The In Vitro Investigation Of The Effect Of Nicotine For Affecting The Expression Of Il-1β、IL-6and Inducing The Th17Differentiation Of HPDLCs

Human periodontitis is a common disease which could seriously harmhuman health and lead to the progressive destruction of the periodontal tissues.As the high risk factor of periodontitis, smoking has much relationship withthe occurrence and progressive of the disease. However the detail mechanismbehind it is unknown. Nicotine is the main toxicity of cigarette which plays aimportant role in the development of smoking related periodontitis. In theprevious study, our research group has found that the specific receptor ofnicotine-alpha7nicotinic acetylcholine receptor, α7nAChR expressed in theperiodontal tissues of rat and in hPDLCs. Nicotine could up regulate theexpression of the receptor and aggravate the inflamed tissues. The resultssignified that nicotine may combine with α7nAChR and lead to a series ofdownstream biological reactions, thus affecting the disease.Recent studies indicated that the immunity reaction determines thedirection of the development of human periodontitis. It comes to the Th17 cells which could specifically secrete IL-17and finally affecting the turnoverand prognosis of periodontitis by its characteristics of proinflammatory andbone destruction. As a result. we suppose that nicotine may combine with theα7nAChR and induce the differentiation of Th17, then give rise to a series ofimmunity reactions and affecting the progressive of smoking relatedperiodontitis in the end.This study taked hPDLCs, periphery blood CD4⁺T cells andhPDLCs-periphery blood CD4⁺T cells co-cultured system as the experimentmodel. Aiming to investigate the changes of expression of IL-1β、IL-6ofhPDLCs and further explore whether these changes could inducing CD4⁺Tcells differentiate into the Th17so as to clarify the detailed mechanisms of therole of α7nAChR in the smoking related periodontitis.Part1cell culturing and identification of hPDLCsMethods: We got healthy premolars without any caries from12¹5year-old patients who came for orthodontic treatment. Then scratched theperiodontal ligament tissues from the middle-third of the root and cultured thehPDLCs in the way of tissue clump. After passaging we got the cells inpassages5to identify their resource through the index of keratin and vimentinusing the technique of immunohistochemistry.Results: After about9days of original generation culturing we could seefibroblast like cells in the shape of long shuttle run out of the round of thetissue clumps. In the procedure of culturing, the long shuttle cells aligned in aradiation way. The fibroblast like cells had a well-distributed density, regularand full-grown kytoplasm and round clear nucleus. Theimmunohistochemistry showed a positive result for the vimentin and a mesenchymal.Conclusion: We successfully set up the model for the experiment. Thecells we get are from mesenchymal and we confirmed them as hPDLCs.Part2The IL-1β, IL-6mRNA expression changes of hPDLCs culturedin vitro under the affecting of nicotineMethods: We took hPDLCs as the experiment model. Investigating theIL-1β, IL-6mRNA expression changes with the affecting of nicotine (10^-5M)and/or antagonist α-BTX (10^-8M) through the technique of PCR gelelectrophoresis and Real-time PCR.Results: Through the technique of PCR gel electrophoresis we could getsingle and clear straps at the anticipated location. Compared with the controlgroup, the IL-1β, IL-6mRNA expression was significantly upregulated in thenicotine group （P<0.05）, the results between the other groups have nostatistically significance （P>0.05）. The real-time PCR further test and verify theresults.Conclusions: Nicotine could significantly upregulate the inflammatoryfactors IL-1β, IL-6mRNA expression of the in vitro cultured hPDLCs aftercombining with the α7nAChR, thus aggravate the inflammatory reaction of theperiodontal tissues.Part3The IL-1β, IL-6protein expression changes of hPDLCs and peripheryblood CD4⁺T cells cultured in vitro under the affecting of nicotineMethods: We cultured hPDLCs in vitro in the way of tissue clumps. Thenseparating human periphery blood CD4⁺T cells in the way of density gradient centrifugation and flow cytometry. Then we cultured the cells in vitro and set upthe co-cultured system for hPDLCs and periphery blood CD4⁺T by Transwell.We further divided the samples into three groups and studied the IL-1β、IL-6protein expression changes of hPDLCs、the co-cultured system and the CD4⁺Tcells under the affecting of nicotine (10^-5M) and/or antagonist α-BTX (10^-8M)with the ELISA.Results: The IL-1β, IL-6protein expression was significantly upregulatedin the nicotine group in the co-cultured system compared with the control group（P<0.05）. The other groups had no significant differences compared with thecontrol group （P>0.05）. We could not detect the IL-1β, IL-6protein expressionin the CD4⁺T cells and there was no significant differences between the groups（P>0.05）. The effect of nicotine(10^-5M)pointed out that the IL-1β, IL-6proteinexpression had no differences between hPDLCs and the co-cultured system（P>0.05）, but much higher than the CD4⁺T cells with statistical significance（P<0.001）.Conclusions: After combining with α7nAChR, nicotine could upregulatethe protein expression level of IL-1β, IL-6of the in vitro cultured hPDLCs, thusaggravate the inflammatory reaction in periodontal tissues. We has furthertestifier the result in the Part2of the whole study in protein level. In ourexperiment, IL-1β, IL-6was and was only secreted by hPDLCs while CD4⁺Tcells had no impact to IL-1β, IL-6protein which was secreted by hPDLCs.Part4The in vitro study of nicotine inducing CD4⁺T cells from humanperiphery blood to Th17cell subgroupMethods: Identify and collect the human periphery blood CD4⁺T cells bythe way of density gradient centrifugation and flow cytometry. Then we set up the co-culture system of hPDLCs and periphery blood CD4⁺T cells and dividedthe samples into two groups. There after we investigated if there were IL-17expression in the co-culture system and the CD4⁺T cells with the stimulating ofnicotine (10^-5M) and the antagonist α-BTX (10^-8M) through the technique ofPCR gel electrophoresis and ELISA.Results: We detected there was no target strap showed in the anticipatedarea, all the groups had no expression of IL-17mRNA. The ELISA test furtherverifier our findings in the protein level.Conclusions: In our experiment we could not test that after combining withα7nAChR, nicotine could stimulate hPDLCs secret IL-1β, IL-6, thus induceCD4⁺T cells differentiate into Th17.