活体枯否细胞靶向性基因敲减GeRPs方法对急性肝衰竭小鼠肝组织Ceslf基因表达的影响  

Effect of in vivo Kupffer Cell-targeted Gene Knockdown with GeRPs on Expression of Ces1f in Liver Tissues of Mice with Acute Liver Failure

作　　者：杨雪[1] 何玉 施青青 熊晓晴 刘亮明[1] YANG Xue;HE Yu;SHI Qingqing;XIONG Xiaoqing;LIU Liangming(Departent o f Infectious Disease,Shanghai Songjiang Clinical Medical College o f Nanjing Medical University,Shanghai,201600,China;Songjiang Branch o f First People’s Hospital Affiliated to Shanghai Jiaotong University,Shanghai,201600,China;The Second Hospital Affiliated to Nanchang University,Nanchang,330006,China)

机构地区：[1]南京医科大学上海松江临床医学院,上海市201600 [2]上海交通大学医学院附属松江医院感染科,上海市201600 [3]南昌大学第二附属医院,南昌市330006

出　　处：《医学分子生物学杂志》2020年第3期221-227,共7页Journal of Medical Molecular Biology

基　　金：国家自然科学基金(No.81770612,81070357,30660066)。

摘　　要：目的研究动物活体肝枯否细胞靶向性基因敲减β-1,3-D葡聚糖包裹Endoprter-siRNA颗粒(B-1,3-D-glucan-encapsulated Endoporter-siRNA purticles,GeRPs)注射方法对脂多糖(lplysacchaide,LPS)/D-氨基半乳糖()galacto-sumine.D-CalIN)诱导急性肝衰竭(ueute liver failure,ALF)小鼠肝组织内羧酸酯酶If(erbonylesteraseIf,Ceslf)表达的影响。方法采用B-I,3-D-葡聚糖包裹Endprer-CesIf-siRNA复合物的方法制备GeRPs。健康雄性C57BL/6小鼠随机分为5组:A为正常对照组[GeRPs(-).LPS/D-GalIN(-)Endoponter(-)],B为模型组[GeRPs(-)LPS/D-GalN(+)Endopoter(-)],C为预处理组[GeRPs(+)LPS/D-GaIN(-)Endoporter(-)].D为预处理模型组[GeRPs(+)LPS/D-GaIN(+)Endoprter(-)],E为空白组[GeRPs(-)LPS/D-GaIN(-)Endopoter(+)]。以上各组分别采用GeRPs或Endoporter或PBS预处理后,再以LPS/D-CGalIN腹腔内注射诱导ALF实验动物模型。采用原位胶原酶灌注和选择性贴壁等方法分离培养小鼠原代肝细胞,并以LPS刺激细胞。Ceslf基因表达采用ral-time PCR方法和荧光原位杂交(fuorescenee in situ hybridizaion FISH)方法检测;肝脏组织病理形态学变化采用光学显微镜观察。结果PCR与FISH结果显示,与A组比较,B组、C组和D组Ceslf基因的表达均显著下调(均为P<0.01);D组Ceslf mRNA的相对表达水平较B组下降(P<0.05)。在原代肝细胞中,LPS刺激较非刺激细胞Ces1f的表达也明显下调(P<0.01);经LPS/D-CalIN刺激后,小鼠肝脏组织病理损伤明显,通过GeRPs预处理可进一步加巫LPS/D-GalIN诱导的急性肝衰竭小鼠肝脏病理损伤效应。结论GeRPs方法能够下调健康小鼠肝组织Ceslf基因表达,同时,GeRP's预处理能进一步下调ALF小鼠肝组织中Ceslf mRNA表达的受抑程度,并加重ALF小鼠肝脏病理损伤效应。Objective To study an ffeet of in riro Kupffer cells(KCs)-targeted gene knockdown by way of injection ofβ-1,3-D-glucan-encapsulated siRNA particles(GeRPs)on the expression of hepatie cearboxylesterase If(Ceslf)in lipopolysaccharide/D-galactosamine(LPS/D-GalN)induced acute liver failure(ALF)mice.Methods GeRP's were prepared by encapsulating Endoporter-CesIf-siRNA complex withβ-1,3-D glucan.Healthy male C57BL/6 mice were ranc dom-ly divided into 5 groups:normal control group[GeRPs(-)LPS/D-GalN(-)Endoporter(-),group A],modcl group[CcRP:(一)LPS/D CalN(+)Endoporter(一),group B],pretreatment group[GeRP's(+)LPS/D-GalN(-)Endoporter(-),group C],pre-treatment model group[GeRPs(+)LPS/D-GalIN(+)Endoporter(-),group D],and blank group[GeRP's(-)LPS/D-GalN(-)Endoporter(+),group E].The above groups were pretreated with GeRPs,Endoporter or PBS,and then iected with LPS/D-GaIN intraprio-neally to induce ALF models.In addition,in situ collagenase perfusion and selective adherence were used to isolate and culture the primary hepatocytes,and cells were then stimulated with LPS.The mRNA expression of Ceslf was detected by real-time PCR and fluorescence in situ hybridization(FISH)assay,respecively.The pathological and morphologeal changes of liver lissues were ob-served under an optical microscope.Results RT-PCR and FISH showed that the expression of Ceslf gene in groups B,C and D was significantly down-regulated when compared with that in group A(all P<0.01).The relative expression level of Ceslf mRNA was significantly decreased in group D as compared with that in group B(P<0.05).In primary hepatoeyles,LPS stimulated Ceslf ex-pression was significantly down-regulated(P<0.01).The pathological damage of mouse liver tissue was signifcantly induced after LPS/D-GalN stimulation,while pretreatment with GeRP's could fur-ther aggravate LPS/D-GalN-induced liver pathological damage in mice with acute liver fail-ure.Conclusion GeRP's can down.regulate the expression of Ceslf gene in healthy mouse liver tis-sue.Meanwhile,GeRPs pretreatment can

关 键 词：基因敲减 GeRPs 急性肝衰竭 Ceslf基因 小鼠 

分 类 号：R575.3[医药卫生—消化系统]