神经因素影响大鼠牙周组织缺损愈合过程的机制探讨
发布时间：2018-09-19 18:10
【摘要】：背景和目的： 如何使因牙周病造成的病变牙周组织,特别是牙槽骨在“保质”的前提下更有效地“增量”,使已松动的患牙得以再次稳固,并能正常行使牙齿的咀嚼功能是目前牙周病治疗领域的研究重点。以牙槽骨吸收破坏为主要特征的牙周病变组织,同时亦存在该部位的神经血管系统结构与功能的破坏,从发育生物学的观点从发,牙周组织再生过程中神经支配或许起到至关重要的作用。牙周病序列治疗中,以各种基础治疗手段,消除炎症,控制发展这一治疗目标已基本实现。但如何促进牙周组织的再生,特别是有质有量的牙槽骨的再生,不论从临床实践,还是理论研究,都是值得不断深化研究与探索的一个牙周学科领域的重要课题。 在现有众多牙周组织再生治疗的策略和相关研究中,以种子细胞、生物支架材料和各种生长因子的研究较为热门,但是由于牙周病病因及相应组织修复机制的复杂性,以及机体调控组织再生的系统性,其所取得的研究结果并没有达到我们所追求牙周组织再生的目标。牙槽骨是全身骨组织中代谢最活跃的骨质,神经血管在牙槽骨改建过程中的作用不容忽视,而现有的研究重点大多集中于如何获得更多的再生组织却忽略了这一过程中神经与血管的再生,其获得的再生牙周组织与天然的生理性牙周组织相比,在组织结构牙槽骨密度及应力方面均有很大的差距。显然这种“简单还原”与组织的“自然形成”相比,忽略了机体生命现象的精确性。探索神经对牙周组织再生过程的影响,在牙周组织再生研究领域中具有重要的意义。目前通过体外相关实验研究证实神经系统可以通过介导成骨细胞以及破骨细胞的活性参与调节骨代谢,在骨的修复改建过程中起着关键性的作用。近年来,随着中枢及周围神经系统对骨折愈合影响研究的深入,发现骨组织也是神经作用的靶器官,周围神经系统在骨折愈合及骨代谢中具有重要作用。并且近期研究发现,其主要是通过肽能神经分泌的神经肽对骨组织起作用的,而降钙素基因相关肽(calcitonin gene related peptide, CGRP)是骨组织中分布最为广泛的一种神经肽,主要分布于骨代谢活跃的部位,CGRP直接或间接地通过调节成骨细胞和破骨细胞分化及活性来影响骨折愈合。早在Hukkanen研究大鼠骨折模型实验过程中,便发现在新生骨组织中有CGRP免疫组织染色阳性纤维长入,随后CGRP的作用及机制也成为研究骨折愈合改建的热点。 研究表明CGRP的阳性神经纤维在颌骨及其骨膜中均有分布,进而推测其参与了生长发育期的颌骨生长形成和后期的修复改建过程。降钙素基因相关肽阳性神经纤维在牙周组织中分布于磨牙牙周组织,牙槽骨以及牙槽骨中的小动脉和小静脉周围均有CGRP阳性神经纤维的环绕,提示CGRP阳性神经纤维在牙周组织再生过程中的神经血管的再生有重要作用。在口腔外科的研究中,针对CGRP在骨折愈合的研究较为广泛,研究显示CGRP可以促进下颌骨骨折的愈合。在正畸医学研究中,CGRP对正畸牙齿移动过程中牙槽骨的改建和重塑具有一定的调节作用。鉴于以上理论基础,我们推测CGRP是牙周组织再生中必不可少的调节因子。本课题组已经通过组织形态学观察证明神经可影响牙周组织再生的质量,并且通过免疫组织化学观察发现CGRP在牙周组织再生过程中的表达变化,本实验在此基础上,通过分子水平检测进一步探讨CGRP在牙周组织再生过程中的作用机制。 方法： 1、离断下牙槽神经后大鼠牙周组织中CGRP及SP的表达变化建立离断大鼠下牙槽神经的动物模型,左侧离断神经为实验组,右侧保留神经为对照组,实验分为六组即0天、3天、7天、14天、21天、28天。采用Realtime-PCR及Western Blot方法检测大鼠牙周组织中CGRP以及SP的表达变化。 2、失下牙槽神经对大鼠牙周组织愈合过程中CGRP、SP及骨改建相关因子表达变化的研究 建立离断大鼠下牙槽神经伴牙周缺损模型,左侧切断大鼠下牙槽神经伴牙周缺损为实验组,对侧保留神经仅做牙周缺损为对照组。实验分为五组即1周、2周、4周、6周和8周。采用Realtime-PCR及Western Blot方法检测大鼠牙周组织中神经相关因子CGRP、SP及骨改建相关因子OPG、RANKL的表达变化。 结果： 1、离断下牙槽神经后大鼠牙周组织中CGRP及SP的的表达变化 Realtime-PCR结果显示失神经后牙龈及牙槽骨中的CGRP和SP均呈现先降低后上升至正常水平的趋势。统计分析结果显示3天、7天和14天组与正常组织表达水平有统计学差异(P0.05),Western Blot检测结果发现,失神经后,大鼠牙周组织中的SP蛋白表达水平总体趋势与SP mRNA的变化基本相同,统计学分析结果显示,术后第3天、7天、14天和21天的表达量与正常对照组相比有统计学差异(P0.05)。Western Blot检测CGRP蛋白结果为阴性。 2、失下牙槽神经对大鼠牙周组织愈合过程中CGRP、SP及骨改建相关因子表达变化的研究 通过Realtime-PCR方法检测方法显示牙周组织中实验组的CGRPmNRA水平在前期表现为下降趋势,从第4周开始逐渐升高,直至第八周CGRPmNRA水平的水平仍处于升高的趋势。而在对照组CGRPmNRA的表达水平在前期明显升高(P0.05),后期逐渐恢复至正常水平。SP的表达变化与CGRP趋势相同。失下牙槽神经导致牙槽骨中OPG持续弱阳性表达而RANKL持续强阳性表达。 结论： 1、离断下牙槽神经后,影响牙龈及牙槽骨中的CGRP及SP的表达 2、失下牙槽神经支配后CGRP及SP的表达变化会影响牙周组织缺损愈合过程中成骨和破骨因子的表达水平,最终影响牙周组织再生。
[Abstract]:Background and purpose:
