Expression Of Choline Acetyltransferase In Human Periodontal Ligament Fibroblasts And Its Role In Destructive Effects Of Nicotine

Tobacco smoking is the major risk factors for periodontal tissue destruction and chronic periodontitis. The prevalence of periodontitis among smokers is much higher than that of non-smokers. Periodontal destructions are much more sever among patients who are also smokers. Nicotine, the main alkaloid in tobacco smoke, can damage the periodontal tissues directly through impeding the normal function of the periodontal immune system. Hovever, the exact mechanisms are largely unknown.Choline acetyltransferase (ChAT) is reported playing an important role in nicotine's destructive effects. To observe the expression of choline acetyltransferase in human periodontal ligament fibroblasts and explore its role in destructive effects of nicotine, we cultured periodontal ligament fibroblasts in vitro and studied the expression and rugulation of ChAT in human periodontal ligament fibroblasts using methodology of semi-quantitative PCR, Real-time quantitative PCR, DNA sequencing and immunohistochemistry. 1 Expression of ChAT in human Periodontal Ligament fibroblastsPrimary PDL cells were obtained from premolars that had been extracted from 12-18 years old volunteers for orthodontic reasons. Periodontal ligament tissues were dissociated from the middle one-third of the root to exclude the intermixing of gingival and dental pulp tissues. PDL cells were primary cultured using modified tissue block method. When the cells surrounding the explants reached confluence, cell layers were idenfied and subcultured until fourth to sixth passage were used for experiments. Expressions of ChAT in PDLF were detected by immunohistochemistry and RT-PCR. Results showed that showed that the kytoplasm of PDLF was positive for ChAT staining. The PCR experiment showed the presence of a positive band at the 299 bp position, which suggested the positive expression of ChAT mRNA in PDLF. Sequence identification revealed that the sequence of the PCR product obtained in this experiment was completely consistent with the data for ChAT in Gene Bank.2 The effect of nicotine on the expression of Choline acetyltransferase in human Periodontal Ligament fibroblastsPDLF were randomly assigned into two groups. with each group receiving one of the following treatments: (1) control, no treatment; (2) nicotine (10–12 M). After 24 hours's incubation, cells were collected and went through immunohistochemistry staining, RT-PCR and Real-time PCR.In the control group, positive staining for ChAT was present in the PDLF. In the nicotine administration group, the expression of ChAT was increased relative to that of the control group. RT-PCR analyses demonstrated that the expression of ChAT was significantly up-regulated (P < 0.05) by the administration of nicotine at the concentration of 1.0×10–12 M. Such results were furtherly confirmed by the Real-time PCR.In conclusion, the results demonstrated that mRNA and protein of ChAT were expressed in human PDLF. Furthermore, nicotine (10–12 M) up regulated the expression of ChAT. These results indicate that nicotine may affect PDLF functions through the cholinergic pathway by regulating expression of ChAT.