿移动模型,运用定量免疫组化、Micro-ct、QRT-PCR、 Westernblot以及敲除PN基因小鼠等方法研究PN在牙周膜改建调控作用及相关信号通路。由于小鼠牙周膜过少,难以进行蛋白水平功能检测。本实验拟通过收集正畸临床患者拔除前磨牙牙周膜,采用QRT-PCR和蛋白组学技术进一步研究PN在正畸牙周组织改建作用的分子信号通路,深入探讨PN在正畸牙齿移动过程中牙周组织改建及其分子机制。为进一步系统性探讨机械力作用下牙周组织改建的分子生物学机制开辟了一条新途径,有助于更好的理解正畸牙齿移动的机理,从而为加快牙齿移动的正畸临床研究提供理论依据。方法： 1.利用自制口腔微型装置建立小鼠正畸牙移动模型利用自制口腔微型装置和口腔显微镜在小鼠切牙和第一磨牙之间用NI-TI拉簧固定、加力20g,分别在加力1、3、5、7d后处死小鼠,固定、脱钙、制作小鼠第一磨牙牙周组织石蜡切片,观察HE染色牙周组织形态学和磨牙间变化。 2.观察小鼠正畸牙齿移动中牙周膜PN表达的变化建立小鼠正畸牙移动模型,分别在加力1、3、5、7d后处死小鼠,制作免疫组化切片,用免疫组织化学和图像定量分析技术检测牙周膜PN表达的变化。 3.PN基因敲除小鼠基因型的鉴定剪取基因敲除小鼠尾部0.5cm放入标记好的EP管内,提取DNA,根据设计引物进行PCR扩增,扩增产物经1.5%琼脂糖凝胶电泳后取出置于紫外灯下观察并拍照。鉴定基因小鼠标准为：PN+/+基因型小鼠PCR扩增产物为691bp, PN-/-基因型小鼠是PCR扩产物为500bp。 4.Micro-ct影像学检查处死C57BL和PN基因敲除小鼠,分离上下颌骨,分别选取各组小鼠上、下颌骨,标记好各组标本,固定于专用扫描管中,使用显微CT成像系统进行断层扫描成像,对牙周组织进行评估。 5.RT-PCR检测小鼠牙移动牙周膜改建中与PN相关信号通路调控因子建立小鼠正畸牙移动模型,分为C57小鼠未加力组、C57小鼠加力组、PN-null小鼠未加力组、PN-null、鼠加力组,分别加力5d后处死,拔出牙齿放于盛有裂解液的EP管中,实时荧光定量PC检测牙周膜中PN、TGF-B1、FAK相关信号通路调控因子的表达,并统计分析各组之间PN、TGF-B1、FAK表达的相关性。 6.RT-PC检测人正畸和未正畸牙周膜中与PN相关信号通路调控因子对照组为重度拥挤病例,实验组为牙列整齐、牙弓前突病例。对照组和实验组均为拔出前磨牙的青少年正畸患者,实时荧光定量PCR检测牙周膜中PN、TGF-B1、FAK相关信号通路调控因子的表达,并统计分析各组之间PN、TGF-B1、FAK表达的相关性。 7Westernblot检测人正畸和未正畸牙周膜中与PN相关信号通路调控因子 Westernblot检测人正畸和未正畸牙周膜中PN、TGF-B1、FAK、P-Akt、AKT相关信号通路调控因子的表达,并统计分析各组之间PN、TGF-B1、FAK、P-Akt、AKT表达的相关性。结果 1.加力过程中牙齿按设定的方向移动,加力组第一磨牙与第二磨牙之间出现间隙。加力组张力侧牙周间隙变宽,牙周膜中纤维排列疏松,细胞成份增多,牙槽表面可见活跃的成骨细胞和新骨沉积,压力侧牙周间隙缩窄。 2.PN在牙周膜呈阳性表达,着色相对较浅,分布较均匀。正畸加力组大鼠牙周膜中PN表达增强,牙周膜阳性染色强。