蠕形螨对共培养HaCaT细胞的TLR2以及炎症相关基因表达的影响

目的通过建立毛囊蠕形螨与HaCaT细胞共培养体系,探讨毛囊蠕形螨与细胞表达TLR2以及炎症相关基因之间的关联性。方法用10只、30只、50只毛囊蠕形螨和空白对照分别与HaCaT细胞共培养24 h,提取细胞RNA,反转录成cDNA;设计特异性引物,对TLR2以及相关的KLK5、IL-1β、IL-6、IL-8和CCL2等炎性因子进行常规PCR扩增、克隆和测序;采用qRT-PCR检测表达量,比较与螨虫数之间的关联性。结果琼脂糖凝胶电泳显示PCR产物为单一清晰条带,序列大小与模板一致,表明引物特异性好。qRT-PCR检测显示,除10只螨虫组与空白组差异均无统计学意义外(t=0. 00~2. 25,P>0. 05),TLR2和IL-6在30只和50只螨虫组与空白组差异有统计学意义(TLR2:t=6. 54和10. 85;IL-6:t=14. 35和17. 52,P<0. 001),且50只螨虫组上调明显大于30只螨虫组;IL-8、CCL2和KLK5在30只和50只螨虫组与空白组的差异也有统计学意义(IL-8:t=5. 34和6. 98;CCL2:t=3. 12和4. 03;KLK5:t=3. 31和4. 05,P<0. 05),但30只与50只螨虫组的差异无统计学意义;而IL-1β只在50只螨虫组与空白组的差异有统计学意义(t=2. 60,P<0. 05),30只螨虫组与空白组的差异无统计学意义。HaCaT细胞TLR2的表达与蠕形螨虫数呈正相关(r=0. 984),与TLR2调控的炎性因子IL-6、IL-8、CCL2、KLK5和IL-1β的表达也呈正相关(r=0. 970、0. 984、0. 985、0. 974和0. 938),尤其是TLR2和IL-6表达量变化最明显。结论本研究成功构建了毛囊蠕形螨与HaCaT细胞共培养体系,首次从细胞水平揭示皮肤免疫反应与蠕形螨感染数量有关,这一探索性研究结果对于揭示蠕形螨寄生诱发面部皮肤损害的分子机制具有重要的科学意义。

Effects of Demodex mites on the expression of TLR2 and inflammation-related genes in co-cultured HaCaT cells

Objective To establish a co-culture system of Demodex folliculorum and HaCaT cells for investigating the relationship between D.folliculorum and the expression of TLR2 and inflammatory genes in cells.Methods A total of 10,30,and 50 D.folliculorum mites and 0 mites(blank control)were co-cultured with HaCaT cells for 24h,respectively.RNA was extracted from the cells,and reversely transcribed into cDNA.Specific primers were designed,and TLR2 and related inflammatory factors including KLK5,IL-1β,IL-6,IL-8,and CCL2 were amplified,cloned,and sequenced.qRT-PCR was performed to detect the expression level and compare the correlation with the number of mites.Results Agarose gel electrophoresis revealed single and clear PCR product band.The sequence size was consistent with the template,indicating good primer specificity.qRT-PCR showed that except for 10 mite group and the blank group(t=0.00~2.25,P>0.05),the expression level of TLR2 and IL-6 in 30 and 50 mite group were significantly different from those in the blank group(TLR2:t=6.54 and 10.85;IL-6:t=14.35 and 17.52,respectively.P<0.001),and the up-regulation in 50 mite group was significantly greater than that in 30 mite group.In IL-8,CCL2 and KLK5,there were statistical differences between the 30 and 50 groups and the blank group(IL-8:t=5.34 and 6.98;CCL2:t=3.12 and 4.03;KLK5:t=3.31 and 4.05,respectively.P<0.05),yet the difference was insignificant between the 30 mite group and the 50 mite group.However,in IL-1β,there was only difference between the 50 mite group and the blank group(t=2.60,P<0.05),and no difference between the 30 mite group and the blank group.The expression of TLR2 in HaCaT cells as well as inflammatory factors(IL-6,IL-8,CCL2,KLK5 and IL-1β)regulated by TLR2 was positively correlated with the number of mites(r=0.984,0.970,0.984,0.985,0.974 and 0.938,respectively),with TLR2 and IL-6 expression being dominant.Conclusion We successfully constructed the co-culture system of D.folliculorum and HaCaT cells,and for the first time confirmed that the skin immune response was related to the number of infected mites at the cellular level.The findings can be of great scientific significance for revealing the molecular mechanism of facial skin damage induced by Demodex mites.