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目的:对体外培养的牙龈上皮细胞和成纤维细胞施加环孢素A(cyclosporin,CsA)刺激,应用免疫组化方法探讨CsA引发药物性牙龈过度生长(gingival overgrowth,GO)的病理机制。方法:对体外培养的牙龈上皮细胞和成纤维细胞分别施加浓度为600、800和1000ng/mL,作用时间为48、72h的CsA刺激。在观察细胞生长曲线及其变化的基础上,通过对细胞铺片的免疫酶染色(ABC法)定量分析和对细胞培养液的酶联免疫吸附检测(ELISA法),分别对牙龈组织细胞IL-6的表达和分泌进行测定,应用SAS 6.0软件包对数据进行统计学分析。结果:牙龈上皮细胞接受CsA刺激后,细胞数量明显增加,与对照组相比具有显著差异(P<0.05)。在接受相同条件CsA刺激下,牙龈上皮细胞和成纤维细胞胞内IL-6表达无显著差异,细胞胞内IL-6表达量与CsA作用时间、浓度间相关。接受CsA刺激的最初24h内,牙龈成纤维细胞分泌IL-6的总量在各CsA浓度实验组及对照组间均无显著差异(P>0.05);牙龈成纤维细胞接受刺激超过24h后,CsA浓度为1000ng/mL的实验组与对照组间在细胞分泌IL-6总量上有显著增加(P<0.05)。牙龈上皮细胞各实验组的IL-6分泌总量极低且无显著差异。结论:在CsA引发GO的过程中,产生的IL-6可能多源自牙龈结缔组织的成纤维细胞;牙龈成纤维细胞接受CsA刺激后,产生促进IL-6生成的作用与之存在浓度和时间相关性。
OBJECTIVE: To investigate the mechanism of CsA-induced gingival overgrowth (GO) induced by cyclosporin A (CsA) on cultured gingival epithelial cells and fibroblasts by immunohistochemistry. Methods: Gingival epithelial cells and fibroblasts cultured in vitro were stimulated by CsA at concentrations of 600, 800 and 1000 ng / mL for 48 and 72 h respectively. On the basis of observing the cell growth curve and its changes, the enzyme immunoassay (ABC method) and enzyme-linked immunosorbent assay (ELISA) were used to detect the cell growth curve of IL- 6 expression and secretion were measured, using SAS 6.0 software package for statistical analysis of the data. Results: After gingival epithelial cells were stimulated by CsA, the number of cells significantly increased compared with the control group (P <0.05). Under the same conditions of CsA stimulation, there was no significant difference in intracellular IL-6 expression between gingival epithelial cells and fibroblasts, and the intracellular IL-6 expression was correlated with the time and concentration of CsA. The total amount of IL-6 secreted by gingival fibroblasts in the first 24 h after CsA stimulation was not significantly different between experimental group and control group (P> 0.05). When Gingival fibroblasts were stimulated for more than 24 h, CsA The concentration of IL-6 secreted by the cells was significantly increased (P <0.05) between the experimental group and the control group at a concentration of 1000ng / mL. The total amount of IL-6 secretion in the gingival epithelial cells in each experimental group was extremely low and no significant difference. CONCLUSIONS: IL-6 produced by CsA is mainly derived from fibroblasts of gingival connective tissue during the process of CsA-induced GO. Gingival fibroblasts, stimulated by CsA, have the effect of promoting the production of IL-6 in a concentration and time Correlation.