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目的探讨发热样温度对朗格汉斯细胞(Langerhans cells,LCs)表面抗原递呈相关分子CD40、CD80、CD86、HLA-DR以及Toll-like受体(Toll-like receptor,TLR)-3、TLR-4表达的影响。方法采用Ficoll淋巴细胞分离液分离PBMCs,CD14免疫磁珠分离CD14+细胞。应用含细胞因子(rhGM-CSF 1 000IU/ml,rhIL-4 1 000IU/ml,TGF-β10ng/ml)的RPMI-1640培养基诱导培养CD14+细胞,第3d补加细胞因子,培养7d后的细胞采用流式细胞术检测表面Langerin,鉴定细胞为LCs。将LCs分别置于37℃、39.5℃、41℃、43℃5%CO2细胞培养箱中处理30min,37℃过夜恢复培养后台盼蓝染色法检测LCs的存活率,流式细胞术检测LCs表面抗原递呈相关分子CD40、CD80、CD86、HLADR以及TLR-3和TLR-4的表达情况。结果细胞因子诱导培养7d后细胞出现典型的树枝状突起,表达Langerin,表明LCs诱导成功。发热样温度处理后LCs存活率从37℃时的96.86%分别降至39.5℃时的90.09%、41℃时的86.11%和43℃时的76.78%。检测两份样品的LCs表面分子的变化。结果显示39.5℃处理的LCs表面CD86、HLADR、TLR-4的表达较37℃对照组显著增加(P<0.05),其阳性细胞百分率均值较对照组均值分别增加1.38、1.3和1.78倍;CD40、CD80、TLR-3的表达差异无统计学意义(P>0.05)。41℃处理的LCs表面TLR-3及TLR-4的表达显著增加(P<0.05),其阳性细胞百分率均值较对照组均值分别增加18.96和15.75倍;CD40、CD80、CD86、HLA-DR的表达差异无统计学意义(P>0.05)。43℃处理的LCs表面CD40、CD80、CD86的表达显著增加(P<0.05),其阳性细胞百分率均值较对照组均值分别增加5.32、1.36和1.63倍;HLA-DR、TLR-3、TLR-4表达显著变化(P>0.05)。结论发热样温度处理能增强LCs部分表面抗原递呈相关分子CD40、CD80、CD86、HLA-DR以及TLR-3、TLR-4的表达。其中43℃处理能显著增加CD40的表达,41℃处理能显著增加TLR-3及TLR-4的表达。
Objective To investigate the effect of fever-like temperature on the expression of antigens on CD40, CD80, CD86, HLA-DR and Toll-like receptor (TLR) -3 on the surface of Langerhans cells (LCs) -4 expression. Methods PBMCs were isolated by Ficoll lymphocyte separation and CD14 cells were isolated by magnetic beads. CD14 + cells were cultured in RPMI-1640 medium containing cytokines (rhGM-CSF 1000 IU / ml, rhIL-4 1000 IU / ml, TGF- β 10 ng / ml), supplemented for 3 days with cytokines, and cultured for 7 days The surface Langerin was detected by flow cytometry and the cells were identified as LCs. The LCs were placed in 37 ℃, 39.5 ℃, 41 ℃, 43 ℃ 5% CO2 cell incubator for 30min, overnight at 37 ℃ to restore culture after trypan blue staining LCs survival rate detected by flow cytometry LCs surface antigen Relevant molecules CD40, CD80, CD86, HLADR and the expression of TLR-3 and TLR-4 were presented. Results Cytokines induced typical dendritic cells after cultured for 7 days, expressing Langerin, indicating successful LCs induction. The survival rate of LCs decreased from 96.86% at 37 ℃ to 90.09% at 39.5 ℃, 86.11% at 41 ℃ and 76.78% at 43 ℃, respectively. Changes in LCs surface molecules of two samples were examined. The results showed that the expression of CD86, HLADR and TLR-4 on the surface of LCs treated with 39.5 ℃ increased significantly (P <0.05) compared with the control at 37 ℃, the average percentage of positive cells increased by 1.38, 1.3 and 1.78 times, CD80, TLR-3 expression was no significant difference (P> 0.05). The expression of TLR-3 and TLR-4 on the surface of LCs treated with 41 ℃ increased significantly (P <0.05), and the average percentage of positive cells increased by 18.96 and 15.75 folds compared with the mean value of the control group, while the expressions of CD40, CD80, CD86 and HLA-DR The difference was not statistically significant (P> 0.05). The expression of CD40, CD80 and CD86 on the surface of LCs treated with 43 ℃ increased significantly (P <0.05), and the positive percentage of positive cells increased by 5.32, 1.36 and 1.63 times than that of the control group respectively. HLA-DR, TLR-3 and TLR-4 The expression was significantly changed (P> 0.05). Conclusion Fever-like temperature treatment can enhance the expression of some surface antigen-presenting molecules CD40, CD80, CD86, HLA-DR, TLR-3 and TLR-4 in LCs. Treatment with 43 ℃ significantly increased the expression of CD40, and the treatment with 41 ℃ significantly increased the expression of TLR-3 and TLR-4.