Rab7负向调控巨噬细胞中TLR3/4信号转导通路的研究
摘要
Toll样受体家族可以识别几乎所有的病原微生物的一些结构组分和代谢产物,通过信号转导通路诱发细胞因子和趋化因子等启动机体的天然免疫,构成了机体抵抗病原微生物的第一道防线,在免疫学研究中一直是一个很受关注的家族。TLRs的活化是一把双刃剑。一方面TLRs的活化在启动天然免疫应答和适应性免疫应答中发挥重要作用,另外一方面过渡活化或者活化异常可能引起急慢性疾病。因此TLR信号转导通路的负相调控成为天然免疫研究中的热点问题。目的:本实验通过建立稳定表达Rab7及其突变体的巨噬细胞系,分析稳定表达细胞系的生物学特性,研究Rab7及其突变体基因过表达后对LPS和Poly I:C刺激的巨噬细胞表达细胞因子的影响。方法:将Rab7及其突变体Rab7(T22N)的真核表达载体通过脂质体法转染RAW264.7细胞,G418选择性培养基筛选,建立稳定表达Rab7及Rab7T22N的细胞系。通过RT-PCR和Western Blot方法鉴定稳定表达细胞系。通过观察细胞形态及MTT法分析稳定表达细胞系的生长特性。以LPS和Poly I:C刺激稳定表达细胞系不同时间后检测细胞因子的表达量变化。结果:与转染空质粒的细胞相比,转染Rab7和Rab7T22N的细胞中Rab7的mRNA和蛋白水平都显著增高。Rab7过表达后引起细胞形态变化并显著抑制了细胞增殖,Rab7T22N过表达后促进细胞增殖。Rab7过表达后,巨噬细胞在LPS和Poly I:C刺激后分泌的细胞因子显著降低,而Rab7T22N过表达后其的分泌又恢复。结论:成功建立了稳定表达Rab7及其突变体的细胞系。Rab7过表达后抑制了细胞增殖,负向调控了TLR信号通路。该研究为进一步阐明Rab7在TLRs信号通路中的作用奠定了基础。
The recoganition of the structural components and metabolites of pathogenic microorganism by Toll-like receptors can iniatiate innate immunity by promoting the production of cytokines and chemokines, and thus constitue the first line of defence to pathogenic microorganism. Study on the Toll like receptor has been a popular concern in immunological research. However, the activation of TLRs is a double edged sword.On one hand, activation of TLRs play a vital role in the innate and adaptive immune responses, on the other hand, overreaction of the signaling of TLRs has reported to be associated with acute and chronic diseases. Therefore,negative regulation on the TLR signaling pathways become a hot issue. Objective: To establish and analyze the biological characteristics of transformed macrophage cell lines stably expressing Rab7and its dominant negative mutant Rab7T22N, and study the production of cytokines in transformed macrophage cell lines after stimulation with LPS and Poly I: C. Methods: the eukaryotic expression vectors of Rab7 and Rab7T22N were transfected into RAW264.7 cells by liposome, and screened with G418.The G418-resistant colonies were obtained and amplified. The transformed cell lines were identified by RT-PCR, Real-Time PCR and Western Blot. The characteristics of transformed cell lines were analyzed by observing the cell morphology and MTT methods. The production of cytokines were measured after transformed cell lines stimulation with LPS and Poly I:C. Results: Compared with the control group, the mRNA and protein expression of Rab7 were significantly improved in cell lines transfected with Rab7 and Rab7T22N. Overexpression of Rab7 changed the cell morphology and inhibited the proliferation of Raw264.7 cells, while overexpression of Rab7T22N promoted the proliferation of Raw264.7 cells. Overexpression of Rab7 significantly inhibited the production of cytokines in LPS and poly I:C stimulated macrophages. The production of cytokines was restored in Rab7T22N cell line. Conclusion: Overexpression of Rab7 inhibits the proliferation of macrophages, and suppresses the production of cytokines in macrophages stimulated with LPS and Poly I: C. The study may lay a foundation for our further study the role of Rab7 in TLRs signaling pathways.
The recoganition of the structural components and metabolites of pathogenic microorganism by Toll-like receptors can iniatiate innate immunity by promoting the production of cytokines and chemokines, and thus constitue the first line of defence to pathogenic microorganism. Study on the Toll like receptor has been a popular concern in immunological research. However, the activation of TLRs is a double edged sword.On one hand, activation of TLRs play a vital role in the innate and adaptive immune responses, on the other hand, overreaction of the signaling of TLRs has reported to be associated with acute and chronic diseases. Therefore,negative regulation on the TLR signaling pathways become a hot issue. Objective: To establish and analyze the biological characteristics of transformed macrophage cell lines stably expressing Rab7and its dominant negative mutant Rab7T22N, and study the production of cytokines in transformed macrophage cell lines after stimulation with LPS and Poly I: C. Methods: the eukaryotic expression vectors of Rab7 and Rab7T22N were transfected into RAW264.7 cells by liposome, and screened with G418.The G418-resistant colonies were obtained and amplified. The transformed cell lines were identified by RT-PCR, Real-Time PCR and Western Blot. The characteristics of transformed cell lines were analyzed by observing the cell morphology and MTT methods. The production of cytokines were measured after transformed cell lines stimulation with LPS and Poly I:C. Results: Compared with the control group, the mRNA and protein expression of Rab7 were significantly improved in cell lines transfected with Rab7 and Rab7T22N. Overexpression of Rab7 changed the cell morphology and inhibited the proliferation of Raw264.7 cells, while overexpression of Rab7T22N promoted the proliferation of Raw264.7 cells. Overexpression of Rab7 significantly inhibited the production of cytokines in LPS and poly I:C stimulated macrophages. The production of cytokines was restored in Rab7T22N cell line. Conclusion: Overexpression of Rab7 inhibits the proliferation of macrophages, and suppresses the production of cytokines in macrophages stimulated with LPS and Poly I: C. The study may lay a foundation for our further study the role of Rab7 in TLRs signaling pathways.
