不同卟啉单胞菌脂多糖对小鼠成骨细胞CD14、TLRs表达的影响

Effects of lipopolysaccharides from various Porphyromonas on the expression of CD14 and TLRs in mouse osteoblast

目的:研究牙髓卟啉单胞菌(P.e)和牙龈卟啉单胞菌(P.g)脂多糖(LPS)对成骨细胞表达CD14和TLRs的影响。方法:10μg/mLP.e-LPS和P.g-LPS刺激MC3T3-E1细胞,应用RT-PCR检测不同时间(0、1、3、6、12及24h)后,细胞CD14、TLR2及TLR4 mRNA表达的变化;流式细胞术检测P.e-LPS和P.g-LPS作用24h后,细胞表面CD14、TLR2和TLR4的表达。采用SPSS11.0软件包对结果进行单因素方差分析和Dunnett t检验。结果:P.e-LPS作用1h后,MC3T3-E1细胞CD14和TLR4 mRNA的表达量明显增加,TLR2 mRNA的表达量随着P.e-LPS作用时间的增加并无明显变化。P.g-LPS作用于MC3T3-E1细胞后,其CD14、TLR2、TLR4 mRNA的表达量均明显增加;流式细胞术分析显示,P.e-LPS作用24h后,与未加LPS组相比,CD14和TLR4蛋白的表达量均显著增加,但TLR2蛋白的表达量无显著增加。P.g-LPS作用24h后,细胞CD14、TLR2、TLR4蛋白的表达均显著增加(P<0.05)。结论:P.e-LPS可能是通过CD14和TLR4受体对成骨细胞发挥作用,而P.g-LPS对成骨细胞的刺激作用则可能依赖于CD14、TLR2和TLR4受体。

PURPOSE：To observe the effect of lipopolysaccharides（LPS） extracted from Porphyromonas endodontalis（P.e） and Porphyromonas gingivals（P.g） on the expression of CD14 and TLRs in osteoblast.METHODS： MC3T3-E1 cells were stimulated with 10μg/mL P.e-LPS and P.g-LPS.The change of CD14,TLR2 and TLR4 mRNA was observed at different time point（0,1,3,6,12,24h） using RT-PCR,and the expression of CD14,TLR2 and TLR4 protein was measured by flow cytometry at 24-hour.Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS11.0 software package.RESULTS： MC3T3-E1 cells were stimulated with 10μg/mL P.e-LPS for 1h,the expression of CD14 and TLR4 mRNA increased significantly.There was no increase of TLR2 mRNA with stimulation of P.e-LPS.The CD14,TLR2 and TLR4 mRNA expression increased significantly after stimulation with 10μg/mL P.g-LPS.Flow cytometry showed that CD14 and TLR4 protein increased significantly after stimulation with 10μg/mL P.e-LPS.CD14,TLR2 and TLR4 protein increased significantly after treatment with 10μg/mL P.g-LPS.CONCLUSIONS： CD14,TLR4 receptors are involved in P.e-LPS effect and CD14,TLR2 and TLR4 receptors are involved in P.g-LPS effect in mouse osteoblast.Supported by Science and Technology Research Projects of Shenyang City（1091175-1-04）.