靶向NF-κB的圈套ODNs拮抗pMφ炎性介质表达的实验研究
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摘要
目的：过度炎症反应不仅在经久难愈的慢性病中严重影响着人类的生活质量，而其过度、失控性的全身炎症反应所导致的广泛而严重的组织结构和生理功能的损害则直接危害着患者的生命，因此，除了原发病的治疗以外，抗炎已成为临床治疗的基本策略。基于NF-κB是控制炎性因子基因表达的瓶颈，靶向NF-κB的抗炎治疗成为国内外众多学者研究的热点，圈套策略以其特异性及高效性有着其他策略无可比拟的优势，成为一种新型的基因治疗方案。本实验设计合成靶向NF-κB的哑铃形圈套ODNs，并验证其抗炎作用。
材料与方法：(1)以阳离子脂质体为载体，将荧光标记的圈套ODNs (2.0μg/ml)转染入纯化的大鼠pMφ细胞,分别在30min、1h，3h，6h，9h，12h，18h，24h用荧光显微镜及激光共聚焦显微镜观察荧光标记的圈套ODNs在细胞内的动态变化。(2)用免疫组化法测大鼠pMφ细胞内NF-κBp65在LPS刺激前及刺激后1h的变化。(3)用ELISA法及RT-PCR法测TNFα、IL-6、IL-10在LPS刺激前及刺激后1、2、6、12、18、24h蛋白及mRNA水平的变化。(4)以阳离子脂质体为载体，将圈套ODNs转染大鼠pMφ细胞6h后用LPS(10μg/ml)刺激，以阳离子脂质体、无关ODNs及单纯LPS刺激组作对照，用ELISA法及RT-PCR法测圈套ODNs的不同浓度(2、4、8μg/ml)及不同时间(转染后8、12、18h)对TNFα、IL-6、IL-10在蛋白及mRNA水平的影响。
结果：(1)圈套寡核苷酸在pMφ细胞中的分布：荧光显微镜下转染30min细胞内便有荧光标记的圈套ODNs出现，随着时间的延长，细胞内的荧光越来越强，转染6～12h细胞内的荧光最强。激光共聚焦显微镜显
示：转染30min细胞内荧光标记的圈套ODNs主要分布在胞浆内，随着时间的延长，细胞内尤其胞核内的荧光越来越强，转染6～12h细胞内的荧光最强。(2)LPS刺激前大鼠pMφ细胞NF-κBp65大多数集中于胞浆，细胞呈"空泡"现象，LPS刺激后NF-κBp65大多数集中于胞核，细胞呈"实心"现象，且NF-κBp65的表达较刺激前明显增加。(3)LPS刺激后TNFα、IL-6、IL-10的表达较刺激前显著增高，且分别以2～6h、12～18h和4～12h时相最明显。(4)LPS刺激后TNFα、IL-6、IL-10mRNA的表达较刺激前显著增高，且分别以2h、12h和6h时相最明显。(5)在蛋白水平及mRNA水平，圈套ODNs 4μg/ml、8μg/ml组可显著降低pMφ表达TNFα、IL-6，且具有剂量依赖性，而只有8μg/ml剂量组对IL-10的抑制有统计学上的差别，且抑制效果随着时间的延长而明显。阳离子脂质体组、无关ODNs组与LPS刺激组相比，无显著性差异。
结论：(1)以阳离子脂质体为载体的哑铃形圈套ODNs可转染入大鼠pMφ细胞并分布于胞浆及胞核(2)LPS可引起NF-κB的激活及TNFα、IL-6、IL-10在mRNA及蛋白水平的显著升高(3)哑铃形圈套ODNs可明显降低TNFα、IL-6在mRNA及蛋白水平的表达，而对IL-10的抑制作用较弱。
Objective Over-inflammatory response severely influence the life quality of patients suffered from chronic diseases, and extensive and severe insult on the tissue and the physical function induced by over-inflammatory response imperil patients' lives. Therefore, besides treatment for primary diseases, anti-inflammation is the basic strategy in clinic. Considering nuclear factor-κB (NF-κB) is the key point to control gene expression of inflammatory factors, decoy-ODNs in dumbbell shape with the nuclear sequence similar to NF-κB cis-elements were synthetized in this study. To measure the anti-inflammatory action of decoy-ODNs targeting NF-κB, decoy-ODNs wrapped up by cationic liposomes were transfected into pMφ cells from rats. Then the effects of decoy-ODNs on expression of inflammatory mediators (TNFαand IL-6) and anti-inflammatory mediators (IL-10) in protein and gene levels were observed with enzyme-linked immunoadsorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR).