引文
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2 Takaoka.A,et al.Integral role of in IRF-5 in the gene induetion programme activated by Toll-like receptors[J]. Nature,2005,434(7030):243-249
3 Burns.K.,et al.Inhibition of interleukin 1 receptor/Toll-Iike receptor signaling through the alternatively splieed,short form of MyD88 is due to its failure to recruit IRAK-4[J]. J Exp Med,2003,197(2):263-268
4 Takeda K,Kaisho T,AkiraS.Toll-like receptors[J]. Annu Rev Immunol,2003,21:335-376
5 Takeuchi.O.,et al.Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins[J]. J Imununol,2002,169(l):10-14
6 Asai.Y.,et al.Baeterial fimbriae and their peptides activate human gingival epithelial cell sthrough Toll-like receptor 2[J]. Infect Immun,2001,69(12):7387-739
7 Lien.E,et al.Toll-like receptor 2 funetions as apattern recognition receptor for diverse baeterial products[J]. J Biol Chem,1999,274(47):33419-33425
8 Alexopoulou,L,et al.Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3[J]. Nature,2001,413(6857):732-738
9 Hoshino,K.,et al.Cutting edge: Toll-like receptor4 (TLR4)-defieient mice are hyporesponsive to lipopolysaeeharide: evidence for TLR4 as the LPS gene product[J]. J Immunol,1999,162(7):3749-3752.
10 Gao,B,and M.F,Tsan.Endotoxin contamination in recombinant human heat shoek Protein70(HsP70) Preparation is responsible for the induction of tumor necrosis factor alpha release by murine macrophages[J]. J Biol Chem,2003,278(l):174-179
11 Smith,K.D.,et al. Toll-like receptor 5 recognizes a conserved site on flagellin require for protofilament formation and baeterial motility[J]. Nat Immunol,2003,4(12): 124
12 Takeuehi,O.,e tal. Diserimination of bacterial lipoproteins by Toll-like receptor 6[J]. Int Immunol,2001,13(7):933-940
13 Diebold,S.S.,et al.Innate antiviral responses by means of TLR7-mediatedrecognition of single-stranded RNA[J]. Science,2004,303(5663):1529-1531
14 Hemmi,H.,et al.A Toll-like receptor recognizes bacterial DNA[J]. Nature,2000,408(6813):740-745
15 Yarovinsky,F.,et al.TLR11 activation of dendritic cells by a protozoan profiling-like protein[J]. Science,2005,308(5728):1626-1629
16 Ozinsky,A.,et al.Cooperative induetion of pro-infiammatory signaling by Toll-like receptors[J]. J Endotoxin Res,2000,6(5):393-396
17 O’Neill,L.A. A. G. Bowie,The family of five: TIR-domain-containing adaptors in To11-like receptor signaling[J]. Nat Rev Immunol,2007,7(5):353-364
18 Pulendran,B.,K. Palueka. Banehereau,Sensing pathogens and tuning immune responses[J]. Seience,2001,293(5528):253-256
19 Rock FL,Hardiman G.Developmental Biology: A family of Human receptors struetuxally related to Drosophila Toll[J]. Proc Natl Acad Sci USA,1998,95(2):588-593
20 Schulz O,Diebold SS.Toll-like receptor 3 promotes cross-primising to virus-infected cells[J]. Nature,2004,33(7028):887-892
21 Takeda K,Akira S.TLR signaling pathways[J]. Semin Immunol,2004,16(l):3-9
22 Kalai M,Van loo G.Tipping the balance between necrosis and Apoptosis in human and murine cell streated with interferon and dsRNA[J]. Cell Death Differ,2002,9(9):981-994.
