Abstract: 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 re- lated 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, indica- ting 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 inflamma- tory 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 domi- nant. 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.

Key words: Demodex folliculorum, HaCaT cell, Co-culture, TLR2, Inflammatory cytokine