Caco-2细胞TLR2和TLR4对微小隐孢子虫刺激信号的识别

Caco-2 cells recognize and respond to Cryptosporidium parvum via TLR2 and TLR4

目的研究Caco-2细胞在微小隐孢子虫感染后TLRs的激活情况。方法微小隐孢子虫子孢子刺激Caco-2细胞后,通过qRT-PCR检测TLRs mRNA的表达水平,采用Western blot检测NF-κB激活情况,采用ELISA检测IL-8的分泌量。同时,在微小隐孢子虫子孢子感染Caco-2细胞前加入TLR2或TLR4的抑制剂,检测NF-κB激活情况以及IL-8的分泌情况。结果将Caco-2细胞感染微小隐孢子虫子孢子2h后,提取的RNA反转成cDNA,采用普通PCR进行检测,结果表明Caco-2能够表达所有10种人TLRs;qRT-PCR检测刺激组与对照组TLRs表达量,经spss分析,p<0.01,刺激组TLR2和TLR4的表达量较对照组显著上调。对Caco-2细胞感染微小隐孢子虫子孢子1h后提取的细胞全蛋白进行Western blot,显示NF-κB激活水平较对照组显著上调;Caco-2细胞感染微小隐孢子虫子孢子12h后收集的细胞培养上清经ELISA检测,IL-8分泌量由刺激前的65.23pg/ml显著上调至488.23pg/ml。而在微小隐孢子虫子孢子感染Caco-2细胞前加入TLR2或TLR4的抑制剂,NF-κB激活水平和IL-8的分泌量较未加抑制剂感染组均显著下调,IL-8的分泌量分别降至204.02pg/ml(抑制TLR2活性后)和142.79pg/ml(抑制TLR4活性后)。结论 Caco-2细胞的TLR2和TLR4参与对微小隐孢子虫子孢子的识别。

Objective To investigate the role of TLRs in host cell responses during Cryptosporidium parvuminfection of Caco-2cells. Methods After Caco-2cells were infected with C.parvum,TLR expression was measured using qRTPCR,and TLR expression,NF-κB activation,and IL-8production were measured.For further analysis of the roles of TLR in response to C.parvum,TLR2 and TLR4were inhibited by TLR inhibitors,and then NF-κB activation and IL-8production were measured. Results Two h after infection with C.parvum,RNA was extracted and then normal PCR and qPCR were performed.Results indicated that Caco-2cells expressed all 10 TLRs.Analysis with SPSS and P〈0.01 indicated up-regulation of TLR2 and TLR4.One h after infection with C.parvum,NF-κB activation was measured using Western blotting and indicated up-regulation in comparison to the control group.Twelve h after infection with C.parvum,IL-8production was measured using ELISA and indicated up-regulation in comparison to the control group,with an increase from 65.23pg/ml to 488.23pg/ml.Upon inhibition of TLR2 or TLR4,however,the IL-8production and NF-κB activation induced in Caco-2cells by C.parvum was suppressed.The IL-8production dropped to 204.02pg/ml（TLR2inhibition）and to 142.79pg/ml（TLR4inhibition）. Conclusion TLR2 and TLR4mediate the recognition of C.parvumby human intestinal epithelial cells and their response to C.parvum.