【作者】 王勇；

【导师】 王小竞；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2010， 硕士

【摘要】 牙周炎以细菌诱发为始动因子,需在宿主、环境、遗传等因素共同作用下导致发病。大量研究已经证明,吸烟能极大地增加牙周破坏的风险,是牙周炎的危险因素。然而,到目前为止,吸烟对牙周组织的破坏机制、对牙周炎发生发展过程的影响仍不明了。尼古丁是烟草的主要成分,有学者推测,尼古丁可能在牙周炎的发生发展中起重要作用。非神经乙酰胆碱能系统包含:乙酰胆碱,乙酰胆碱酯酶,胆碱乙酰基转移酶,烟碱型乙酰胆碱受体和毒蕈碱型乙酰胆碱受体及乙酰胆碱转运体。研究认为,尼古丁作为烟草的主要化学物质之一,是非神经乙酰胆碱能系统中烟碱型乙酰胆碱受体的特异性配体。尼古丁与细胞膜上的烟碱型乙酰胆碱受体特异性结合后,经细胞内外信号转导,产生效应。也就是说,作为烟碱型乙酰胆碱受体特异性配体的尼古丁,可以代替乙酰胆碱,侵入局部非神经乙酰胆碱能系统的网络,发挥生物学效应。已有研究报道在人牙龈上皮细胞中存在非神经乙酰胆碱能系统。我们前期研究证实,人牙周膜成纤维细胞(human periodontal ligament fibroblasts,人PDLF)中存在烟碱型乙酰胆碱受体,且尼古丁可上调该受体表达。然而,人PDLF中是否存在完整的非神经乙酰胆碱能系统,尼古丁是否通过该系统发挥作用,目前尚不清楚。本实验所用药物为尼古丁以及-银环蛇毒素( -BTX)。其中, -BTX是烟碱型乙酰胆碱受体7亚型( 7nAChR)的特异性拮抗剂,能够特异性结合人体内7nAChR。本研究采用细胞培养、免疫荧光、蛋白印迹等方法观察人PDLF中是否存在非神经乙酰胆碱能系统的另一重要成员乙酰胆碱酯酶,以及尼古丁对该酶的影响,旨在从另一方面探讨尼古丁对非神经乙酰胆碱能系统的作用,有助于阐明吸烟相关性牙周炎发生发展的作用机制。第一部分实验人牙周膜成纤维细胞的体外培养及鉴定1主要方法取因正畸治疗需要拔除的12-14岁健康人前磨牙,无菌条件下刮取根中1/3牙周膜组织,采用组织块法进行原代培养,待原代培养成功后,常规传代,免疫组化方法进行抗波形丝蛋白、抗角蛋白染色,显微镜下观察。2主要结果倒置显微镜下观察细胞形态和生长情况。细胞铺满培养瓶底,密度均匀,呈星形或长梭形,细胞胞浆均匀,细胞质丰满,细胞核为圆形,细胞排列呈旋涡状或放射状。免疫组化结果显示培养细胞抗波形丝蛋白染色阳性,抗角蛋白染色为阴性。3主要结论本实验所培养细胞为来源于中胚层的人PDLF。第二部分实验免疫荧光检测人牙周膜成纤维细胞中是否表达乙酰胆碱酯酶1主要方法选取第5代人PDLF接种于24孔板,加液(低糖DMEM,5%小牛血清),于37℃,5﹪CO2条件下在孵箱内培养24小时,选取人表皮上皮细胞作为阳性对照。免疫荧光法检测细胞中乙酰胆碱酯酶的表达情况。2主要结果作为对照组的人表皮上皮细胞中,图片绿色区域为阳性表达,主要分布在胞浆内。而人PDLF中细胞胞浆内也有阳性表达。3主要结论免疫荧光检测结果表明人PDLF存在乙酰胆碱酯酶的表达,表达部位在细胞胞浆。提示在人PDLF中存在乙酰胆碱酯酶。结合前期和同期的研究,人PDLF中存在7nAChR和胆碱乙酰基转移酶的表达,证实在人牙周膜组织中可能存在非神经乙酰胆碱能系统,尼古丁作为7nAChR的激动剂,可能通过作用于该系统,对牙周组织产生影响。第三部分实验蛋白印迹检测尼古丁及α-银环蛇毒素对人牙周膜成纤维细胞中乙酰胆碱酯酶表达的影响1主要方法选第5代人PDLF和人表皮上皮细胞接种于75cm2培养瓶中,分5组:尼古丁组(N组,尼古丁10-12mol∕L),尼古丁+ -BTX组(N+组,10-12mol∕L尼古丁+10-9mol∕L -BTX), -BTX组(组,10-9mol∕L -BTX),空白对照组(B组)和阳性对照组(P组)。各组给予相应浓度的药物,培养24小时后,提取细胞总蛋白,进行蛋白定量、转膜、SDS聚丙烯酞胺凝胶电泳,封闭,孵育,最后进行显色。2主要结果各组均在82kDa位置呈现条带。根据软件分析,各组乙酰胆碱酯酶表达由强至弱依次是N组、P组、N+组、B组、组。3主要结论正常人PDLF中存在乙酰胆碱酯酶,一定浓度的尼古丁可上调该酶蛋白的表达, -BTX可拮抗其表达。结合前期和同期的实验结果,尼古丁的介入,改变了人PDLF中非神经乙酰胆碱能系统各成员的表达,打破了非神经乙酰胆碱能系统的平衡。由于该系统在疾病发生发展中起到一定作用,提示尼古丁很可能通过作用于该系统来影响牙周炎的发生发展。更多还原

