RNAi下调凝血酶敏感蛋白-1对脓毒症损伤影响的实验研究
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摘要
目的:利用RNA干扰(RNAi)方法探讨凝血酶敏感蛋白-1(TSP-1)对脓毒症肝损伤的影响及其作用机制。方法:雄性Balb/c小鼠通过盲肠结扎穿刺成功制作脓毒症模型,观察腹腔及肝脏大体解剖,检测血清转氨酶含量、肝脏TSP-1mRNA表达水平及肝脏TSP-1蛋白表达水平,探讨脓毒症时肝脏TSP-1的变化规律;利用RNAi技术靶向降解TSP-1 mRNA,引起转录后基因沉默,从而下调肝脏TSP-1表达水平,检测血清转氨酶含量、血清TNF-α含量,观察肝细胞凋亡情况,检测肝脏p38 MAPK蛋白表达变化,探讨TSP-1在脓毒症肝损伤中的作用及其机制。结果:与对照组相比,制作脓毒症模型后6小时肝脏TSP-1 mRNA及TSP-1蛋白表达明显增高,同时伴有肝脏炎症加重、血清ALT和AST含量明显升高,之后在制模24小时观察期内TSP-1维持于较高水平;利用流体尾静脉注射法予以siRNA-TSP1有效片段进行RNA干扰,制作脓毒症模型后血清ALT和AST含量、血清TNF-α含量均明显降低,肝细胞凋亡明显减少,同时肝脏磷酸化p38 MAPK蛋白表达减少。结论:脓毒症时肝脏TSP-1表达增加,作为炎症介质可能参与介导脓毒症肝损伤发生,导致血清转氨酶含量升高、血清TNF-α含量升高、肝细胞凋亡增加,同时磷酸化p38 MAPK蛋白活化,TSP-1可能通过p38 MAPK信号转导通路的激活介导其肝损伤作用。
Objective:To explore the impact and mechanism of the thrombospondin-1 in septic liver injury by application of RNA interference.Methods:The model for sepsis was developed by cecal ligation and puncture(CLP) in male Balb/c mice.Then the general anatomical feature of abdominal cavity and liver was observed.Serum aminotransferase,expression level of liver TSP-1 mRNA and TSP-1 protein were detected.The pattern of liver TSP-1 in sepsis was investigated.By application of RNA interference targeted degradation of TSP-1 mRNA to induce post transcriptional gene silencing(PTGS),the downregulation of liver TSP-1 mRNA and protein was achieved.Serum aminotransferase and serum TNF-αcontents were detected.Apoptosis of liver cell was observed.The change of liver p38 mitogen activated protein kinase was detected.The role and mechanism of TSP-1 in septic liver injury were investigated.Results:In comparison with control group,the expression of liver TSP-1 mRNA and TSP-1 protein was significantly increased accompanied with more severe liver inflammation as well as dramatically increasing serum ALT and AST contents 6 hours after the CLP model for sepsis.TSP-1 maintained a relatively high level during 24 hours observation period.Through hydrodynamic tail vein injection effective fragments of siRNA specific for TSP-1 were deliveried to make RNA inference,and then the CLP model for sepsis was developed.Serum ALT,AST and TNF-αcontents were significantly decreased,apoptosis of liver cell reduced markedly,and the expression of liver phospho-p38 MAPK was decreased.Conclusion:In sepsis the expression of liver TSP-1 is increased which may induce septic liver injury as a mediator of inflammation.This results in increased serum aminotransferase,serum TNF-αcontents and increased apoptosis of liver cell,with the activation of phospho-p38 MAPK.TSP-1 may induce liver injury by the activation of p38MAPK signal transduction pathway.
