热带病与寄生虫学

热带病与寄生虫学 ›› 2020, Vol. 18 ›› Issue (1): 5-11.

• 论著 • 上一篇    下一篇

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

 胡丽1 , 赵亚娥1∗, 熊国典2 , 张又仁3 , 王昊若2 , 刘博2
  

  1. 1. 西安交通大学基础医学院病原生物学与免疫学系,西安 710061; 2. 西安交通大学医学部本科生; 3. 西安交通大学经济与金融学院本科生
  • 收稿日期:2020-01-20 出版日期:2020-03-10 发布日期:2020-03-10
  • 通信作者: 赵亚娥,E-mail:zhaoyae@ xjtu.edu.cn
  • 作者简介:胡丽,女,博士,讲师,研究方向:人体寄生虫的致病机制与宿主免疫。 E-mail:huli2017@ xjtu. edu. cn
  • 基金资助:
    国家自然科学基金( 81271856),中国博士后科学基金( 2017M623189),西安交通大学新教师科研支持计划 (YX6K007)

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

HU Li1 , Zhao Ya-e1∗ , XIONG Guo-dian2 , ZHANG You-ren3 , WANG Hao-ruo2 , LIU Bo2   

  1. 1. Department of Pathogen Biology and Immunology, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an 710061, China; 2. Health Science Center, Xi'an Jiaotong University; 3. School of Economics and Finance,Xi'an Jiaotong University
  • Received:2020-01-20 Online:2020-03-10 Published:2020-03-10
  • Contact: Corresponding author, E-mail:zhaoyae@xjtu.edu.cn

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摘要： 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。

关键词: 毛囊蠕形螨, HaCaT细胞, 共培养, TLR2, 炎性因子

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

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