| Periodontal disease is one of the most prevaten in human race. Gingivitis and periodontitis are two main parts of the periodontal disease. Statistics data shows that the incidence rate of gingivitis is up to 70-90% all over the world, and the incidence rate of periodontitis can reach 25-40%, which is the main reason for adult teeth loss.It has been validated that all sorts of stress of mentality and social mentality can influence both the severity degree and the disease process of the periodontal disease (Freeman & Goss 1993; Linden et al, 1996). It is suggested that the local release of neuropeptide may display some neural adjusting function to inflammation changes (Bartold et al, 1994). Substance P (SP) is the high level content in most of the oral tissues especially in the dental pulp and the peridentium. The action of neurotransmitter and neuroregulator make SP to be the remarkable sign of tissue inflammation. Kvinnsland also thought about that the SP level of the tissues was the important parament of the recovery mechanism of the patients, although the definite meaning was not known about thoroughly. As a result, it is very meaningful to find out the expression of the substance P in the periodontal tissues and the meanings in the process of directing the systemic treatment of the periodontal diseases.In this study, we used a routine method of tissue culture to raise the human gingival fibroblast (HGF) in vitro. Cells were identified by the methods of morphology, immunohistochemistry staining. Effect of SP onintracellular free calcium of the HGF was measured by using laser scanning confocal microscope (LSCM) to analyze the significance of SP in the process of the periodontitis. The difference of SP content levels in gingival crevicular fluid (GCF) at the molar sites of periodontal healthy and diseased subjects were tested by radioimmunoassay to study the the relationship between SP level in GCF and the tissues destruction degree of periodontitis.Part I: Culture the human gingival fibroblast (HGF) in vitro [Materials and Methods] Patients who need to be teeth extraction for orthotherapy or dental impaction were randomly selected from the department of surgery in Affiliated Stomatologial Hospotal of Nanjing Medical University. Healthy human gingival tissues were obtained from above mentioned patients with their informed consent by a minimally invasive procedure causing little postoperative discomfort with complete regeneration of tissue occurring within 4 weeks. Isolated gingiva were further cut into pieces with the size of 1mm x 1mm x 1mm under sterile status and transferred to culture bottles. The cells were maintained in RPMI 1640 containing 10% heat-inactivated fetal bovine serum (FBS), 2 mmol/L L-glutamine, 100 U/ml penicillin, and 100/xg/ml streptomycin at 37癈 in a humidified, 5% CO2 atmosphere. Cells were passaged with 0.25% trypsin. Immunohistochemistry staining assay ( IHC ) were performed to identify vimentin and keratin expressed in the 3rd generation cells.[Results] The results from IHC indicate that the human gingival fibroblast cells steadily expressed vimentin, however, did not express any keratin.Part n : Effect of Substance P on Intracellular Free Calcium Concentration in Cultured Human Gingival Fibroblast[Materials and Methods] The cells forementioned were cultured to the fourth generation and were digested by 0.25% trypsin. Cells concentration were adjusted to 105/L. The cells were inoculated into six-cell-plate whose each hole was put into a 24mm x 24mm coverglass and were cultured for more 24 hours to be tested. The cells were marked by fluorescence probe of fluo-3/AM and were put onto object stage of the laser scanning confocal microscope. Substance P was added to the coverglass and diffused onto the cells. The last concentration of substance P was 10"7mol/L. The cells adhering better were selected to be tested. Laser scanning confocal microscope was used to scan the cells before and after substance P being added. The optimum excitation wavelength was 488nm and the emission w... |
PDF Full Text Request