Materials and Methods 1. Using cationic liposomes as carriers, decoy-ODNs (0.2 μg/ml) labeled with fluorescein were transfected into the purified pMφ cells from rats, and the dynamic intracellular changes of decoy-ODNs were tested at 30 minutes, 1, 3, 6, 9, 12, 18 and 24 hours after transfection by fluorescent microscope and confocal laser scanning microscope. 2. The changes of NF-κB p65 in pMφ cells were measured at 1 hour before and after LPS stimulation, respectively, with immunohistochemistry method. 3.
The changes of TNFα, IL-6 and IL-10 in protein and gene levels were measured before LPS stimulation and at 1, 2, 4, 6, 12, 18 and 24 hours after LPS stimulation. 4. With carriers of cationic liposomes, decoy-ODNs were transfected into pM cells from rats. The pMφ transfected with ODNs of unrelated sequence or stimulated by LPS only to be as control. Then the inhibiting effects of the decoy-ODNs with different concentrations on protein and gene of TNFα, IL-6 and IL-10 were tested at the different intervals after stimulation of LPS (10 ug/ml).
Results 1. Decoy-ODNs labeled with fluorescein could be observed in cells under the fluorescent microscope at 30 minutes after transfection. With time passing by, the intracellular fluorescent light became brighter and brighter, reached to the brightest at 6-12 hours after transfection. The confocal laser scanning microscope showed decoy-ODNs was distributed mainly in plasma at 30 minutes after transfection. With time passing by, the intracellular especially the nuclear fluorescent light became brighter and brighter, and it also reached the brightest at 6-12 hours after transfection. 2. Before stimulation of LPS, NF-κBp65 mainly existed in nucleus, and cells showed "vacuole" phenomenon. After stimulation, NF-κBp65 mainly existed in nucleus and cells showed "solid" phenomenon. 3. After stimulation of LPS, the expressions of TNFα, IL-6 and IL-10 were higher significantly compared with that before stimulation (P < 0.01), especially at 2-6, 12-18 and 4-12 hours after stimulation, respectively. 4. The gene expressions of TNFα, IL-6 and IL-10 were higher significantly compared with that before stimulation (P < 0.01), especially at 2, 12 and 6 hours after stimulation, respectively. 5. Decoy-ODNs could decrease the expression of TNFα and IL-6 by dose-dependent fashion. There existed statistical inhibiting significance in IL-10 expression and gene expression when only 0.8μg/ml decoy-ODNs was used. And the inhibiting effect increased with time passing by.
Conclusions 1. With carriers of cationic liposomes, Decoy-ODNs in dumbbell shape can be transfected into the pMφ cells from rats and distributed in plasma and nucleus. 2. LPS can activate NF-κB and significantly increased the expressions of TNFα, IL-6 and IL-10 in protein and gene levels. 3. Decoy-ODNs in dumbbell shape can significantly decrease the expressions of TNFαand IL-6 in protein and gene levels, but have weaker inhibiting effect on IL-10.
