【摘要】：背景和目的： 如何使因牙周病造成的病變牙周組織,特別是牙槽骨在“保質(zhì)”的前提下更有效地“增量”,使已松動(dòng)的患牙得以再次穩(wěn)固,并能正常行使牙齒的咀嚼功能是目前牙周病治療領(lǐng)域的研究重點(diǎn)。以牙槽骨吸收破壞為主要特征的牙周病變組織,同時(shí)亦存在該部位的神經(jīng)血管系統(tǒng)結(jié)構(gòu)與功能的破壞,從發(fā)育生物學(xué)的觀點(diǎn)從發(fā),牙周組織再生過(guò)程中神經(jīng)支配或許起到至關(guān)重要的作用。牙周病序列治療中,以各種基礎(chǔ)治療手段,消除炎癥,控制發(fā)展這一治療目標(biāo)已基本實(shí)現(xiàn)。但如何促進(jìn)牙周組織的再生,特別是有質(zhì)有量的牙槽骨的再生,不論從臨床實(shí)踐,還是理論研究,都是值得不斷深化研究與探索的一個(gè)牙周學(xué)科領(lǐng)域的重要課題。 在現(xiàn)有眾多牙周組織再生治療的策略和相關(guān)研究中,以種子細(xì)胞、生物支架材料和各種生長(zhǎng)因子的研究較為熱門(mén),但是由于牙周病病因及相應(yīng)組織修復(fù)機(jī)制的復(fù)雜性,以及機(jī)體調(diào)控組織再生的系統(tǒng)性,其所取得的研究結(jié)果并沒(méi)有達(dá)到我們所追求牙周組織再生的目標(biāo)。牙槽骨是全身骨組織中代謝最活躍的骨質(zhì),神經(jīng)血管在牙槽骨改建過(guò)程中的作用不容忽視,而現(xiàn)有的研究重點(diǎn)大多集中于如何獲得更多的再生組織卻忽略了這一過(guò)程中神經(jīng)與血管的再生,其獲得的再生牙周組織與天然的生理性牙周組織相比,在組織結(jié)構(gòu)牙槽骨密度及應(yīng)力方面均有很大的差距。顯然這種“簡(jiǎn)單還原”與組織的“自然形成”相比,忽略了機(jī)體生命現(xiàn)象的精確性。探索神經(jīng)對(duì)牙周組織再生過(guò)程的影響,在牙周組織再生研究領(lǐng)域中具有重要的意義。目前通過(guò)體外相關(guān)實(shí)驗(yàn)研究證實(shí)神經(jīng)系統(tǒng)可以通過(guò)介導(dǎo)成骨細(xì)胞以及破骨細(xì)胞的活性參與調(diào)節(jié)骨代謝,在骨的修復(fù)改建過(guò)程中起著關(guān)鍵性的作用。近年來(lái),隨著中樞及周圍神經(jīng)系統(tǒng)對(duì)骨折愈合影響研究的深入,發(fā)現(xiàn)骨組織也是神經(jīng)作用的靶器官,周圍神經(jīng)系統(tǒng)在骨折愈合及骨代謝中具有重要作用。并且近期研究發(fā)現(xiàn),其主要是通過(guò)肽能神經(jīng)分泌的神經(jīng)肽對(duì)骨組織起作用的,而降鈣素基因相關(guān)肽(calcitonin gene related peptide, CGRP)是骨組織中分布最為廣泛的一種神經(jīng)肽,主要分布于骨代謝活躍的部位,CGRP直接或間接地通過(guò)調(diào)節(jié)成骨細(xì)胞和破骨細(xì)胞分化及活性來(lái)影響骨折愈合。早在Hukkanen研究大鼠骨折模型實(shí)驗(yàn)過(guò)程中,便發(fā)現(xiàn)在新生骨組織中有CGRP免疫組織染色陽(yáng)性纖維長(zhǎng)入,隨后CGRP的作用及機(jī)制也成為研究骨折愈合改建的熱點(diǎn)。 研究表明CGRP的陽(yáng)性神經(jīng)纖維在頜骨及其骨膜中均有分布,進(jìn)而推測(cè)其參與了生長(zhǎng)發(fā)育期的頜骨生長(zhǎng)形成和后期的修復(fù)改建過(guò)程。降鈣素基因相關(guān)肽陽(yáng)性神經(jīng)纖維在牙周組織中分布于磨牙牙周組織,牙槽骨以及牙槽骨中的小動(dòng)脈和小靜脈周圍均有CGRP陽(yáng)性神經(jīng)纖維的環(huán)繞,提示CGRP陽(yáng)性神經(jīng)纖維在牙周組織再生過(guò)程中的神經(jīng)血管的再生有重要作用。在口腔外科的研究中,針對(duì)CGRP在骨折愈合的研究較為廣泛,研究顯示CGRP可以促進(jìn)下頜骨骨折的愈合。