CRP敲除通过介导Kupffer细胞分化对大鼠血清总胆固醇水平的影响  

Effect of CRP knockout on serum total cholesterol level in rats via regu⁃lating Kupffer cell differentiation

作　　者：刘芳英 张雪娣 王帅 黄长浩 宋业鼎 穆云萍 李芳红 赵子建 LIU Fangying;ZHANG Xuedi;WANG Shuai;HUANG Changhao;SONG Yeding;MU Yunping;LI Fanghong;ZHAO Allan Zijian(School of Biomedical and Pharmaceutical Sciences,Guangdong University of Technology,Guangzhou,510006,China)

机构地区：[1]广东工业大学生物医药学院,广东广州510006

出　　处：《中国病理生理杂志》2023年第6期1030-1036,共7页Chinese Journal of Pathophysiology

基　　金：国家重点研发计划(No.2018YFA0800603);广东省重点领域研发计划“新药创制”专项(No.2019B020201015);广东省创新团队(No.2016ZT06Y432);国家自然科学基金青年项目(No.82100064);广州市基础与应用基础研究基金(No.202201010167)。

摘　　要：目的:研究C-反应蛋白(CRP)基因全身敲除介导肝巨噬细胞[库普弗(Kupffer)细胞]分化以及对大鼠血清总胆固醇水平的影响。方法:利用TALEN技术构建CRP基因敲除大鼠模型,PCR技术鉴定其基因型,筛选纯合子后代(纯合敲除为CRP^(-/-),野生型为CRP+/+),随机选择10只SPF级雄性CRP^(-/-)大鼠(8周龄)为模型组,10只SPF级雄性CRP+/+大鼠(8周龄)为正常对照组。用Western blot、RT-qPCR和免疫组织化学技术验证CRP在CRP^(-/-)大鼠重要脏器中的表达情况;胆固醇试剂盒检测大鼠血清总胆固醇水平;RT-qPCR检测CRP^(-/-)大鼠肝脏M1型巨噬细胞中CCL2、CCL3和IL-6表达水平,以及M2型巨噬细胞中IL-4、IL-10和转化生长因子β(TGF-β)表达水平;用分子生物学技术检测CRP^(-/-)大鼠肝脏中M1型巨噬细胞标志物CD68、M2型巨噬细胞标志物CD163及胆固醇逆向转运(RCT)关键蛋白B族1型清道夫受体(SR-B1)的表达水平。结果:与CRP+/+大鼠相比,CRP^(-/-)大鼠心、肝、肾、脾等重要脏器未检测到CRP表达(P<0.01),表明CRP^(-/-)大鼠模型构建成功。CRP^(-/-)大鼠血清总胆固醇显著低于CRP+/+大鼠(P<0.01),肝脏中IL-6、CCL2和CCL3水平显著降低,而IL-4、IL-10和TGF-β表达显著增高(P<0.01);M1型巨噬细胞标志物CD68表达水平显著下降,而M2型巨噬细胞标志物CD163表达水平显著上升(P<0.01);CRP^(-/-)大鼠肝脏内SR-B1水平显著高于CRP+/+大鼠(P<0.05)。结论:CRP基因全身敲除影响Kupffer细胞分化并降低大鼠血清总胆固醇水平,具体表现为:促进大鼠肝脏内单核细胞分化为M2型巨噬细胞,并抑制其向M1型巨噬细胞分化,显著促进大鼠肝脏内RCT从而降低大鼠血清总胆固醇。AIM:To investigate the effect of systemic knockout of C-reactive protein(CRP)gene on serum total cholesterol level and the differentiation of liver macrophages(Kupffer cells)in rats.METHODS:The CRP knockout rat model was constructed using TALEN technology,and the genotypes were identified by PCR(hereafter CRP^(-/-)for the knockout group and CRP+/+for the wild-type group).Ten SPF-grade male CRP^(-/-)rats(8 weeks of age)were used as the model group,with ten SPF-grade male littermate CRP+/+rats(8 weeks of age)as the normal control group.Further validation of CRP expression in the organs of CRP^(-/-)rats was performed using Western blot,RT-qPCR and immunohistochemistry(IHC)assays.Serum total cholesterol level was measured with a cholesterol kit.RT-qPCR was used to quantify the hepatic expression levels of chemokines CCL2 and CCL3,and inflammatory factor interleukin-6(IL-6)in M1 macrophages.The expression levels of anti-inflammatory factors,such as IL-4,IL-10 and transforming growth factor-β(TGF-β),in M2 macrophages were also analyzed.The expression of CD68(M1 macrophage marker),CD163(M2 macrophage marker),and scavenger receptor class B type 1[SR-B1;the crucial protein of cholesterol reverse transport(RCT)]in the liver was quantified by RT-qPCR,IHC and Western blot.RESULTS:Compared with CRP+/+rats,CRP gene was successfully knocked out in the liver,heart,kidney and spleen of CRP^(-/-)rats(P<0.01).The expression levels of IL-6,CCL2 and CCL3 were dramatically decreased in CRP^(-/-)rats compared with CRP+/+rats,while the IL-4,IL-10 and TGF-βexpression levels were significantly up-regulated in CRP^(-/-)rats(P<0.01).In comparison to CRP+/+rats,the expression level of CD68 showed a significant reduction,while CD163 was increased in CRP^(-/-)rats(P<0.01).The SR-B1 level was significantly higher in the liver of CRP^(-/-)rats than that in CRP+/+rats(P<0.01).CONCLUSION:Deletion of CRP greatly enhances the RCT in the liver by driving Kupffer cells to differentiate into M2 macrophages while suppressing their maturation into M1

关 键 词：C-反应蛋白 库普弗细胞 细胞分化 胆固醇逆向转运 

分 类 号：R364.5[医药卫生—病理学] R363.2[医药卫生—基础医学]