与Od对照组比较,加力实验各组牙周膜PN表达均增强(P0.05),与1d组比较,3d、5d组牙周膜PN表达均增强(P0.05),7d加力实验组PN表达无统计学意义(P0.05)。与3d加力组比较,5d、7d加力实验组(P0.05)差异无统计学意义(P0.05)；与5d加力实验组比较,7d加力实验组牙周膜PN表达显著减弱,(P0.01)。5d组是小鼠整个牙齿移动改建周期中PN表达量最高。 3.1泳道只有一个目的条带,亮度强,条带范围在691bp处,根据判定标准判定为WT小鼠。2泳道也只有一个目的条带,亮度较强,条带范围在500bp左右,因此根据判定标准判定为PN-/-小鼠。 4.小鼠Micro-ct三维重建图显示：与对照组C57小鼠相比,PN-null、鼠牙周组织破坏,牙槽骨出现明显的水平吸收,磨牙根分叉区完全暴露。小鼠Micro-ct曲面断层图显示：与对照组C57小鼠相比,PN-nul1小鼠牙周组织破坏,牙槽骨出现明显的吸收,牙槽脊顶消失,牙周膜连续性中断破坏。 5.与C57未加力对照组相比,C57小鼠牙加力实验组牙周膜中PNmRNA表达增强,(P0.05)差值有统计学意义；FAK mRNA表达显著增强,(P0.05)差值有统计学意义；TGF-B1mRNA表达显著增强(P0.05)差值有统计学意义。与PN-null未加力对照组相比,PN-nul1加力实验组牙周膜中FA mRNA表达增强(P0.05)差值有统计学意义,TGF-B1mRNA表达显著增强,(P0.01)差值有统计学意义。与PN-null未加力对照组相比,C57未加力组牙周膜TGF-B1mRNA表达显著减少,(P0.01)差值有统计学意义,FAKmRNA表达显著增强,(P0.01)差值有统计学意义。与PN-nul1加力组相比,C57加力实验组牙周膜TGF-BmRNA表达减少,(P0.05)差值有统计学意义；FAKmRNA表达显著增强,(P0.01)差值有统计学意义。 6.与人正畸未加力对照组相比,人正畸加力实验组牙周膜中PNmRNA表达显著增强,(P0.01)差值有统计学意义；FAKmRNA表达显著增强,(P0.01)差值有统计学意义；TGF-B1mRNA表达显著增强,(P0.05)差值有统计学意义。 7.与未正畸组相比,人正畸组牙周膜PN、TGF-B1、FAK、P-Akt的蛋白表达均显著增高,(P0.01)差值均有统计学意义。与未正畸组相比,人正畸组牙周膜Ak、a-tublin的蛋白表达均没有明显差异,(P0.05)差值无统计学意义结论 1.首次成功建立了小鼠正畸加力模型,小鼠正畸牙齿移动过程中,牙周膜中PN表达增强且贯穿牙周膜改建的全过程,从而确定PN参与了小鼠牙齿移动中的牙周膜改建。 2.PN是保持牙周膜的结构和功能完整性所必需的,是正畸作用下牙周膜的改建过程一个重要分子环节,发挥着转导调控作用。 3.TGF-B1-PN-FAK信号通路参与调控了正畸作用下牙周膜的改建过程,FAK可能通过除TGF-B1-PN-FAK信号通路之外的其他途径参与了牙周膜改建的过程。 4.TGF-B1-PN-FAK可能通过激活下游的Akt磷酸化信号通路,对正畸力牙周膜改建中进行调控。
[Abstract]:Background and purpose of the study :

