慢性乙型肝炎病毒感染树鼩Kupffer细胞TLR2和TLR4的表达及其意义  

Expressions and significance of TLR2 and TLR4 in Kupffer cells of tree shrews chronically infected with hepatitis B virus

作　　者：阮萍[1,2,3] 杨春 苏建家 欧超 曹骥 骆成漂 唐艳萍 秦虹[1,2] 孙雯[1,2] 李瑗 

机构地区：[1]广西肿瘤医院实验研究部 [2]广西医科大学研究生院,广西南宁530021 [3]广西中医药大学附属瑞康医院病理科,广西南宁530011

出　　处：《细胞与分子免疫学杂志》2014年第7期686-690,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(30660171);广西自然科学基金项目(桂科自0447087)

摘　　要：目的探索慢性乙型肝炎病毒(HBV)感染树鼩Kupffer细胞Toll样受体(TLR)家族中的TLR2和TLR4在mRNA水平的表达情况及其对Kupffer细胞功能的影响。方法树鼩分为确定慢性感染HBV的树鼩、疑似慢性感染HBV的树鼩和未接种HBV的正常对照树鼩。全部动物定期抽血和进行肝活检手术,采用实时荧光定量PCR(qRT-PCR)分析血清和肝组织的HBV DNA水平;对手术切取的树鼩肝组织进行Kupffer细胞的分离、纯化和原代培养,采用qRT-PCR检测TLR2、TLR4以及TNF-α的mRNA表达水平;采用迁移实验及溶酶体荧光探针等方法分析TLR2和TLR4对Kupffer细胞迁移能力及溶酶体数量的影响。结果确定慢性感染HBV的树鼩TLR2 mRNA和TLR4 mRNA表达水平均低于疑似慢性感染HBV的树鼩和未接种HBV的正常对照树鼩(P<0.05),表达水平均与动物肝组织的HBV DNA拷贝数呈负相关(P<0.05),与Kupffer细胞的细胞迁移数、溶酶体密度及TNF-αmRNA表达水平呈正相关(P<0.05)。结论 Kupffer细胞中的TLR2和TLR4可能通过影响Kupffer细胞功能而参与树鼩HBV感染后肝脏病变的慢性化发展过程。Objective To investigate the mRNA expression levels of Toll-like receptors 2 （TLR2） and TLR4 in Kupffer cells of tree shrews that were chronically infected with hepatitis B virus （ HBV）, and the effects of these receptors on the function of Kupffer cells. Methods The tree shrews were divided into tree shrews proved with chronic HBV infection, tree shrews suspected with chronic HBV infection, and normal control tree shrews without hepatitis B vaccination. The samples of serum and liver biopsy were collected periodically, and the levels of HBV DNA in serum and liver tissues were detected by fluorescence-based quantitative real-time PCR （qRT-PCR）. Meanwhile, Kupffer cells were isolated from the biopsied liver tissues, and then purified and primarily cultured. Afterwards, qRT-PCR was applied to detect the mRNA expression levels of TLR2, TLR4 and TNF-α in the Kupffer cells. Cell migration assay and lysosome-specific fluorescent probe were adopted to analyze the effects of TLR2 and TLR4 on the migration capacity of Kupffer cells and the quantity of lysosomes in these cells. Results The mRNA expression levels of TLP,2 and TLR4 in tree shrews proved with chronic HBV infection were lower than those in the ones suspected with chronic HBV infection and normal controls without hepatitis B vaccination （ P 〈 0.05）, and these expression levels were all negatively correlated with the level of HBV DNA in liver tissues of the animals （ P 〈 0.05 ）, but were positively correlated with the number of migrated Kupffer cells, the density of lysosomes and the mRNA expression level of TNF-α （P 〈0.0.5）. Conclusion TLR2 and TLR4 in Kupffer cells may play important roles in the chronic process of hepatic pathological changes in tree shrews infected with HBV through their influence on the function of Kupffer cells.

关 键 词：TOLL样受体 KUPFFER细胞 乙型肝炎病毒 树鼩 

分 类 号：R392.11[医药卫生—免疫学] R373.21[医药卫生—基础医学]