材料与方法：(1)以阳离子脂质体为载体，将荧光标记的圈套ODNs (2.0μg/ml)转染入纯化的大鼠pMφ细胞,分别在30min、1h，3h，6h，9h，12h，18h，24h用荧光显微镜及激光共聚焦显微镜观察荧光标记的圈套ODNs在细胞内的动态变化。(2)用免疫组化法测大鼠pMφ细胞内NF-κBp65在LPS刺激前及刺激后1h的变化。(3)用ELISA法及RT-PCR法测TNFα、IL-6、IL-10在LPS刺激前及刺激后1、2、6、12、18、24h蛋白及mRNA水平的变化。(4)以阳离子脂质体为载体，将圈套ODNs转染大鼠pMφ细胞6h后用LPS(10μg/ml)刺激，以阳离子脂质体、无关ODNs及单纯LPS刺激组作对照，用ELISA法及RT-PCR法测圈套ODNs的不同浓度(2、4、8μg/ml)及不同时间(转染后8、12、18h)对TNFα、IL-6、IL-10在蛋白及mRNA水平的影响。
结果：(1)圈套寡核苷酸在pMφ细胞中的分布：荧光显微镜下转染30min细胞内便有荧光标记的圈套ODNs出现，随着时间的延长，细胞内的荧光越来越强，转染6～12h细胞内的荧光最强。激光共聚焦显微镜显
示：转染30min细胞内荧光标记的圈套ODNs主要分布在胞浆内，随着时间的延长，细胞内尤其胞核内的荧光越来越强，转染6～12h细胞内的荧光最强。(2)LPS刺激前大鼠pMφ细胞NF-κBp65大多数集中于胞浆，细胞呈"空泡"现象，LPS刺激后NF-κBp65大多数集中于胞核，细胞呈"实心"现象，且NF-κBp65的表达较刺激前明显增加。(3)LPS刺激后TNFα、IL-6、IL-10的表达较刺激前显著增高，且分别以2～6h、12～18h和4～12h时相最明显。(4)LPS刺激后TNFα、IL-6、IL-10mRNA的表达较刺激前显著增高，且分别以2h、12h和6h时相最明显。(5)在蛋白水平及mRNA水平，圈套ODNs 4μg/ml、8μg/ml组可显著降低pMφ表达TNFα、IL-6，且具有剂量依赖性，而只有8μg/ml剂量组对IL-10的抑制有统计学上的差别，且抑制效果随着时间的延长而明显。阳离子脂质体组、无关ODNs组与LPS刺激组相比，无显著性差异。
结论：(1)以阳离子脂质体为载体的哑铃形圈套ODNs可转染入大鼠pMφ细胞并分布于胞浆及胞核(2)LPS可引起NF-κB的激活及TNFα、IL-6、IL-10在mRNA及蛋白水平的显著升高(3)哑铃形圈套ODNs可明显降低TNFα、IL-6在mRNA及蛋白水平的表达，而对IL-10的抑制作用较弱。
Objective Over-inflammatory response severely influence the life quality of patients suffered from chronic diseases, and extensive and severe insult on the tissue and the physical function induced by over-inflammatory response imperil patients' lives. Therefore, besides treatment for primary diseases, anti-inflammation is the basic strategy in clinic. Considering nuclear factor-κB (NF-κB) is the key point to control gene expression of inflammatory factors, decoy-ODNs in dumbbell shape with the nuclear sequence similar to NF-κB cis-elements were synthetized in this study. To measure the anti-inflammatory action of decoy-ODNs targeting NF-κB, decoy-ODNs wrapped up by cationic liposomes were transfected into pMφ cells from rats. Then the effects of decoy-ODNs on expression of inflammatory mediators (TNFαand IL-6) and anti-inflammatory mediators (IL-10) in protein and gene levels were observed with enzyme-linked immunoadsorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR).
Materials and Methods 1. Using cationic liposomes as carriers, decoy-ODNs (0.2 μg/ml) labeled with fluorescein were transfected into the purified pMφ cells from rats, and the dynamic intracellular changes of decoy-ODNs were tested at 30 minutes, 1, 3, 6, 9, 12, 18 and 24 hours after transfection by fluorescent microscope and confocal laser scanning microscope. 2. The changes of NF-κB p65 in pMφ cells were measured at 1 hour before and after LPS stimulation, respectively, with immunohistochemistry method. 3.
