脂多糖诱导高迁移率族蛋白B1在人牙髓细胞中转位的研究

Study on lipopolysaccharide-induced translocation of high mobility group protein B1 in human dental pulp cells

目的:观察低浓度脂多糖(lipopolysaccharide, LPS)诱导人牙髓细胞(human dental pulp cells, hDPCs)中高迁移率族蛋白B1(high-mobility group box 1,HMGB1)在细胞中的转位及其hDPCs细胞活性的影响。方法:采用原代组织块培养法,培养人牙髓细胞,取第3~6代细胞用于实验。分别用含不同质量浓度(0、10、50、100、500、1 000 ng/mL)LPS的培养液培养hDPCs 3 d,采用CCK-8法检测细胞活性;100 ng/mL LPS刺激hDPCs后,免疫荧光检测0 h、6 h、12 h及24 h HMGB1在hDPCs中的表达定位,RT-PCR检测各时间点hDPCs细胞TNF-α,IL-1及HMGB1的mRNA表达,ELISA试剂盒分析细胞上清中HMGB1的含量。结果:低浓度LPS刺激hDPCs后,细胞活性并无明显改变,提示hDPCs具有抵抗低浓度LPS的能力。RT-PCR结果示LPS浓度在10 ng/mL、50 ng/mL和100 ng/mL时,IL-1和HMGB1的mRNA水平无显著变化;500 ng/mL和1 000 ng/mL时,TNF-α、IL-1和HMGB1的mRNA水平显著上调。免疫荧光示LPS诱导后,在6 h、12 h及24 h均可见hDPCs细胞浆中HMGB1的表达,ELISA结果示HMGB1能分泌到细胞上清中。结论:低浓度LPS可对hDPCs活性基本无影响,且可诱导HMGB1在hDPCs中从细胞核转位到细胞浆,并释放到细胞外。

Objective:To investigate the effect of lipopolysaccharide(LPS)on the translocation and mRNA expression of high-mobility group box 1(HMGB1)on human dental pulp cells(hDPCs).Methods:Cell viability of hDPCs was examined using CCK-8 after culturing primary hDPCs in the presence of LPS with different doses(0,10,50,100,500,1 000 ng/mL)for 3 days.An immunoflurescence staining and real time PCR was used to detect the translocation and mRNA expression of HMGB1 in the primary hDPCs after stimulated by LPS on 0 h, 6 h, 12 h, 24 h, respectively.Results:LPS had no effects on cell viability of hDPCs on concentration from 10 ng/mL to 100 ng/mL,while significantly affected the cell viability of hDPCs on concentration from 500 ng/mL to 1 000 ng/mL.LPS on concentration of 100 ng/mL induced the HMGB1 translocation from nucleus to cytoplast and was released into the supernatant.mRNA level of HMGB1 was no significantly change after hDPCs stimulated by LPS on different times.Conclusion:LPS had no cytotoxicity on hDPCs at the low concentration from 10 to 100 ng/mL.HMGB1 was detected translocation from nucleus to cytoplast on hDPCs stimulated by LPS on the concentration of 100 ng/mL,and was released into the supernatant.