Objective:To explore the impact and mechanism of the thrombospondin-1 in septic liver injury by application of RNA interference.Methods:The model for sepsis was developed by cecal ligation and puncture(CLP) in male Balb/c mice.Then the general anatomical feature of abdominal cavity and liver was observed.Serum aminotransferase,expression level of liver TSP-1 mRNA and TSP-1 protein were detected.The pattern of liver TSP-1 in sepsis was investigated.By application of RNA interference targeted degradation of TSP-1 mRNA to induce post transcriptional gene silencing(PTGS),the downregulation of liver TSP-1 mRNA and protein was achieved.Serum aminotransferase and serum TNF-αcontents were detected.Apoptosis of liver cell was observed.The change of liver p38 mitogen activated protein kinase was detected.The role and mechanism of TSP-1 in septic liver injury were investigated.Results:In comparison with control group,the expression of liver TSP-1 mRNA and TSP-1 protein was significantly increased accompanied with more severe liver inflammation as well as dramatically increasing serum ALT and AST contents 6 hours after the CLP model for sepsis.TSP-1 maintained a relatively high level during 24 hours observation period.Through hydrodynamic tail vein injection effective fragments of siRNA specific for TSP-1 were deliveried to make RNA inference,and then the CLP model for sepsis was developed.Serum ALT,AST and TNF-αcontents were significantly decreased,apoptosis of liver cell reduced markedly,and the expression of liver phospho-p38 MAPK was decreased.Conclusion:In sepsis the expression of liver TSP-1 is increased which may induce septic liver injury as a mediator of inflammation.This results in increased serum aminotransferase,serum TNF-αcontents and increased apoptosis of liver cell,with the activation of phospho-p38 MAPK.TSP-1 may induce liver injury by the activation of p38MAPK signal transduction pathway.
引文
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[37]American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference:definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis.Crit Care Med,1992,20(6):864-874
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[41]朱雪琦,刘清泉,姚咏明.脓毒症动物模型的研究进展.中国危重病急救医学,2006,18:114-116
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[43]Warren HS.Strategies for the treatment of sepsis.N Engl J Med,1997,336(8):952-953
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[45]孟宪钧,王平,张平.肝脏在多器官功能衰竭中的作用.中华外科杂志,1988,26(1):13-15
[46]吴荣谦,宋旭华,徐迎新等.促炎症和抗炎症细胞因子基因表达与脓毒症小鼠肝损伤的关系.中华普通外科杂志,2001,16(2):103-105
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[48]Cohen JJ,Duke RC.Apoptosis and programmed cell death in immunity.Ann Rev Immunol,1992,10(2):267-273
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[1]Fire A,Xu S,Montgomery MK,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature,1998,391(6669):806-811
[2]Reinhart BJ,Slack FJ,Basson M,et al.The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans.Nature,2000,403(6772):901-906
[3]Parrish S,Mello C,Fire A,et al.Functional anatomy of a dsRNA trigger:differential requirements for the two trigger strands in RNA interference.Molec Cell,2000,6:1077-1087
[4]Sugimoto A.Throughput RNAi in Caenorhabditis elegans:genome-wide screens and functional genomic s.Differentiation,2004,72(2-3):81-91
[5]Kuttenkeuler D,Boutros M.Genome-wide RNAi as a route to gene function in Drosophila.Brief Funct Genomic Proteomic,2004,3(2):168-176
[6]McCaffrey AP,Meuse L,Pham TT,et aI.RNA interference in adult mice.Nature,2002,418(6893):38-39
[7]Lewis DL,Hagstrom JE,Loomis AG,et al.Efficient delivery of siRNA for inhibition of gene expression in postnatal mice.Nat Genet,2002,32(1):107-108
[8]Zhang G,Budker V,Williams P,et al.Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates.Hum Gene Ther,2001,12(4):427-438
[9]Eastman SJ,Baskin KM,Hodges BL,et al.Development of catheter-based procedures for transducing the isolated rabbit liver with plasmid DNA.Hum Gene Ther,2002,13(17):2065-2077
[10]Danialou G,Comtois AS,Matecki S,et al.Optimization of regional intraarterial naked DNA-mediated transgene delivery to skeletal muscles in a large animal model.Molec Ther,2005,11(2):257-266
[11]Mittal V.Improving the efficiency of RNA interference in mammals.Nat Rev Genet,2004,5(5):355-365
