發(fā)布時(shí)間：2018-09-18 14:04

【摘要】：牙周病是一種影響廣泛的高發(fā)病,主要損傷牙齒支持組織,最終導(dǎo)致牙齒的松動(dòng)脫落。理想的牙周再生,應(yīng)同時(shí)包括牙骨質(zhì)、牙周膜和牙槽骨的再生。牙骨質(zhì)的再生是牙周組織的重要組成部分,是目前的牙周組織復(fù)合體恢復(fù)研究中的熱點(diǎn)和難點(diǎn)。在牙周組織再生時(shí),如果缺乏牙周膜和／或牙骨質(zhì)再生,新生的牙槽骨將占據(jù)牙周膜間隙與形成直接結(jié)合,從而引起骨粘連。這種病理性、缺乏彈性的結(jié)合方式會(huì)導(dǎo)致牙齒支持功能的缺失,最終引起牙根吸收。牙骨質(zhì)再生是牙骨質(zhì)-牙周膜界面結(jié)構(gòu)再生的先決條件,因此必須采用特異性生長因子誘導(dǎo)牙骨質(zhì)再生。工程化牙周組織就是在這個(gè)條件基礎(chǔ)上實(shí)現(xiàn)的牙周組織重構(gòu)。前期研究表明牙骨質(zhì)蛋白1是一種牙骨質(zhì)所特有的非膠原蛋白,從組織或真核細(xì)胞中提取的人牙骨質(zhì)蛋白具有促進(jìn)礦化的作用。因此我們猜想CEMP1正是誘導(dǎo)牙骨質(zhì)再生最理想的特異性生長因子。牙骨質(zhì)蛋白1自身的昂貴價(jià)格局限了其研究及應(yīng)用。在蛋白表達(dá)中最基本的兩種系統(tǒng)分別是原核表達(dá)系統(tǒng)和真核表達(dá)系統(tǒng)。原核表達(dá)使用時(shí)間最早,也是目前研究最清楚、最經(jīng)濟(jì)實(shí)惠的表達(dá)系統(tǒng),同時(shí)也是美國FDA批準(zhǔn)通過的基因工程表達(dá)系統(tǒng)。本實(shí)驗(yàn)通過大腸桿菌表達(dá)系統(tǒng)得到可與CEMP1抗體特異性結(jié)合的rhCEMP1,分子量為34kDa。此蛋白誘導(dǎo)人牙周膜細(xì)胞后可使其CEMP1 mRNA表達(dá)量升高。實(shí)驗(yàn)證明了大腸桿菌原核表達(dá)和Ni柱純化可以獲得濃度及純度較高并具有生物活性的CEMP1蛋白。本研究在仿生生物礦化體系觀察濃度范圍為0-100 μg/ml的CEMP1對(duì)HA晶體48小時(shí)生長過程的調(diào)控作用。結(jié)果發(fā)現(xiàn)CEMP影響HA的晶體成核與生長,當(dāng)CEMP濃度較低(0-50 μg/ml)時(shí),只有短小束狀HA晶體生成,礦化效果較差。當(dāng)CEMP濃度提高到100μg/ml時(shí),大部分晶體的形態(tài)變?yōu)橐?guī)整的長針狀結(jié)構(gòu)。FTIR顯示HA的特征峰1106 cm-1,557cm-1和598 cm-1存在。因此確定CEMP1在體外可以通過提高局部濃度誘導(dǎo)排列整齊的HA晶體形成。為了解決rhCEMP1在體內(nèi)應(yīng)用時(shí)難以緩慢長效釋放的難題,結(jié)合牙骨質(zhì)基質(zhì)的特點(diǎn),選用ACP緩釋載藥系統(tǒng)合成rhCEMP1/ACP納米緩釋顆粒。研究采用PEG穩(wěn)定的方法獲得ACP,并在低溫濕化學(xué)反應(yīng)中加載rhCEMP1因子,使得磷酸鈣與細(xì)胞因子形成穩(wěn)定的結(jié)合。同時(shí)通過調(diào)節(jié)載藥量比例,得到了載藥量分別為1,2,5,8%的rhCEMP1/ACP納米緩釋顆粒。分析四種rhCEMP1/ACP納米緩釋顆粒的載藥量和包封率的關(guān)系以及釋放譜,數(shù)據(jù)表明主要有三種釋放方式,首日爆釋、第一周慢型釋放以及一周后的后期快型釋放。因此可以針對(duì)牙骨質(zhì)再生的實(shí)際需要選用合適的釋放譜型的rhCEMP1/ACP納米緩釋顆粒。本研究用靜電紡絲的方法獲得了具有多孔納米結(jié)構(gòu),表面粗糙度高,親水性良好的復(fù)相ACP/PCL/COL支架。本實(shí)驗(yàn)發(fā)現(xiàn)rhCEMP1/ACP/PCL/COL支架在體外與PDLC共同培養(yǎng)7天,可抑制PDLC增殖；促進(jìn)成牙骨質(zhì)細(xì)胞分化因子CAP和CEMP1表達(dá),抑制成骨細(xì)胞分化因子OCN和OPN表達(dá)；將rhCEMP1/ACP/PCL/COL支架在大鼠顱骨標(biāo)準(zhǔn)缺損模型培養(yǎng)4周可誘導(dǎo)類牙骨質(zhì)組織形成。綜上所述,本研究證實(shí)了CEMP1在調(diào)節(jié)HA晶體生長的過程中存在著劑量效應(yīng),確定了CEMP1體內(nèi)牙骨質(zhì)誘導(dǎo)再生作用。為CEMP1的作用及機(jī)制提供了思路,為rhCEMP1在臨床中應(yīng)用于促牙骨質(zhì)再生提供的研究基礎(chǔ)。
[Abstract]:Periodontal disease is a widespread and high-incidence disease, which mainly damages the supporting tissues of the teeth and eventually leads to the loosening of the teeth. The ideal periodontal regeneration should include the regeneration of cementum, periodontal ligament and alveolar bone. In periodontal tissue regeneration, if periodontal ligament and/or cementum regeneration are absent, the new alveolar bone will occupy the periodontal ligament space and form a direct bond, resulting in bone adhesion. This pathological, inelastic bond will