活体靶向羧酸酯酶1f基因敲减对急性肝衰竭小鼠枯否细胞极化活性的影响  

Effects of in vivo targeted carboxylesterase 1f gene knockdown on the Kupffer cells polarization activity in mice with acute liver failure

作　　者：赵赛[1] 杨雪[1] 于倩 刘亮明[1,2] Zhao Sai;Yang Xue;Yu Qian;Liu Liangming(Departent of Infectious Disease,Shanghai Songjiang Clinical Medical College of Nanjing Medical University,Shanghai 201600,China;Departent of Infectious Disease,Songjiang Hospital Affiliated to Shanghai Jiaotong University School of Medicine,Shanghai 201600,China)

机构地区：[1]南京医科大学上海松江临床医学院感染科,上海201600 [2]上海交通大学医学院附属松江医院感染科,上海201600

出　　处：《中华肝脏病杂志》2023年第6期582-588,共7页Chinese Journal of Hepatology

基　　金：国家自然科学基金项目(81770612、81070357、30660066)。

摘　　要：目的探讨靶向羧酸酯酶1f(Ces1f)基因敲减对脂多糖/D-半乳糖胺(LPS/D-GalN)诱导的急性肝衰竭小鼠枯否细胞(KC)极化活性的影响。方法将携带靶向Ces1f干扰序列的小RNA(siRNA)与多肽转运载体(Endoporter)结合形成的复合物siRNA-EndoPorter包裹在β-1,3-D葡聚糖壳中形成复合体颗粒(GeRPs)。将30只雄性C57BL/6小鼠随机分为正常对照组、模型组(LPS/D-GalN)、预处理组(GeRPs)、预处理模型组(GeRPs+LPS/D-GalN)、空载体组(EndoPorter)。采用实时荧光定量PCR和蛋白质印迹(western blot)技术检测各组小鼠肝组织Ces1f mRNA及蛋白表达水平;采用实时荧光定量PCR技术检测各组小鼠KC M1极化表型分化簇86(CD86)mRNA以及KC M2极化表型分化簇163(CD163)mRNA表达水平;采用免疫荧光双染技术检测KC中Ces1f蛋白以及M1/M2极化表型CD86/CD163蛋白表达情况;采用苏木精-伊红染色观察肝组织病理损伤情况。多组间均数比较采用单因素方差分析,或方差不齐时采用独立样本非参数秩和检验。结果正常对照组与模型组、预处理组和预处理模型组肝组织Ces1f mRNA/蛋白相对表达水平分别为:1.00±0.00、0.80±0.03/0.80±0.14、0.56±0.08/0.52±0.13、0.26±0.05/0.29±0.13,各组间差异均有统计学意义(F值分别为9.171/3.957、20.740/9.315、34.530/13.830,P值均<0.01)。正常对照组与模型组、预处理组和预处理模型组KC Ces1f阳性细胞百分比分别为91.42%±3.79%、73.85%±7.03%、48.70%±5.30%、25.68%±4.55%,各组间差异均有统计学意义(F值分别为6.333、15.400、23.700,P值均<0.01)。正常对照组与模型组、预处理模型组CD86 mRNA相对表达水平分别为1.00±0.00、2.01±0.04、4.17±0.14,各组间差异均有统计学意义(F值分别为33.800、106.500,P值均<0.01)。正常对照组与模型组、预处理模型组CD163 mRNA相对表达水平分别为1.00±0.00、0.85±0.01、0.65±0.01,各组间差异均有统计学意义(F值分别为23.360、55.350,P值均<0.01)。正�Objective To investigate the effect of targeted carboxylesterase 1f(Ces1f)gene knockdown on the polarization activity of Kupffer cells(KC)induced by lipopolysaccharide/D-galactosamine(LPS/D-GalN)in mice with acute liver failure.Methods The complex siRNA-EndoPorter formed by combining the small RNA(siRNA)carrying the Ces1f-targeting interference sequence and the polypeptide transport carrier(Endoporter)was wrapped inβ-1,3-D glucan shell to form complex particles(GeRPs).Thirty male C57BL/6 mice were randomly divided into a normal control group,a model group(LPS/D-GalN),a pretreatment group(GeRPs),a pretreatment model group(GeRPs+LPS/D-GalN),and an empty vector group(EndoPorter).Real-time fluorescent quantitative PCR and western blot were used to detect Ces1f mRNA and protein expression levels in the liver tissues of each mouse group.Real-time PCR was used to detect the expression levels of KC M1 polarization phenotypic differentiation cluster 86(CD86)mRNA and KC M2 polarization phenotypic differentiation cluster 163(CD163)mRNA in each group.Immunofluorescence double staining technique was used to detect the expression of Ces1f protein and M1/M2 polarization phenotype CD86/CD163 protein in KC.Hematoxylin-eosin staining was used to observe the pathological damage to liver tissue.A one-way analysis of variance was used to compare the means among multiple groups,or an independent sample nonparametric rank sum test was used when the variances were uneven.Results The relative expression levels of Ces1f mRNA/protein in liver tissue of the normal control group,model group,pretreatment group,and pretreatment model group were 1.00±0.00,0.80±0.03/0.80±0.14,0.56±0.08/0.52±0.13,and 0.26±0.05/0.29±0.13,respectively,and the differences among the groups were statistically significant(F=9.171/3.957,20.740/9.315,34.530/13.830,P<0.01).The percentages of Ces1f-positive Kupffer cells in the normal control group,model group,pretreatment group,and pretreatment model group were 91.42%,±3.79%,73.85%±7.03%,48.70%±5.30%,and 25.68%

关 键 词：肝衰竭 羧酸酯酶1f 枯否细胞极化 活体基因敲减 

分 类 号：R575.3[医药卫生—消化系统]