For a long time , one of the outstanding problems of orthodontic treatment is the long duration of clinical treatment , and the key factors for determining whether to correct the orthodontic tooth movement under the condition of no reversible damage to periodontal tissue , root and pulp .

in that proces of orthodontic treatment , the mechanical force stimulate the transmission of teeth to the periodontal membrane , initiate and activate the biological metabolic activity of various effector cells in the periodontal ligament , thereby influencing the shaping and reconstruction of the periodontal tissue , and finally moving the teeth into place .
The periodontal ligament is composed of compact connective tissue , which is composed of dense connective tissue , the two ends of which are respectively embedded in the bone wall of the alveolar cavity of the alveolar bone , so that the teeth are fixed in the alveolar cavity .

Periostin ( PN ) is a newly discovered extracellular matrix protein with a molecular weight of 90kD . It is expressed in bone and periodontal ligament . The bone membrane protein can be expressed by transforming growth factor - 尾 ( TGF - 尾 ) , which supports cell adhesion , collagen cross - linking and regulation of fiber formation , plays an important role in the tissue repair and regeneration of periodontal tissues , tooth development , eruption and mechanical stress .
It is believed that extracellular matrix protein plays a key role in the steady state regulation of periodontal ligament , which may play a key role in periodontal disease and orthodontic tooth movement .

The relationship between PN and TGF - 尾1 is more and more concerned , and many researches have shown that PN is involved in the development of multiple system diseases , and its expression is closely related to TGF - 尾1 . The secretion of PN in airway epithelial cells can activate TGF - 尾1 , thus promoting airway fibrosis and reducing airway elasticity leading to asthma .
The study found that PN was involved in the synthesis and maturation of collagen in cardiac muscle cells . After stimulation of TGF - 尾1 , the collagen synthesis was only 32 % of the wild type mouse collagen synthesis .
In the experimental group , the periodontal tissues of the experimental group were significantly damaged , the periodontal attachment decreased and the alveolar bone resorption , indicating that the membrane protein plays an indispensable role in maintaining the integrity and function of the periodontal ligament , and the change of the expression level in the hypofunctional group may be regarded as an adaptive form of environmental change .

The results showed that the expression of PNmRNA in periodontal ligament fibroblasts was significantly decreased by treatment with TGF - 尾1 . The results suggested that the expression of PNmRNA in periodontal ligament fibroblasts was significantly decreased by treatment with TGF - 尾1 .
Otherwise , the periodontal tissue is destroyed , the remodeling of the orthodontic periodontal tissue , the alteration of the bone tissue and the movement of the orthodontic tooth are never started .

PN , as an important extracellular matrix protein , has attracted extensive attention and plays an important role in the development of periodontal ligament cells .

Method :

1 . Establishment of mouse orthodontic tooth movement model by self - made oral micro - device

Using a self - made oral micro - device and an oral microscope , a NI - TI tension spring was used to fix the teeth of the mouse and the first molar , and 20 g were added , and the mice were sacrificed after 1 , 3 , 5 and 7 days respectively , and the paraffin sections of the first molar periodontal tissues of the mice were made , and the morphological and molar changes of the periodontal tissues were observed with HE staining .

2 . Observe the change of periodontal ligament PN expression in orthodontic tooth movement in mice

The mice were sacrificed after 1 , 3 , 5 and 7 days respectively , and the mice were sacrificed after 1 , 3 , 5 and 7 days respectively , and immunohistochemistry and image analysis were used to detect the change of PN expression in periodontal ligament .

3 . Identification of Gene Knockout Mice

The gene knockout mice were subjected to PCR amplification by 1.5 % agarose gel electrophoresis . The PCR amplified products were 691bp in PN + / + genotype mice and 500 bp for PN - / - genotype mice .

4 . Micro - ct imaging examination

C57BL and PN gene knockout mice were sacrificed and the mandible was isolated . Each group of mice were divided into two groups : the mandible , the marked group and the special scanning tube . The CT imaging system was used to perform the tomographic imaging , and the periodontal tissues were evaluated .

5 . RT - PCR Detection of Regulation Factors of PN - related Signal Pathway in Dental Movement of Mouse Teeth

The model of mouse orthodontic tooth movement was established , which was divided into C57 mice without stress group , C57 mouse additive group , PN - null mice without force group , PN - null and mouse additive force group . After 5 days of addition of PN - null mice , the expression of PN , TGF - B1 in the periodontal ligament was detected by real - time fluorescence quantitative PC , and the correlation between the expression of PN , TGF - B1 and focal adhesion in periodontal ligament was detected by real - time fluorescence quantitative PC .

6 . RT - PC Detection of the Regulation Factors of PN - related Signal Pathway in Orthodontist and Unorthodontic periodontal ligament

In the control group , there were severe congestion cases in the experimental group . The control group and the experimental group were orthodontic patients with premolar teeth , and the expression of PN , TGF - B1 in the periodontal ligament was detected by real - time fluorescence quantitative PCR , and the correlation between the expression of PN , TGF - B1 and focal adhesion in the periodontal ligament was analyzed by real - time quantitative PCR .