lead to the loss of tooth support function and eventually cause root resorption. Cementum regeneration is cementum. The engineered periodontal tissue is based on this condition to achieve periodontal tissue remodeling. Previous studies have shown that cementin-1 is a non-collagen protein unique to cementum, extracted from tissue or eukaryotic cells. Cementoprotein 1 is an ideal specific growth factor for inducing cementum regeneration. The high price of cementoprotein 1 limits its research and application. The two basic systems in protein expression are prokaryotic expression system and eukaryotic expression system. It is the earliest and most economical expression system that has been studied and approved by FDA in the United States. In this study, rhCEMP1, which can specifically bind to CEMP1 antibody, was obtained by E. coli expression system, and its molecular weight is 34 kDa. The results showed that CEMP could regulate the 48-hour growth of HA crystals in a biomimetic mineralization system. CEMP could affect the crystal growth of HA. When the concentration of CEMP increased to 100 ug/ml, the morphology of most of the crystals changed into regular long needle-like structure. FTIR showed that the characteristic peaks of HA were 1106 cm-1,557 cm-1 and 598 cm-1. Therefore, CEMP1 could be enhanced in vitro. In order to solve the problem of slow and long-term release of rhCEMP1 in vivo, combined with the characteristics of cementum matrix, rhCEMP1/ACP nanoparticles were synthesized by ACP sustained-release drug delivery system. ACP was obtained by PEG stabilization method and loaded with rhCEMP1 in low-temperature wet chemical reaction. RhCEMP1/ACP nanoparticles with 1,2,5,8% drug loading were obtained by adjusting the drug loading ratio. The relationship between drug loading and encapsulation efficiency and release spectra of four kinds of rhCEMP1/ACP nanoparticles were analyzed. In this study, the composite ACP/PCL/COL scaffolds with porous nanostructures, high surface roughness and good hydrophilicity were prepared by electrospinning. It was found that rhCEMP1/ACP/PCL/COL scaffold could inhibit the proliferation of PDLC in vitro for 7 days, promote the expression of CAP and CEMP1, and inhibit the expression of OCN and OPN. RhCEMP1/ACP/PCL/COL scaffold could induce the morphology of cementoid tissue after 4 weeks of culture in rat standard skull defect model. In conclusion, this study confirmed the dose-effect of CEMP1 in regulating the growth of HA crystals, and confirmed the role of CEMP1 in inducing cementum regeneration in vivo.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R783.1

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本文編號(hào)：2248146