How to make the periodontal tissues, especially alveolar bone, more effectively "increment" under the premise of "quality preservation" to make the loose teeth stabilize again, and can normally exercise the masticatory function of the teeth is the research focus in the field of periodontal disease treatment. From the viewpoint of developmental biology, innervation may play an important role in the regeneration of periodontal tissues. In the sequential treatment of periodontal diseases, the goal of eliminating inflammation and controlling the development of periodontal tissues has been basically achieved. How to promote the regeneration of periodontal tissue, especially the regeneration of alveolar bone with abundant quality, is an important subject in the field of periodontal science, which is worthy of further study and exploration, whether from clinical practice or theoretical research.
Seed cells, scaffolds and various growth factors are the most popular methods for periodontal tissue regeneration. However, due to the complexity of periodontal disease etiology and the mechanism of tissue repair, and the systemic regulation of periodontal tissue regeneration, the results have not reached me. Alveolar bone is the most active bone metabolism in the whole body. The role of nerves and blood vessels in alveolar bone remodeling can not be ignored. However, most of the existing research focuses on how to obtain more regenerated tissues but neglects the regeneration of nerves and blood vessels in this process. Compared with natural physiological periodontal tissues, periodontal tissues have great differences in alveolar bone mineral density and stress. Obviously, this "simple reduction" ignores the accuracy of life phenomena compared with the "natural formation" of tissues. It has been proved that the nervous system can regulate bone metabolism by mediating the activity of osteoblasts and osteoclasts, and plays a key role in bone repair and remodeling. Bone tissue is also a target organ of nervous function. Peripheral nervous system plays an important role in fracture healing and bone metabolism. Recent studies have found that it mainly acts on bone tissue through neuropeptides secreted by peptidergic nerves, and calcitonin gene related peptide (CGRP) is a bone tissue component. One of the most widely distributed neuropeptides, mainly distributed in sites with active bone metabolism, CGRP affects fracture healing directly or indirectly by regulating the differentiation and activity of osteoblasts and osteoclasts. Early in Hukkanen's study of rat fracture models, it was found that CGRP immunostaining positive fibers grew into new bone tissues. Subsequently, the role and mechanism of CGRP also become a hot spot in the study of fracture healing and reconstruction.
