脂多糖通过TLRs信号途径诱导树突状细胞成熟机理初探

目的探讨脂多糖(LPS)通过TLRs信号途径进一步促进人外周血单核细胞来源树突状细胞(DC)成熟的机理。方法从外周血分离单核细胞,加入rhGM-CSF及rhIL-4,在培养的第6d,实验组加入LPS刺激24h,对照组不加。分别提取细胞总RNA,行半定量RT-PCR,产物进行琼脂糖凝胶电泳分析。激光扫描共聚焦显微镜检测DC内NF-κB表达情况。结果单核细胞经GM-CSF及IL-4诱导7d后的DC表达较高水平TLR2、3、4。LPS刺激24h后,TLR2、3、4表达下降,TLR4几乎测不到。激光扫描共聚焦显微镜检测到,未刺激组DC胞浆染色阳性,胞核为阴性。LPS刺激组的细胞核染色为强阳性,胞浆染色为阳性。结论DC在受LPS刺激前后TLRs的表达有显著变化,NF-κB也从胞浆移位至核内。这标志着经LPS刺激后DC在功能上进一步成熟。为下一步研究DC的功能奠定了基础。

On Mechanism of How Lipopolysaccharide Induces Dendritic Cells to Mature Through TLRs Signal Pathway

OBJECTIVE: To find out how lipopolysaccharide (LPS) induces dendritic cells (DC) to be more mature through Toll-like receptors (TLRs) signal pathway. METHODS: Monocytes were isolated from heparinized whole blood. 7 days later the monocytes were induced into dendritic cells by adding rhGM-CSF and rhIL-4. Total cellular RNA of unstimulated and stimulated DC were extracted by LPS 24 h later. Semi-quantitative RT-PCR and agarose gel electrophoresis were conducted to analyze the PCR products. The expression of NF-kappaB was detected by laser scanning confocal microscopy (LSCM). RESULTS: Expressions of TLR2, 3, 4 were significantly higher in monocytes-derived DC induced for 7 days, the plasm of DC was stained positive and the nucleus of DC was stained negative. After being stimulated by LPS for 24 h, the expressions of TLR2, 3, 4 decreased, and that of TLR4 was almost undetectable; the nucleus of DC was stained strong positive, the plasm of DC was stained positive. CONCLUSION: The expressions of TLR2, 3, 4 on DC change significantly after being stimulated by LPS. And NF-kappaB shifts from the plasm to the nucleus. This signifies that LPS induces DC to be more mature in function.