The Expression Of Acetylcholinesterase In Human Periodontal Ligament Fibroblasts And The Affect Of Nicotine And Its Antagonists To The Enzyme

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.