【作者】 谢晓莉；

【导师】 凌天牖；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 目的探讨脂磷壁酸(LTA)和／或脂多糖(LPS)对人牙周膜细胞(hPDLC)凋亡及分泌炎性细胞因子的影响。方法组织贴壁法体外培养hPDLC,以hPDLC为受试对象,LTA、LPS为施加因素。1.利用透射电镜观察LTA和／或LPS作用后hPDLC超微结构的变化及凋亡小体,用MTT法检测LTA和LPS对hPDLC的抑制作用,ELISA检测TNF-α的表达,流式细胞术对凋亡细胞进行定量分析；2.采用Re 1-time PCR技术检测hPDLC Fas、Bax、Cytochrome C、Caspase-3 mRNA的表达,Western-blot技术检测相应蛋白的表达；3.抗体芯片技术检测炎性细胞因子的改变。结果1.凋亡相关改变：透射电镜下，LTA和／或LPS作用hPDLC12h后,细胞质浓缩,空泡及凋亡小体出现；且同一时间点10μg/ml LTA与1μg/ml LPS刺激hPDLC分泌TNF-α的量无统计学差异；流式细胞结果表明,各实验组细胞早期／晚期凋亡率及死亡率明显增加。2. Real-time PCR技术检测LTA和／或LPS作用于hPDLC 12h后,Fas、Cytochrome C、Caspase-3 mRNA表达均明显上调,BAX表达下降,Western-blot技术检测相应蛋白表达与mRNA结果一致。3.炎性细胞因子的改变：LTA和／或LPS作用于hPDLC后,上清液中多种细胞因子高表达,其中与细胞凋亡密切相关的IL-1在各实验组中均呈高表达,LTA+LPS组与LPS组、LTA组比较,其凋亡相关细胞因子TNF-α、TNF-β, IL-1表达均明显增加。结论1.LTA和LPS作用于hPDLC后,其细胞凋亡率升高。2.TNF-α的分泌量与LTA及LPS作用呈明显时间正相关。3.LTA和LPS作用后,hPDLC Fas、Cytochrome C、Caspase-3、mRNA表达与相应蛋白表达均升高,Bax mRNA表达下降。4.LTA和LPS作用后,hPDLC分泌的炎性因子高表达种类相似,但LTA所引起的炎性因子总体强度较弱。5.LTA和LPS共同作用于牙周膜细胞后,分泌的炎性因子种类及量均明显增多,且大于二者分别作用之和,表明两者有协同效应。更多还原

【Abstract】 Objective To investigate the effects of LTA and/or LPS on human periodontal ligament cells’apoptosis and inflammatory cytokines production.Methods Human periodontal ligament cells (hPDLC) that are prepared from the middle 1/3 periapical tissues were maintained in high glucose DMEM medium with or without LTA and/or LPS (1) Ultra-structure and apoptotic body were observed by transmission electron microscope; growth and proliferation were examined by MTT colorimetric assay; the expression of TNF-a was examined by enzymelinked immunosorbent assay (ELISA); quantitative analysis of apoptotic cells were evaluated by flow cytometry. (2) Real-time PCR was used to detect Fas, Bax, Caspase-3 and Cytochrome C mRNA expression, and Western Blot was utilized to determine the corresponding proteins in human periodontal ligament cells. (3) Inflammatory cytokines were measured by Antibody chip technology.Results (1) Condensesd and vacuolited cytoplasm and apoptotic body were observed in each group throμgh transmission electron microscope. Both LTA and LPS could stimulate apoptosis of hPDLC. TNF-a secretion can be detected 12h after stimulation with LTA and/or LPS and increasingly escalate within 48h. There are no statistic difference between 100μg/ml LTA group and 1μg/ml LPS group at the same time point. Apoptosis rate of early and late stage in each groups were obviously enhanced compared with control group throμgh flow cytometry technology. (2) Cytochrome C, Fas, and Caspase-3 mRNA were up-regulated throμgh real-time PCR after incubated with LTA and/or LPS, similarly, the production of corresponding proteins were also increased. Expression of Bax mRNA decreased. (3) Various cytokines were released by hPDLC after LTA and/or LPS stimulation, especially IL-1, which was closely related to the apoptosis process. LTA, LPS have synergistic effect, their combination can greatly enhance the expression of apoptosis-related cytokines such as TNF-a, p and IL-1.Conclusions (1) Both LTA and LPS can induce apoptosis of human periodontal ligament cells. (2) LTA and LPS can enhance the expression of TNF-a. (3) The production of Fas, Cytochrome C, Caspase-3 mRNA and protein were up-regulated when induced by LTA and/or LPS in hPDLC. Expression of Bax mRNA decreased. (4) The types of up-regulated cytokines were similar when induced by LTA and LPS respectively, but the effect of the former was relatively inferior. (5) LTA and LPS have synergistic effect; their combination can greatly enhance the expression of apoptosis-related cytokines.更多还原