在正畸醫(yī)學(xué)研究中,CGRP對(duì)正畸牙齒移動(dòng)過(guò)程中牙槽骨的改建和重塑具有一定的調(diào)節(jié)作用。鑒于以上理論基礎(chǔ),我們推測(cè)CGRP是牙周組織再生中必不可少的調(diào)節(jié)因子。本課題組已經(jīng)通過(guò)組織形態(tài)學(xué)觀察證明神經(jīng)可影響牙周組織再生的質(zhì)量,并且通過(guò)免疫組織化學(xué)觀察發(fā)現(xiàn)CGRP在牙周組織再生過(guò)程中的表達(dá)變化,本實(shí)驗(yàn)在此基礎(chǔ)上,通過(guò)分子水平檢測(cè)進(jìn)一步探討CGRP在牙周組織再生過(guò)程中的作用機(jī)制。 方法： 1、離斷下牙槽神經(jīng)后大鼠牙周組織中CGRP及SP的表達(dá)變化建立離斷大鼠下牙槽神經(jīng)的動(dòng)物模型,左側(cè)離斷神經(jīng)為實(shí)驗(yàn)組,右側(cè)保留神經(jīng)為對(duì)照組,實(shí)驗(yàn)分為六組即0天、3天、7天、14天、21天、28天。采用Realtime-PCR及Western Blot方法檢測(cè)大鼠牙周組織中CGRP以及SP的表達(dá)變化。 2、失下牙槽神經(jīng)對(duì)大鼠牙周組織愈合過(guò)程中CGRP、SP及骨改建相關(guān)因子表達(dá)變化的研究 建立離斷大鼠下牙槽神經(jīng)伴牙周缺損模型,左側(cè)切斷大鼠下牙槽神經(jīng)伴牙周缺損為實(shí)驗(yàn)組,對(duì)側(cè)保留神經(jīng)僅做牙周缺損為對(duì)照組。實(shí)驗(yàn)分為五組即1周、2周、4周、6周和8周。采用Realtime-PCR及Western Blot方法檢測(cè)大鼠牙周組織中神經(jīng)相關(guān)因子CGRP、SP及骨改建相關(guān)因子OPG、RANKL的表達(dá)變化。 結(jié)果： 1、離斷下牙槽神經(jīng)后大鼠牙周組織中CGRP及SP的的表達(dá)變化 Realtime-PCR結(jié)果顯示失神經(jīng)后牙齦及牙槽骨中的CGRP和SP均呈現(xiàn)先降低后上升至正常水平的趨勢(shì)。統(tǒng)計(jì)分析結(jié)果顯示3天、7天和14天組與正常組織表達(dá)水平有統(tǒng)計(jì)學(xué)差異(P0.05),Western Blot檢測(cè)結(jié)果發(fā)現(xiàn),失神經(jīng)后,大鼠牙周組織中的SP蛋白表達(dá)水平總體趨勢(shì)與SP mRNA的變化基本相同,統(tǒng)計(jì)學(xué)分析結(jié)果顯示,術(shù)后第3天、7天、14天和21天的表達(dá)量與正常對(duì)照組相比有統(tǒng)計(jì)學(xué)差異(P0.05)。Western Blot檢測(cè)CGRP蛋白結(jié)果為陰性。 2、失下牙槽神經(jīng)對(duì)大鼠牙周組織愈合過(guò)程中CGRP、SP及骨改建相關(guān)因子表達(dá)變化的研究 通過(guò)Realtime-PCR方法檢測(cè)方法顯示牙周組織中實(shí)驗(yàn)組的CGRPmNRA水平在前期表現(xiàn)為下降趨勢(shì),從第4周開(kāi)始逐漸升高,直至第八周CGRPmNRA水平的水平仍處于升高的趨勢(shì)。而在對(duì)照組CGRPmNRA的表達(dá)水平在前期明顯升高(P0.05),后期逐漸恢復(fù)至正常水平。SP的表達(dá)變化與CGRP趨勢(shì)相同。失下牙槽神經(jīng)導(dǎo)致牙槽骨中OPG持續(xù)弱陽(yáng)性表達(dá)而RANKL持續(xù)強(qiáng)陽(yáng)性表達(dá)。 結(jié)論： 1、離斷下牙槽神經(jīng)后,影響牙齦及牙槽骨中的CGRP及SP的表達(dá) 2、失下牙槽神經(jīng)支配后CGRP及SP的表達(dá)變化會(huì)影響牙周組織缺損愈合過(guò)程中成骨和破骨因子的表達(dá)水平,最終影響牙周組織再生。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R781.4

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本文編號(hào)：2250926