Jia Ge, Qiu Li-Hong, Li Ren, Lü You, Yu Ya-Qiong, Zhong Ming
Department of Endodontics, School of Stomatology, China Medical University, Shenyang.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2011 Sep;46(9):531-6. doi: 10.3760/cma.j.issn.1002-0098.2011.09.006.
To evaluate the effect of cluster of differentiation 14 (CD-14) and Toll like receptors (TLR) on the expression of interleukin-6 (IL-6) mRNA induced by Porphyromonas endodontalis (Pe) lipopolysaccharides (LPS).
MC3T3-E1 cells were treated with 10 mg/L Pe-LPS for different hours, and the cells uninvolved by anything as the blank group. The expression of IL-6 was detected by reverse transcription polymerse chain reaction (RT-PCR) and enzyme-liked immunosorbent assay (ELISA). The expression of CD-14, TLR-2 and TLR-4 mRNA was observed at different time point (0 - 24 h) by RT-PCR. The protein of CD-14, TLR-2 and TLR-4 was analyzed with a flow cytometer. MC3T3-E1 cells were pretreated with anti-CD-14, anti-TLR-2 and anti-TLR-4 antibody for 1 h, and then cells were stimulated with 10 mg/L Pe-LPS for 6 h. The expression of IL-6 mRNA was examined by RT-PCR. Statistical analysis was performed using one-way ANOVA Dunnett-t test with SPSS 11.0 software package.
The IL-6 mRNA and proteins increased significantly after treatment with Pe-LPS. When MC3T3-E1 cells treated by Pe-LPS for 6 h, the expression of proteins soared from (11.696 ± 0.672) ng/L to (36.534 ± 0.574) ng/L (P < 0.01); In the control group, the CD-14 and TLR-4 mRNA are ambly-expression, and the ratios of CD-14 and TLR-4 positive cells were (39.038 ± 3.131)% and (11.438 ± 0.385)% respectively in MC3T3-E1. After treatment by Pe-LPS, the expression of CD-14 and TLR-4 mRNA increased significantly, and the ratios of CD-14 and TLR-4 positive cells markedly increased to (62.407 ± 1.800)% and (21.367 ± 2.271)%. TLR-2 expression did not change apparently after Pe-LPS treatment. The expression of IL-6 mRNA was partly inhibited by anti-CD-14 or anti-TLR-4 antibody, but not by TLR-2.
Pe-LPS can induce the expression of IL-6 in osteoblast MC3T3-E1 through CD-14 and TLR-4, but not TLR-2.
评估分化簇14（CD - 14）和Toll样受体（TLR）对牙髓卟啉单胞菌（Pe）脂多糖（LPS）诱导的白细胞介素-6（IL - 6）mRNA表达的影响。
用10mg/L的Pe - LPS处理MC3T3 - E1细胞不同时间，未作任何处理的细胞作为空白组。通过逆转录聚合酶链反应（RT - PCR）和酶联免疫吸附测定（ELISA）检测IL - 6的表达。通过RT - PCR在不同时间点（0 - 24小时）观察CD - 14、TLR - 2和TLR - 4 mRNA的表达。用流式细胞仪分析CD - 14、TLR - 2和TLR - 4的蛋白。用抗CD - 14、抗TLR - 2和抗TLR - 4抗体预处理MC3T3 - E1细胞1小时，然后用10mg/L的Pe - LPS刺激细胞6小时。通过RT - PCR检测IL - 6 mRNA的表达。使用SPSS 11.0软件包进行单向方差分析Dunnett - t检验进行统计分析。
用Pe - LPS处理后，IL - 6 mRNA和蛋白显著增加。当MC3T3 - E1细胞用Pe - LPS处理6小时时，蛋白表达从（11.696±0.672）ng/L飙升至（36.534±0.574）ng/L（P < 0.01）；在对照组中，CD - 14和TLR - 4 mRNA表达较弱，在MC3T3 - E1中CD - 14和TLR - 4阳性细胞比例分别为（39.038±3.131）%和（11.438±0.385）%。用Pe - LPS处理后，CD - 14和TLR - 4 mRNA表达显著增加，CD - 14和TLR - 4阳性细胞比例显著增加至（62.407±1.800）%和（21.367±2.271）%。Pe - LPS处理后TLR - 2表达无明显变化。抗CD - 14或抗TLR - 4抗体可部分抑制IL - 6 mRNA的表达，但抗TLR - 2抗体无此作用。
Pe - LPS可通过CD - 14和TLR - 4诱导成骨细胞MC3T3 - E1中IL - 6的表达，但不能通过TLR - 2诱导。