The changes of TNFα, IL-6 and IL-10 in protein and gene levels were measured before LPS stimulation and at 1, 2, 4, 6, 12, 18 and 24 hours after LPS stimulation. 4. With carriers of cationic liposomes, decoy-ODNs were transfected into pM cells from rats. The pMφ transfected with ODNs of unrelated sequence or stimulated by LPS only to be as control. Then the inhibiting effects of the decoy-ODNs with different concentrations on protein and gene of TNFα, IL-6 and IL-10 were tested at the different intervals after stimulation of LPS (10 ug/ml).
Results 1. Decoy-ODNs labeled with fluorescein could be observed in cells under the fluorescent microscope at 30 minutes after transfection. With time passing by, the intracellular fluorescent light became brighter and brighter, reached to the brightest at 6-12 hours after transfection. The confocal laser scanning microscope showed decoy-ODNs was distributed mainly in plasma at 30 minutes after transfection. With time passing by, the intracellular especially the nuclear fluorescent light became brighter and brighter, and it also reached the brightest at 6-12 hours after transfection. 2. Before stimulation of LPS, NF-κBp65 mainly existed in nucleus, and cells showed "vacuole" phenomenon. After stimulation, NF-κBp65 mainly existed in nucleus and cells showed "solid" phenomenon. 3. After stimulation of LPS, the expressions of TNFα, IL-6 and IL-10 were higher significantly compared with that before stimulation (P < 0.01), especially at 2-6, 12-18 and 4-12 hours after stimulation, respectively. 4. The gene expressions of TNFα, IL-6 and IL-10 were higher significantly compared with that before stimulation (P < 0.01), especially at 2, 12 and 6 hours after stimulation, respectively. 5. Decoy-ODNs could decrease the expression of TNFα and IL-6 by dose-dependent fashion. There existed statistical inhibiting significance in IL-10 expression and gene expression when only 0.8μg/ml decoy-ODNs was used. And the inhibiting effect increased with time passing by.
Conclusions 1. With carriers of cationic liposomes, Decoy-ODNs in dumbbell shape can be transfected into the pMφ cells from rats and distributed in plasma and nucleus. 2. LPS can activate NF-κB and significantly increased the expressions of TNFα, IL-6 and IL-10 in protein and gene levels. 3. Decoy-ODNs in dumbbell shape can significantly decrease the expressions of TNFαand IL-6 in protein and gene levels, but have weaker inhibiting effect on IL-10.
引文
1. Koch T,et al. Origin and mediators involved in sepsis and the systemic inflammatory response syndrome.Kidney Int,1998,53(Suppl 64):S66-S69.
2. Abraham E,Wunderink R,Silverman H,et al.Efficacy sand safety of monoclonal antibody to human necrosis factor-alpha in patients with sepsis syndrome:a randomized,controlled,doubleblind,multic enter clinical trial.JAMA,1995,273-934.
3. Perenboom R M,Beckers P,Van-Der-Meer J W,et al.Proinflammatory cytokines in lung and blood during steroid-induced pneumocystis carinii pneumonia in rats.J Leukoc Biol,1996,60:710-715.
4. Marik P E,Zaloga G P,et al.Hypothermia and cytokines in septic shock.Norasept study investigators.North American study of the safety and efficacy of murine monoclonal antibody to tumor necrosis factor for the treatment of septic shock.Intensive Care Med,2000,26:716-721.
5. Ohlsson K,Bjork P,Bergenfeldt M,et al.An interleukin-1 receptor antagonist reduce mortality from endotoxin shock.Nature,1990,348-550.
6. Barzilay E,Kessler D,Lesmes C,et al.Sequential plasma filter dialysis with slow,continuous hemofiltration additional treatment for sepsis induced AOSF treatment.J Crit Care,1988,3-163.
7. Sen R,baltimore D, et al.Multiple nuclear factors interact with the
immunoglobulin enhancer sequences.Cell,1986,46(5):705-716.
8. Marumo T,Schini-kerth V B,et al.Vascular endothelial growth factor activates nuclear factor-kappaB and induces monocyte chemoattractant protein-1 in bovine retinal endothelial cells.Diabetes,1999，48(5):1131-1137.
9. Baldwin A S,et al.Series introduction:the transcription factor NF-kappaB and human disease.J Clin Invest,2001,107(1):3-6.