23 Groskreutz D J,Monick M.Respiratory syncytial virus induces TLR3 protein and protein kinase R, 1eading to increased double-stranded RNA responsiveness in airway epithelial cells[J]. J Immunol,2006,176:1733-1740
24王磊,覃少华.聚肌胞对小鼠的急性毒性和蓄积毒性阴[J].中国组织工程研究与临床康复,2007,11(15):2826-2828,2835
25 Medhzitov R,Preston-Hurlburt R. A human homologue of the Drosophila Toll protein singals activation of dapative immnunity [J]. Nuatre,1997,388:394-397
26 Mulhto G,Hihgtower L E.Cell surface expression of heat shock proteins and the immune response[J]. Cell Stress Chaperone,1996,1:167-176
27陈凌.Toll受体与肿瘤免疫[J].国外医学肿瘤学分册,2004,31(6):403-406
28 Jack C S, Arbour N. TLR signaling tailors innate immune responses in human microglia and astrocytes[J]. J Immunol,2005,175∶4320-4330.
29 Manetti R,Annunziato F.Polyinosinic acid:polycytidylic acid promotes T helper type 1-specificimmune responses by stimulating macrophage production of interferon-alpha and interleukin-12. Eur [J]. Immunol,1995,25:2656-2660
30 Marrack P,Kappler J. Type I interferons keep activated T cells alive[J]. Exp Med,1999,189:521-530
31方艳秋,宋燕.IFN-γ对体外培养系统中IL-18诱导的前列腺癌快速杀伤效应的影响[J].吉林大学学报:医学版,2003,29(4):428-432
32田原僮,潘玉琢.polyI:C对小鼠前列腺癌的抑制作用研究[J].吉林大学学报(医学版),2006,32(5):819-21
33蒋建新主编.《细菌内毒素基础与临床》[M].人民军医出版社,2004
34 Pereira-LealJBetal [J]. J MolBiol,2001,313:889
35 Dale LB,Seachrist JL. Regulation of angiotensin II type 1A receptor intracellular retention, degradation,and recycling by Rab5, Rab7, and Rab11 GTPases[J]. J Biol Chem,2004,279:13110-13118
36 Seabra MC,Mules EH.Rab GTPases, intracellular traffic and disease[J]. Trends Mol Med,2002 Jan,8(1):23-30
37 Hoepfner S,Severin F. Modulation of receptor recycling and degradation by the endosomal kinesin KIF16B[J]. Cell,2005,121(3):437-50
38 Rink J,Ghigo E. Rab conversion as a mechanism of progression from early to late endosomes [J]. Cell,2005,122(5):735-49
39 Bucci C,Chiariello M. Signal transduction gRABs attention[J]. Cell Signal,2006,18(1):1-8
40 Jordens I,Fernandez. The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors[J]. Curr Biol,2001,11(21):1680-1685
41 Fiory,F.,et al.Protein kinase Czeta and protein kinase B regulate distinct steps of insulin endocytosis and intracellular sorting[J]. J Biol Chem,2004,279(12):11137-45
42 Ceresa BP,Bahr SJ. Rab7 activity affects epidermal growth factor: epidermal growth factor receptor degradation by regulating endocytic trafficking from the late endosome[J]. J Biol Chem,2006,281:1099-1106
43 Husebye H,Halaas O. Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity[J]. EMBO J,2006,25:683-692.
44 Saxena S,Bucci C. The small GTPase Rab7 controls the endosomal trafficking and neuritogenic signaling of the nerve growth factor receptor TrkA[J]. J Neurosci,2005,25:10930-10940.
45 Dale LB,Seachrist JL. Regulation of angiotensin II type 1A receptor intracellular retention, degradation,and recycling by Rab5, Rab7, and Rab11 GTPases[J]. J Biol Chem,2004,279:13110-13118
46 Ceresa BP,Bahr SJ.Rab7 activity affects epidermal growth factor:epidermal growth factor receptor degradation by regulating endocytic trafficking from the late endosome[J]. J Biol Chem,2006,281:1099-1106
47 Dmitry Poteryaev1,Hanna Fares.Caenorhabditis elegans SAND-1 is essential forRAB-7 function in endosomal traffic[J]. The EMBO Journal,2007,26:301–312
48 Jun Zhang,Karen L.Thirty-One Flavors of Drosophila Rab Proteins Genetics [J]. J Biol Chem,2007,176:1307–1322
49 Homan ER,Zendzian RP. Studies on Polyl:C toxieity in experiental animals[J]. Toxicology and applied pharmacology,1972,23(4):579-588
50程书权.聚肌胞引起流感样症状5例报道[J].中华实验和临床病理学杂志,1994,3(01):108
51蒋廷旺,吴传勇.PolyI:C诱导C57BL/6小鼠产生自身免疫样病变(Autoimmune Characteristics Induced by PolyI: C in C57BL/6 M ice)[J].临床军医杂志(Clin JMed Offic),2007,35(4):489-490
52 Edward L,Kraw it T. Autoimmune hepatitis[J]. N ENGL JMED,2006, 354:54-66
53 Hutehens M,Luker KE.TLR3 increases disease morbidity and mortality from vaccine infection[J]. J Immunol,2008,180(1):483-491
54 Diamond MS,Klein RS. West Nile virus: crossing the blood-brain barrier[J]. Nat Med,2004,10(12):1294-1295
55 Gowen BB,Hoopes JD.TLR3 deletion limits mortality and disease severity due to Phlebovirus infection[J]. J Immunol,2006,177(9):6301-6307
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