NEK7在慢性肝脏炎症及纤维化形成中的作用
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国家自然科学基金项目(81670570);江苏省重点病种规范化诊疗研究项目(BE2016789)


NEK7 in chronic liver inflammatory and liver fibrosis
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    摘要:

    目的:探讨肝脏巨噬细胞中NIMA相关蛋白激酶7(never in mitosis gene a-related kinase 7,NEK7)在肝脏纤维化组织中的表达及其通过对NOD样受体家族3(nucleotide-binding oligomerization domain-like receptors,NLRP3)炎症小体信号通路调控肝脏纤维化(liver fibrosis)的作用机制。方法:通过HE和Masson染色以及qRT-PCR技术观察对比正常人和肝纤维化患者之间肝脏纤维化程度的差异;采用Western blot和qRT-PCR技术检测正常人和肝纤维化患者肝脏组织中NEK7的表达;运用免疫荧光染色技术观察CD68阳性细胞中NEK7的表达情况。通过腹腔注射四氯化碳(carbon tetrachloride,CCl4)8周的方法建立小鼠肝脏纤维化模型,采用巨噬细胞特异性干扰RNA(small interfering RNA,siRNA)敲低巨噬细胞中NEK7的表达,取组织标本,通过HE、Masson和天狼星红染色技术以及qRT-PCR技术,观察小鼠肝脏组织纤维化程度的差异;同时利用组织免疫化学染色和组织免疫荧光染色技术,观察小鼠肝脏炎症反应中中性粒细胞和巨噬细胞聚集程度的差异;进一步运用Western blot技术检测其对NLRP3炎症小体信号通路的影响。结果:NEK7在肝纤维化肝脏组织中表达明显高于正常肝脏组织;在体内特异性敲低巨噬细胞中NEK7的表达有助于减轻肝脏纤维化的程度和炎症反应以及抑制NLRP3炎症小体信号通路的激活。结论:抑制巨噬细胞中NEK7的表达能通过抑制NLRP3炎症小体信号通路,减轻炎症反应,阻止肝纤维化病变。

    Abstract:

    Objective:This study aims to investigate the never in mitosis gene a-related kinase 7(NEK7)expression in liver fibrosis and its effect on nucleotide-binding oligomerization domain-like receptors(NLRP3)signal pathway to regulate liver fibrosis. Methods:HE staining,Masson staining and quantitative reverse transcription PCR(qRT-PCR)were used to evaluate those degree of hepatic fibrosis in normals and patients with cirrhosis. The expression of NEK7 in liver tissues of non-cirrhotic and cirrhotic patients was detected by Western blot and qRT-PCR. CD68-positive cells was determined by immunofluorescence tissue staining. To induce hepatic fibrosis,C57BL/6J mice were injected intraperitoneally with carbon tetrachloride(CCL4)twice a week for 8 weeks. C57BL/6J mice were injected through tail vein with macrophage-specific siRNA to knockdown the expression of NEK7 in macrophages. HE staining,Masson staining and sirius red staining were used to observe the changes of liver fibrosis in mice liver tissues. The level of the migration and aggregation of neutrophils and macrophages were monitored by immunohistochemistry tissue staining and immunofluorescence tissue staining. The impacts of NEK7 on NLRP3 signaling pathway were measured by Western blot. Results:In the fibrotic liver tissues,NEK7 expression was significantly higher than that in the normal tissues. Specific blockade of NEK7 in macrophages obviously attenuated liver fibrosis and hepatic inflammation in vivo. Specific blockade of NEK7 in macrophages also inhibited the activation of NLRP3 inflammasome signaling pathway. Conclusion:Inhibition of NEK7 may alleviate macrophage inflammation reaction and liver fibrosis through inhibiting NLRP3 inflammasome signaling pathway.

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王艺睿,王 皓,刘硕琛,倪 鸣,李相成. NEK7在慢性肝脏炎症及纤维化形成中的作用[J].南京医科大学学报(自然科学版),2021,(5):663-669

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  • 收稿日期:2021-02-27
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  • 在线发布日期: 2021-06-02
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