The Effects Of Nicotine On The Proliferation And The Synthesis Of Fibronectin Of Human Periodontal Ligament Cells Cultured In Vitro

There are some hundred millions of smokers in our country.Smoking affects oral environment, and is good for the growth of anaerobic bacterium.Therefore smokers are easy to infect definite periodontic pathogen and increase the chance of infecting definite subgingival pathogen. Smoking can activate mononuclear macrophages secrete much of mediators of inflammation,aggrevate inflammation reaction,stimulate alveolar bone resorption. Smoking can damage gingival fibroblasts and periodontal ligament cells directly or indirectly,and inhibits their proliferation , adhesion , synthesis and the function of secreting collagens. The symptoms of smokers with periodontitis are more serious than the non-smokers'such as depth of pocket,attachment loss,alveolar resorption,the prevalence of root furcation and the effect of periodental non-surgical treatment and so on. Some investigations indicate that there are 35.2% smokers with serious alveolar loss but 7.5% in non-smokeres. Other researchs indicate that smoking aggravates the periodontal pathogenetic condition,and cuts down the drugs'therapeutic effect to prevent the reduction of the PD and the restoration of attachment level. So the study of the effect of nicotine to PDLCs directs the clinical work from theory,and it's significant of suggesting smokers stop smoking and pay attention to dental hygiene.Periodontal ligament cells are the most important cells in periodontal membrane.Its principal function is the synthesis of collagens,and have the activity of swallowing and hydrolyzing with enzyme to degrade old collagen fibers.Otherwise , periodontal ligament cells have the characteristic of stem cells.It can differentiate to osteoblasts and cementoblasts. The most idea healing way of periodontal surgery is that:PDLCs grow to the crown in precedence,occupy the roots,and progenitor cells of them can differentiate to cementoblasts,osteoblasts and fibroblasts.The cells deposite to the roots forming new cementum and new periodontal membrane fibers to implant to the former.The other extreme of the collagen fibers implant to the new alveolar bones and form new attachment. So it will destroy the periodontal supporting tissues and affect the therapeutic efficacy when PDLCs are damaged by any reason.Fn is a major extracellular matrix.It is synthesized and secreted by fibroblasts, vascular endothelial cells,macrophages and parts of epithelial cells. Its main function is linked through the different peptide domain help cells attached to the extracellular matrix, and participation in the transitional cell differentiation and growth process.Fn educes complex biological function through combinating with many collagens , proteoglycans , celluloses ,integrins,heparins,DNA and many kinds of cells.Fn has chemotactic effect to PDLCs,and it can stimulate the migration,coherence and multiplication of PDLCs;it also stimulates the synthesis ofⅠ,Ⅲcollagens significantly,maintains the periodontal spaces,and forms the new periodontal attachment.Fn stimulates the multiplication of gingival fibroblasts and PDLCs.Its mechanism may be that Fn is a signaling molecule,activates the corresponding protein kinase,and causes the cells to produce reaction to the informational molecule and speeding up the synthesis of DNA and the multiplication of the cells.Methods:Collect the healthy premolars of 12～18 year teenagers in oraloutpatient surgery,and flush the teeth 2～3 times with stroke-physiological saline solution to clean blood and bacterium of the crown and the root of teeth.In the super-clean bench we scraped periodontal membrane of the 1/3 dental root with subgingival curettes,and culture PDLCs in vitro with enzyme digestion.After convertional culture and passage we do the research with the fifth generation PDLCs.First of all we used immunohistochemistry of SP to stain the culturing cells of antivimentin and antikeratin.The identification outcome showed that vimentin with masculine and keratine with negative,and it indicated that the cells are PDLCs.Then we allocated different densities of nicotine to stimulate PDLCs,the proliferation of cells was measured with MTT chromatometry,and the synthesis of Fn was measured with ELISA and RT-PCR.Result:nicotine of 250ng/ml～3ug/ml inhibited the proliferation of PDLCs.Among the total nicotine of 1ug/ml showed the best inhibition,and the nicotine of 50ng/ml didn't have statistically significant compare with the control group.Meanwhile 50ng/ml～3ug/ml nicotine inhibited the synthesis of Fn with concentration dependent.After nicotine stimulated PDLCs , the express level of mRNA of Fn degraded.The inhibition of genetic expression of Fn enhanced with the advancement of the concentration of nicotine.The results coincidenced to the results of ELISA.Our findings show that nicotine inhibite the proliferation and the synthesis of PDLCs cultured in vitro.It provides theory to the effects of nicotine to periodontitis.And it demonstrates that smoking is one of the principal risk factors to periodontitis.Smoking can aggrate the periodontitis ,affect the therapeutic efficacy and the regeneration of periodontal tissue.Therefore it's necessary to teach the patients with oral hygiene,advise them to stop smoking when operating systematic periodontal therapy.At present there are many questions unclear and demanding to confirm between the relationship of smoking and periodontitis.It needs us to approach and compromise.It can form substantial rationale to guide the clinical work.