CGRP-positive nerve fibers were found in the mandible and periosteum, which may be involved in the growth and development of the jaw and the repair and remodeling of the jaw. Calcitonin gene-related peptide-positive nerve fibers were distributed in the periodontal tissues of molars, alveolar bones and small arteries and small vessels in alveolar bones. CGRP-positive nerve fibers surround the veins, suggesting that CGRP-positive nerve fibers play an important role in the regeneration of nerves and blood vessels during periodontal tissue regeneration. In oral surgery, the study of CGRP in fracture healing is more extensive, studies have shown that CGRP can promote the healing of mandibular fractures. In orthodontic medical research. In view of the above theoretical basis, we speculate that CGRP is an indispensable regulatory factor in periodontal tissue regeneration. Our team has demonstrated that nerves can affect the quality of periodontal tissue regeneration through histomorphological observation and immunization. The changes of CGRP expression during periodontal tissue regeneration were observed by histochemistry. On this basis, the mechanism of CGRP in periodontal tissue regeneration was further explored by molecular level detection.
Method:
1. The expression of CGRP and SP in the periodontal tissues of rats after inferior alveolar nerve transection was used to establish the animal model of inferior alveolar nerve transection. The left nerve was used as experimental group and the right nerve was used as control group. The experiment was divided into six groups: 0 days, 3 days, 7 days, 14 days, 21 days and 28 days. Changes in expression of CGRP and SP.
2. Changes of CGRP, SP and bone remodeling related factors during periodontal healing in rats with inferior alveolar nerve loss
Rats were divided into five groups: one week, two weeks, four weeks, six weeks and eight weeks. CG was detected by Realtime-PCR and Western Blot. Expression changes of RP, SP and bone remodeling related factors OPG and RANKL.
Result:
1, the expression of CGRP and SP in periodontal tissues of rats after dissecting inferior alveolar nerve.
The results of Realtime-PCR showed that CGRP and SP in gingiva and alveolar bone decreased at first and then increased to normal level after denervation. The overall trend of reaching the level was similar to that of SP mRNA. Statistical analysis showed that the expression of CGRP protein was negative on the 3rd, 7th, 14th and 21st day after operation.
2. Changes of CGRP, SP and bone remodeling related factors during periodontal healing in rats with inferior alveolar nerve loss
The level of CGRPmNRA in the periodontal tissues of the experimental group showed a downward trend in the early stage, and gradually increased from the 4th week to the 8th week. The level of CGRPmNRA in the control group increased significantly in the early stage (P 0.05), and gradually returned to positive in the late stage. The change of SP expression was the same as that of CGRP. Loss of inferior alveolar nerve resulted in persistent weak positive expression of OPG and persistent strong positive expression of RANKL in alveolar bone.
Conclusion:
1, after dissecting the inferior alveolar nerve, the expression of CGRP and SP in gingiva and alveolar bone was affected.
2. The expression of CGRP and SP after the loss of inferior alveolar innervation will affect the expression of osteogenesis and osteoclast factor in the process of periodontal tissue defect healing, and ultimately affect the regeneration of periodontal tissue.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.4
本文编号：2250926
[Abstract]:Background and purpose:
How to make the periodontal tissues, especially alveolar bone, more effectively "increment" under the premise of "quality preservation" to make the loose teeth stabilize again, and can normally exercise the masticatory function of the teeth is the research focus in the field of periodontal disease treatment. From the viewpoint of developmental biology, innervation may play an important role in the regeneration of periodontal tissues. In the sequential treatment of periodontal diseases, the goal of eliminating inflammation and controlling the development of periodontal tissues has been basically achieved. How to promote the regeneration of periodontal tissue, especially the regeneration of alveolar bone with abundant quality, is an important subject in the field of periodontal science, which is worthy of further study and exploration, whether from clinical practice or theoretical research.