[12]Reynolds A,Leake D,Boese Q,et al.Rational SiRNA design for RNA interference.Nat Biotechnol,2004,22(3):326-330
[13]Kumar R,Conklin DS,Mittal V.High-throughput selection of effective RNAi probes for gene silencing.Genome Res,2003,13(10):2333-2340
[14]Hsieh AC,Bo R,Manola J,et al.A library of SiRNA duplexes targeting the phosphoinositide 3-kinase pathway:determinants of gene silencing for use in cell-based screens.Nucleic Acids Res,2004,32(3):893-901
[15]Song E,Lee SK,Wang J,et al.RNA interference targeting Fas protects mice from fulminant hepatitis.Nat Med,2003,9(3):347-351
[16]Giladi H,Ketzinel-Gilad M,Rivkin L,et al.Small interfering RNA inhibits hepatitis B virus
[116]Huang F,Khvorova A,Marshall W,et al.Analysis of clathrin-mediated endocytosis of epidermal growth factor receptor by RNA interference.J Biol Chem,2004,279(16):16657-16661
[117]Shu X,Wu W,Mosteller RD,et al.Sphingosine kinase mediates vascular endothelial growth factor-induced activation of ras and mitogen-activated protein kinases.Mol Cell Biol,2002,22(22):7758-7768
[118]Lassus P,Opitz-Araya X,Lazebnik Y.Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization.Science,2002,297(5585):1352-1354
[119]Chen Z,Indjeian VB,McManus M,et al.CP110,a cell cycle-dependent CDK substrate,regulates centrosome duplication in human cells.Dev Cell,2002,3(3):339-350
[120]Buckingham SD,Esmaeili B,Wood M,et al.RNA interference:from model organisms towards therapy for neural and neuromuscular disorders.Hum Mol Genet,2004,13(Spec No 2):R275-R288
[121]Karlas A,Kurth R,Denner J.Inhibition of porcine endogenous retroviruses by RNA interference:increasing the safety of xenotransplantation.Virology,2004,325(1):18-23
[122]Hommel JD,Sears RM,Dileone RL,et al.Local gene knockdown in the brain using viral-mediated RNA interference.Nature Med,2003,9:1539-1544
[123]Maeda Y,Fukushima K,Nishizaki K,et al.In vitro and in vivo suppression of GJB2 expression by RNA interference.Hum Mol Genet,2005,14(12):1641-1650
[124]Fraser AG,Kamath RS,Zipperlen P,et al.Functional genomic analysis of C.elegans chromosome Ⅰ by systematic RNA interference.Nature,2000,408(6810):325-330
[125]Gonczy P,Echeverri C,Oegema K,et al.Functional genomic analysis of cell division in C.elegans using RNAi of genes on chromosome Ⅲ.Nature,2000,408(6810):331-336
[126]袁婺洲,Bodmer R,朱传炳等.利用RNAi技术研究果蝇心脏发育基因的功能.遗传学报,2002,29(1):34-38
[127]Novina CD,Murray MF,Dykxhoom DM,et al.siRNA-directed inhibition of HIV-1infection.Nat Med,2002,8(7):681-686
[128]Randall G,Grakoui A,Rice CM.Clearance of replicating hepatitis C vires replicon RNAs in cell culture by small interfering RNAs.Proc Natl Acad Sci USA,2003,100(1):235-240
[129]Yang ZG,Chen Z,Ni Q,et al.Inhibition of hepatitis B virus surface antigen expression by small hairpin RNA in vitro.World J Gastroenterol,2005,11(4):498-502
[130]唐霓,黄爱龙,张秉强等.应用RNA干扰技术抑制乙型肝炎病毒抗原表达的实验研究.中国医学杂志,2002,83(15):1309-1312
[131]Wang Z,Ren L,Zhao X,et al.Inhibition of severe acute respiratory syndrome vires replication by small interfering RNAs in mammalian ceils.J Virol,2004,78(14):7523-7527
[31]McNeil PL,Kirchhausen T.An emergency response team for membrane repair.Nat Rev,Mol Cell Biol,2005,6(6):499-505
[32]Fisher JL,Levitan I,Margulies SS.Plasma membrane surface increases with tonic stretch of alveolar epithelial cells.Am J Respir Cell Mol Biol,2004,31(2):200-208
[33]Wang JG,Miyazu M,Xiang P,et al.Stretch-induced cell proliferation is mediated by FAK-MAPK pathway.Life Sci,2005,76(24):2817-2825
[34]Denk H,Stumptner C,Zatloukal K.Mallory bodies revisited.J Hepatol,2000,32(4):689-702
[35]Gandhi CR,Murase N,Subbotin VM,et al.Portacaval shunt causes apoptosis and liver atrophy in rats despite increases in endogenous levels of major hepatic growth factors,J Hepatol,2002,37(3):340-348
[36]Ohrt T,Merkle D,Birkenfeld K,et al.In situ fluorescence analysis demonstrates active siRNA exclusion from the nucleus by Exportin 5,Nucleic Acids Res,2006,34(5):1369-1380
[37]Lund E,Guttinger S,Calado A,et al.Nuclear export of microRNA precursors.Science,2004,303(5654):95-98
[38]Zender L,Hutker S,Liedtke C,et al.Caspase 8 small interfering RNA prevents acute liver failure in mice.Proc Natl Acad Sci USA,2003,100(13):7797-7802
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