【Abstract】 Periodontitis is a common oral disease. Bacteria is the initiating factor of periodontitis, but it doesn’t work alone. And periodontitis is caused by host and other factors. Numerous studies have shown that tobacco can greatly increase the risk of periodontal destruction. Therefore, smoking is a risk factor of periodontitis. However, the mechanism of damage of smoking on periodontal tissue in the development and progression of periodontitis is still not clear. Nicotine is the main component of tobacco. Some scholars have speculated that nicotine may play an important role in the development and progression of periodontitis.A non-neuronal cholinergic system includes: acetylcholine, acetylcholinesterase, choline acetyltransferase, nicotinic acetylcholine receptors, muscarinic acetylcholine receptor and acetylcholine transporter. It is said that nicotine is not only a main chemical substances of tobacco but also the specific ligand of nicotinic acetylcholine receptor which is included in non-neural cholinergic system. Therefore, the specific binding of nicotine and the nicotinic acetylcholine receptor may have some effect through signal transduction. In other words, nicotine, which is the specific ligands of nicotinic acetylcholine receptor, can invasive local non-neuronal cholinergic system and play biological effect in the network instead of acetylcholine. Studies have shown that there is a non-neuronal cholinergic system existing in human oral gingival epithelial cells. In addition, our preliminary studies have shown that there is the expression of nicotinic acetylcholine receptors in human periodontal ligament fibroblasts and nicotine can upregulate the expression of this receptor. However, whether there is the same system in human periodontal ligament fibroblasts and nicotine play a role through this system is not clear.Nicontie and -bungarotoxin are used in this study. -bungarotoxin is one of antagons of 7 nicotinic acetylcholine receptor.Therefore, cell culture, immunofluorescence and western blot were used in this study. The aim is to observe that whether there is acetylcholinesterase or not in human periodontal ligament fibroblasts and the impact of nicotine as well as its antagonist to the enzyme. And the investigation to the effect of nicotine to non-neuronal cholinergic system will help clarify the mechanism of nicotine- related periodontitis.PartⅠ: The culture and identification of human periodontal ligament fibroblasts in vitro1 Main methodsTake for the premolar normal from patients 12-14 years which are extracted because of orthodontic treatment. In sterile conditions scrape periodontal ligament tissue for primary culture and Subculture. Immunohistochemical method was used for vimentin staining and cytokeratin staining.2 Main results The human periodontal ligament fibroblasts were tested Vimentin positive and cytokeratin negative.3 Main conclusionsThe cultured cells derived from the mesoderm. They are human periodontal ligament fibroblasts.PartⅡ: Investigation about the expression of acetylcholinesterase in human periodontal ligament fibroblasts through immunofluorescence1 Main methodsThe 5th generation human periodontal ligament cells cultured in vitro were inoculated in 24-well plate ( 37℃, 5% CO2).And make the human epithelial cells be control cells.24 hours later, the expression of AChE in these cells were detected by immunofluorescence.2 Main resultsThe blue region of the picture of human periodontal ligament fibroblasts is positive and it is mainly in the cytoplasm. It is the same as the one in control cells.3 Main conclusionsResults of immunofluorescence showed that there is positive expression of AChE in human periodontal ligament fibroblasts and the protein mainly exists in the cytoplasm. According to preliminary and earlier research, 7nAChR and CHAT also exists in human periodontal ligament fibroblasts. There may be a non-neuronal cholinergic system in Periodontal tissue. The affect of nicotine which is the agonistof 7nAChR the system may have an impact on periodontal tissues. PartⅢ: Investigation about the affect of nicotine andα-bungarotoxin to the expression of acetylcholinesterase in human periodontal ligament fibroblasts through Western blot1 Main methodsThe 5th generation of human periodontal ligament fibroblasts were divided into 4 groups and human epithelial cells is the control group. They are N group (nicotine 10-12mol∕L),N+ group (nicotine 10-12mol∕L,10-9mol∕L -BTX), group (10-9mol∕L -BTX),black control group, positive control group.2 Main resultsEach group are all presented in the 82kDa band. According to the result of analysis, The expression of AChE from the strong to the weak group is followed by N group,P group,N + group, black control group, group.3 Main conclusionsAChE exists in human periodontal ligament fibroblasts. A certain concentration of nicotine increases the expression of the protein, while the nicotine receptor antagonist -bungarotoxin could antagonize the expression.According to early and earlier experimental results nicotine changes the expression of NNAs members in human periodontal ligament fibroblasts. It breaks the balance of NNAs. Because of their functions and role in development of diseases nicotine is likely to influence the development of periodontitis by acting on the NNAs.更多还原