Seed cells, scaffolds and various growth factors are the most popular methods for periodontal tissue regeneration. However, due to the complexity of periodontal disease etiology and the mechanism of tissue repair, and the systemic regulation of periodontal tissue regeneration, the results have not reached me. Alveolar bone is the most active bone metabolism in the whole body. The role of nerves and blood vessels in alveolar bone remodeling can not be ignored. However, most of the existing research focuses on how to obtain more regenerated tissues but neglects the regeneration of nerves and blood vessels in this process. Compared with natural physiological periodontal tissues, periodontal tissues have great differences in alveolar bone mineral density and stress. Obviously, this "simple reduction" ignores the accuracy of life phenomena compared with the "natural formation" of tissues. It has been proved that the nervous system can regulate bone metabolism by mediating the activity of osteoblasts and osteoclasts, and plays a key role in bone repair and remodeling. Bone tissue is also a target organ of nervous function. Peripheral nervous system plays an important role in fracture healing and bone metabolism. Recent studies have found that it mainly acts on bone tissue through neuropeptides secreted by peptidergic nerves, and calcitonin gene related peptide (CGRP) is a bone tissue component. One of the most widely distributed neuropeptides, mainly distributed in sites with active bone metabolism, CGRP affects fracture healing directly or indirectly by regulating the differentiation and activity of osteoblasts and osteoclasts. Early in Hukkanen's study of rat fracture models, it was found that CGRP immunostaining positive fibers grew into new bone tissues. Subsequently, the role and mechanism of CGRP also become a hot spot in the study of fracture healing and reconstruction.
CGRP-positive nerve fibers were found in the mandible and periosteum, which may be involved in the growth and development of the jaw and the repair and remodeling of the jaw. Calcitonin gene-related peptide-positive nerve fibers were distributed in the periodontal tissues of molars, alveolar bones and small arteries and small vessels in alveolar bones. CGRP-positive nerve fibers surround the veins, suggesting that CGRP-positive nerve fibers play an important role in the regeneration of nerves and blood vessels during periodontal tissue regeneration. In oral surgery, the study of CGRP in fracture healing is more extensive, studies have shown that CGRP can promote the healing of mandibular fractures. In orthodontic medical research. In view of the above theoretical basis, we speculate that CGRP is an indispensable regulatory factor in periodontal tissue regeneration. Our team has demonstrated that nerves can affect the quality of periodontal tissue regeneration through histomorphological observation and immunization. The changes of CGRP expression during periodontal tissue regeneration were observed by histochemistry. On this basis, the mechanism of CGRP in periodontal tissue regeneration was further explored by molecular level detection.
Method:
1. The expression of CGRP and SP in the periodontal tissues of rats after inferior alveolar nerve transection was used to establish the animal model of inferior alveolar nerve transection. The left nerve was used as experimental group and the right nerve was used as control group. The experiment was divided into six groups: 0 days, 3 days, 7 days, 14 days, 21 days and 28 days. Changes in expression of CGRP and SP.
2. Changes of CGRP, SP and bone remodeling related factors during periodontal healing in rats with inferior alveolar nerve loss
Rats were divided into five groups: one week, two weeks, four weeks, six weeks and eight weeks. CG was detected by Realtime-PCR and Western Blot. Expression changes of RP, SP and bone remodeling related factors OPG and RANKL.
Result:
1, the expression of CGRP and SP in periodontal tissues of rats after dissecting inferior alveolar nerve.
The results of Realtime-PCR showed that CGRP and SP in gingiva and alveolar bone decreased at first and then increased to normal level after denervation. The overall trend of reaching the level was similar to that of SP mRNA. Statistical analysis showed that the expression of CGRP protein was negative on the 3rd, 7th, 14th and 21st day after operation.
2. Changes of CGRP, SP and bone remodeling related factors during periodontal healing in rats with inferior alveolar nerve loss
The level of CGRPmNRA in the periodontal tissues of the experimental group showed a downward trend in the early stage, and gradually increased from the 4th week to the 8th week. The level of CGRPmNRA in the control group increased significantly in the early stage (P 0.05), and gradually returned to positive in the late stage. The change of SP expression was the same as that of CGRP. Loss of inferior alveolar nerve resulted in persistent weak positive expression of OPG and persistent strong positive expression of RANKL in alveolar bone.
Conclusion:
1, after dissecting the inferior alveolar nerve, the expression of CGRP and SP in gingiva and alveolar bone was affected.
2. The expression of CGRP and SP after the loss of inferior alveolar innervation will affect the expression of osteogenesis and osteoclast factor in the process of periodontal tissue defect healing, and ultimately affect the regeneration of periodontal tissue.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.4
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