包虫囊液体外诱导小鼠脾脏淋巴细胞TLR2和IDO表达的实验研究

An experimental study of the expression of TLR2 and IDO in mouse spleen lymphocytes induced by hydatid cyst fluid

目的研究细粒棘球蚴囊液对体外培养小鼠脾脏淋巴细胞TLR2和IDO表达的影响。方法制备小鼠脾脏淋巴细胞悬液,接种于24孔培养板中进行培养,用不同浓度比的包虫囊液(1∶10、1∶100)进行干预。分别在干预后12、24、36 h收集2份细胞及1份细胞培养上清,一份细胞用于Trizol法提取总RNA,通过实时荧光定量PCR检测TLR2、IDO、IL-5的mRNA相对表达水平,另一份细胞采用Western blot方法检测TLR2、IDO蛋白。细胞上清中的IL-6、TGF-β含量采用ELISA法检测。对小鼠脾脏淋巴细胞中IDO与TLR2 mRNA的表达进行线性相关分析。结果实时荧光定量PCR检测HCF为1∶10、1∶100时均可上调小鼠脾脏淋巴细胞TLR2、IDO、IL-5 mRNA的表达,其中1∶100稀释的HCF在24h时分别为0.282±0.144、0.218±0.036、0.648±0.148,与对照组0.006±0.003、0.002±0.000、0.008±0.001比较差异均有统计学意义(P<0.05);Western blot检测TLR2和IDO的表达均可被HCF上调,且在低浓度HCF(1∶100)上调更为显著;ELISA检测HCF干预24 h后细胞上清中的IL-6、TGF-β含量升高,其中在HCF1∶100时,相应含量分别为(296.495±12.495)pg/ml和(449.700±5.350)pg/ml,与对照组(67.328±5.839)pg/ml和(324.200±8.065)pg/ml比较差异均有统计学意义(P<0.05)。线性相关分析显示,小鼠脾脏淋巴细胞IDO与TLR2 mRNA表达水平呈正相关(R^2=0.9887,P<0.01)。结论包虫囊液对小鼠脾脏淋巴细胞TLR2和IDO的表达具有上调作用,故认为在CE慢性感染过程中TLR2和IDO共同发生负性免疫调控作用以促进Th2型反应,导致包虫感染过程中的免疫逃避。

Objectives To investigate the effects of the expressions of TLR2 and IDO in mouse spleen lymphocytes treated with hydatid cyst fluid in vitro.Methods Mouse spleen lymphocyte suspensions were prepared and cultured on 24-well culture plates.Mouse spleen lymphocytes then were treated with different concentrations of hydatid cyst fluid(1:10 and 1:100).Cells and supernatants were collected at 12,24,and 36 h.Total RNA was extracted from one group of cells using the Trizol method,and the relative levels of expression of TLR2,IDO,and IL-5 mRNA were detected using FQ-RT-PCR.The other group of cells was used to detect TLR2 and IDO proteins with Western blotting.The levels of IL-6 and TGF-βexpression in cell supernatants were detected using ELISA.The correlations between IDO and TLR2 were determined using linear correlation analysis.Results FQ-RT-PCR indicated that the levels of expression of TLR2,IDO,and IL-5 mRNA were up-regulated by HCF(1:10,1:100).The relative levels of expression of TLR2,IDO,and IL-5 mRNA were 0.282±0.144,0.218±0.036,and 0.648±0.148 when HCF was 1:100 at 24 h,and those levels differed significantly compared to levels in the non-treated group,which were 0.006±0.003,0.002±0.000,and 0.008±0.001(P<0.05 for both).Western blotting indicated that the levels of expression of TLR2 and IDO were up-regulated by HCF,and especially at a low concentration of HCF(1:100).ELISA indicated that the levels of expression of IL-6 and TGF-βin supernatants were increased by HCF after 24 h,and the corresponding levels were 296.495±12.495 pg/ml and 449.700±5.350 pg/ml when HCF was 1:100.This represented a significant difference compared to levels in the non-treated group,which were 67.328±5.839 pg/ml and 324.200±8.065 pg/ml(P<0.05).Linear correlation analysis revealed a correlation between the level of IDO and TLR2 mRNA expression in mouse spleen lymphocytes(R^2=0.9887,P<0.01).Conclusion Hydatid cyst fluid up-regulated the expression of TLR2 and IDO in mouse spleen lymphocytes.This suggests that TLR2 and IDO may have a negative immuno-regulatory role by promoting a Th2 response,leading to immune evasion during the process of Echinococcus infection.