Regulation factors of PN related signal pathway in orthodontic and orthodontic periodontal ligament by 7 Western blot

Western blot was used to detect the expression of PN , TGF - B1 , focal adhesion molecule - 1 , P - 3 - 3 in human orthodontist and non - orthodontic periodontal ligament , and to analyze the correlation between the expression of PN , TGF - B1 , focal adhesion kinase , P - 3 - 3 and P - 3 - 3 in each group .

Results

1 . When the teeth are moved in the set direction during the addition process , there is a gap between the first molar and the second molar in the addition force . The periodontal gap in the tension side of the force group becomes wider , the fibrous arrangement in the periodontal ligament is loose , the cell components are increased , the active osteoblasts and new bone deposits can be seen on the surface of the alveolar groove , and the pressure side periodontal gap is narrowed .

2 . PN was positive in the periodontal ligament , the color was relatively shallow and the distribution was more uniform . Compared with the control group , the expression of PN in periodontal ligament was enhanced ( P0.05 ) . Compared with the control group , the expression of PN in periodontal ligament increased significantly ( P0.05 ) .
Compared with the 5d group , the expression of PN in the periodontal ligament of the experimental group decreased significantly after 7 days ( P0.01 ) , and the expression of PN was the highest in the 5d group .

3.1 The lane has only one target band , the intensity is strong , the band range is 691bp , it is judged as WT mice according to the determination standard . The lane also has only one target band , the brightness is stronger , the band range is about 500bp , and therefore , it is judged as PN - / - mouse according to the determination standard .

4 . Mouse Micro - ct 3 - D reconstruction showed that PN - null mice were destroyed and alveolar bone was completely exposed when compared with control group C57 mice . The mouse micro - ct surface tomographic image showed that PN - nul1 mice were destroyed by periodontal tissue and alveolar ridge disappeared and periodontal ligament continuity was disrupted compared with control group C57 mice .

5 . Compared with the C57 untreated control group , the expression of PNmRNA in the periodontal ligament of C57 mice increased significantly ( P0.05 ) .
The mRNA expression was significantly increased , ( P0.05 ) , and the difference was statistically significant .
Compared with the control group , there was a significant difference in the expression of TGF - B mRNA in the periodontal ligament ( P0.05 ) . Compared with the control group , the expression of TGF - B1 mRNA was significantly increased ( P0.01 ) . Compared with the PN - null unenhanced control group , the expression of TGF - B mRNA in the untreated group was significantly increased ( P0.01 ) .
The expression of FAKmRNA was significantly increased ( P0.01 ) .

6 . Compared with the untreated control group , PNmRNA expression was significantly increased in the periodontal ligament of the experimental group ( P0.01 ) .
The expression of FAKmRNA was significantly increased ( P0.01 ) .
The expression of TGF - B 1 mRNA was significantly increased ( P0.05 ) .

7 . Compared with non - orthodontic group , the expression of protein in periodontal ligament PN , TGF - B1 , focal adhesion kinase and P - 3 in orthodontic group was significantly higher than that in the untreated group ( P0.01 ) . There was no significant difference in protein expression in the periodontal ligament , Ak , a - tufa in the orthodontic group compared with the unorthodontist group ( P0.05 ) .

Conclusion

1 . For the first time , the mouse orthodontic force model was successfully established . During orthodontic tooth movement , the PN expression in periodontal ligament was enhanced and through the whole process of periodontal ligament reconstruction , so it was determined that PN was involved in the reconstruction of periodontal ligament in mouse tooth movement .

2 . PN is necessary to maintain the structural and functional integrity of periodontal ligament . It is an important element in the remodeling process of periodontal ligament under the action of orthodontic treatment .

3 . The TGF - B1 - PN - focal adhesion pathway is involved in the alteration of periodontal ligament during orthodontic treatment , which may be involved in the reconstruction of periodontal ligament by other ways than TGF - B1 - PN - focal adhesion pathway .

4 . TGF - B1 - PN - PKP may regulate the remodeling of orthodontic periodontal ligament by activating the downstream phosphorylation signaling pathway .
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R783.5

【参考文献】