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目的 探讨牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)的致病因子脂多糖(lipopolysaccharide,LPS)刺激巨噬细胞和破骨细胞后的免疫应答反应.方法 获取C57BL/6小鼠骨髓单核细胞,利用条件培养基将其分别诱导为骨髓来源的巨噬细胞(bone marrow derivedmacrophages,BMM)和破骨细胞.实验分为空白对照组和实验组,空白对照组为不加Pg-LPS组,实验组采用Pg-LPS(10 μg/ml)分别刺激BMM和破骨细胞,检测两种细胞表达Toll样受体2(Toll-like receptor-2,TLR-2)和TLR-4的mRNA水平、蛋白水平以及分泌炎症因子的情况,并观察Pg-LPS对BMM分化为破骨细胞过程的影响.结果 小鼠骨髓单核细胞成功诱导为BMM及成熟的破骨细胞.在Pg-LPS的作用下,BMM组的TLR-2 mRNA水平(41.41±13.07)较空白对照组(1.03±0.31)显著上调(P＜0.01),TLR-4 mRNA无明显改变;破骨细胞组的TLR-2 mRNA (2.24±0.23)和TLR-4 mRNA水平(4.83±1.07)均较空白对照组显著上调(P＜0.05,P＜0.01).流式细胞术检测结果显示,BMM组的TLR-2蛋白表达显著上调(P＜0.01),平均荧光强度空白对照组为39.85±5.27,实验组为221.57±13.13;破骨细胞组的TLR-2亦有显著增加(P＜0.01):平均荧光强度空白对照组为83.31±2.69,实验组为108.65±6.32;两组细胞的TLR-4均无明显激活(P＞0.05).BMM和破骨细胞分泌的肿瘤坏死因子α和白细胞介素6水平均显著升高(P＜0.01),其中破骨细胞的炎症因子增幅水平显著低于BMM(P＜0.01).此外,Pg-LPS显著抑制破骨细胞形成的数目及大小,面积百分比较对照组减少47％.结论 Pg-LPS能激活BMM产生明显的免疫炎症反应,而破骨细胞对Pg-LPS的免疫应答较弱;Pg-LPS可抑制BMM分化为破骨细胞.“,”Objective To analyze the immune responses of bone-marrow derived macrophages and osteoclasts to lipopolysaccharide from Porphyromonas gingivalis (Pg-LPS) so as to provide reference for host immunomodulatory therapy of periodontitis.Methods Bone marrow mononuclear cells from C57BL/6 mice were obtained and induced into bone marrow macrophages (BMM) and osteoclasts (OC) by conditioned medium.The BMMs and OCs were divided into blank control group and Pg-LPS treated experimental group.Pg-LPS (10 μg/ml) was used to stimulate BMM and OC.The mRNA and protein expression of TLR-2 and TLR-4 and secretion of inflammatory cytokines were detected,respectively.Furthermore,the effect of Pg-LPS on the differentiation of BMM into OC was evaluated.Results Mouse bone marrow mononuclear cells were successfully induced into BMM and mature OC.In the presence of Pg-LPS,the gene level of TLR-2 in BMM was significantly up-regulated by (41.41 ± 13.07) folds (P＜0.01),while the level of TLR-4 mRNA was not significantly changed.TLR-2 mRNA and TLR-4 mRNA levels in OC increased by (2.24± 0.23) times (P＜0.05) and (4.83 ± 1.07) times (P＜0.01),respectively.Flow cytometry results showed that TLR-2 protein expression in BMM was robustly enhanced (P＜0.01),as mean fluorescence intensities (MFI) were (39.85±5.27) in blank group and (221.57 ± 13.13) in experimental group,respectively.The MFI of TLR-2 in OC also increased as (83.31 ±2.69) in blank group and (108.65 ±6.32) in experimental group,respectively (P＜0.01).However,no significant TLR-4 expressions were observed in both BMM and OC (P＞ 0.05).Moreover,the productions of tumor necrosis factor-α and interleukin-6 in response to LPS in BMM and OC remarkably increased (P＜0.01).Lower cytokine expression was observed in OC than that in BMM (P＜0.01).In addition,Pg-LPS obviously inhibited the number and size of OC formation,with a reduction in area percentage of approximately 47％.Conclusions Pg-LPS generated a robust immune inflammatory response to BMM,while OC had a relatively weaker immune response to the Pg-LPS.Pg-LPS might inhibite the differentiation of BMM into OC.