华支睾吸虫ESP刺激胆管上皮细胞产生IL-17A促进肝纤维化的机制研究
The mechanism of stimulating the secretion of IL-17A from bile duct epithelial cells by Clonorchis sinensis to activate hepatic stellate cells to promote hepatic fibrosis
刘桐彤 1朱浩 1王楠 1王新月 1胡凤爱 1曹璋 2高洪莲 1刘乃国 1柏雪莲1
作者信息
- 1. 滨州医学院附属医院医学研究中心,山东滨州 256603
- 2. 滨州医学院附属医院病理科
- 折叠
摘要
目的 探讨华支睾吸虫感染刺激胆管上皮细胞产生IL-17A激活HSC导致肝纤维化的机制.方法 从流行区获取华支睾吸虫感染的患者血清,ELISA法检测IL-17A的表达.人工消化麦穗鱼,分离华支睾吸虫囊蚴,分别灌胃感染新西兰大白兔和BALB/c小鼠,无菌分离小鼠肝脏和血清.ELISA法、Western blot和免疫组化法检测小鼠血清和肝脏中IL-17A的表达;ESP刺激人胆管癌细胞RBE,分别应用qRT-PCR和Western blot检测RBE细胞IL-17A的表达.收集RBE细胞培养上清加入LX-2细胞培养体系,qRT-PCR和Western blot检测LX-2细胞的α-SMA和Collagen-Ⅰ的表达.结果 华支睾吸虫感染患者血清IL-17A水平显著高于健康体检者(t=6.536,P＜0.01).被感染的小鼠血清和肝脏中IL-17A的表达水平显著高于正常对照组(t=12.98,P＜0.01;t=4.155,P＜0.05).免疫组化结果显示,感染小鼠的肝胆管上皮中IL-17A表达量增多(t=10.80,P＜0.01).ESP刺激后,RBE细胞的IL-17A表达水平显著升高(均P＜0.01).RBE细胞培养上清加入LX-2细胞培养体系后,α-SMA和Collagen-Ⅰ的表达水平显著高于对照组(均P＜0.01).结论 华支睾吸虫可以刺激胆管上皮细胞产生IL-17A,进一步促进炎症并激活HSC产生胶原沉积,从而促进肝纤维化的进展.
Abstract
Objective To investigate the mechanism of HSC activation through IL-17A production by bile duct epithelial cells stimulated by Clonorchis sinensis infection.Methods IL-17A expression level in sera of patients infected with C.sinensis from endemic areas was detected by ELISA.New Zealand white rabbits and BALB/c mice were infected by gavage with C.sinensis metacercariae isolated from freshwater fish.Liver tissue and peripheral blood were collected aseptically.ELISA and western blot were applied to detect the IL-17A expression in mouse sera and liver respectively.Immunohistochemical staining was used to detect the expression of IL-17A in the bile duct epithelium.C.sinensis adults were obtained from rabbits'liver and cultured in PBS.The culture supernatant was saved as ESP.After RBE cells were stimulated with ESP,IL-17A level was analyzed by qRT-PCR,western blot,and ELISA respectively.Subsequently,LX-2 cells were cultured by adding the culture supernatant of RBE cells.The expression levels of α-SMA and Collagen-Ⅰ were assessed by qRT-PCR and western blot.Results The IL-17A levels were significantly elevated in patients serum infected with C.sinensis than in healthy individuals(t=6.536,P＜0.01).The expression of IL-17A in the peripheral blood and liver of infected mice was also significantly increased compared with normal mice(t=12.98,P＜0.01;t=4.155,P＜0.05).Immunohistochemical staining showed that more IL-17A signals were detected in the epithelium of intrahepatic bile ducts of infected mice(t=10.80,P＜0.01).The IL-17A levels in RBE cells were significantly increased after ESP stimulation(P＜0.01).The mRNA and protein levels of α-SMA and Collagen-I in LX-2 cells were significantly increased after cultured with ESP-stimulated RBE cells supernatant(P＜0.01).Conclusion C.sinensis may promote the activation of HSC and hepatic fibrosis by stimulating bile duct epithelial cells to produce IL-17A and consequent inflammation.
关键词
华支睾吸虫/分泌代谢产物/白介素17A/肝纤维化Key words
Clonorchis sinensis/excretory-secretory products/IL-17A/hepatic fibrosis引用本文复制引用
出版年
2025
NETL
NSTL
万方数据