10. Meng L,Mohan R,Kwok B H,et al.Epoxomicin,a potent and selective proteasome inhibitor,exhibits in vivo antiinflammatory activity.Proc Natl Acad Sci USA,1999，96(18):10403-10408.
11. Mattson M P,Camandola S,et al.NF-kappaB in neuronal plasticity and neurodegenerative disorders.J Clin Invest，2001，107(3):247-254.
12. Zandi E,Chen Y,Carin M,et al.Direct phosphorylation of IκB by IκKalpha and IκKbeta:discrimination between free and NF-kappaB-bound substrate. Science,1998，281:1360-1363.
13. May M J,D′ Acquisto F,Madge L A,et al.Selective inhibition of NF-κB activation by a peptide that blocks the interaction of NEMO with the IκB kinase complex.Science,2000,289(5485):1550-1554.
14. Bowie A, O'Neill L.A.J,et al.Oxidative stress and Nuclear factor-κB Activation. Biochemical pharmacology ,2000,59：13-24
15. Epstcin FH,et al. Nuclear factor-k B-A pivotal transcription factor in chronic inflammatory diseases. New Eng J Mrd, 1993;14:436-441
16. Schreck R, Rieber P, Baeuerle PA,et al. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription fact or and HIV-1.EMBO J, 1991;10:2247-2258.
17. Newton R,Adcock I M,barnes P J,et al.Superinduction of NFkappa b by actinomycin D and cycloheximide in epityelial cell.biochem biophys Res Commum,1996,218(2):518-523.
18. Christman J W,Lancaster L H,blackwell T S,et al.Nuclear factorκb:a pivotal role in the systemic inflammatory response syndrome and new target for therapy.Intensive Care Med,1998,24:1131-1138.
19. Miyajima T,Kotake Y.Spin trapping agent,phenyl Ntertbutyl nitrone, inhibits induction of nitric oxide synthase in endotoxininduced shock in mice.biochem biophys Res Commum,1995,215(1):114-121.
20. Epstein F H,et al.Nuclear factorκbA pivotal transcription factor in chronic inflammatory diseases.New eng J Med,1993,14:436-441.
21. Blackwell TS, Christman JW,et al.The role of nuclear factor-kappa B in cytokine gene regulation. Am J Respir Cell Mol Biol, 1997;17:3-9
22. Goebeler M,Gillitzer R,Kilian K,et al.Multiple signaling pathways regulate NF-kappaB-dependent transcription of the monocyte chemoattractant protein-1 gene in primary endothelial cells.Blood，2001，97(1):46-55.
23. Yamamoto Y,Gaynor R B,et al.Therapeutic potential of inhibition of the NF-kappaB pathway in the treatment of inflammation and cancer.J Clin Invest，2001，107(2):135-142.
24. Chung K C，Park J H,Kin C H,et al.Novel biphasic effect of pyrrolidine dithiocarbamate on neuronal cell viability is mediated by the differential regulation of intracellular zinc and copper ion levels,NF-kappaB,and MAP kinases.J Neurosci Res,2000，59(1):117-125.
25. Goebeler M,Gillitzer R,Kilian K,et al.Multiple signaling pathways regulate NF-kappaB-dependent transcription of the monocyte chemoattractant protein-1 gene in primary endothelial cells.Blood，2001，97(1):46-55.
26. Sha W C,Liou H C,Tuomanen EI,et al.Targeted disruption of the p50/p65 subunits of NF-κB leads to multifocal defects in immune responses. Cell,1995,80(2):321-330.
27. Marumo T,Schini-kerth V B,et al.Vascular endothelial growth factor activates nuclear factor-kappaB and induces monocyte chemoattractant protein-1 in bovine retinal endothelial cells.Diabetes,1999,48(5): 1131-1137.
28. Patrick DA etal.The inflammatory profile of interleukin-6, interleukin-8, and soluble intercellular adhesion molecule-1 in postinjury multiple organ failure.Am J Surg.1996;172:425-31
Van Zee KJ,etal. Effects of intravenous IL-8 administration in nonhuman
29. primates.J Immunol 1992;148:1746-52
30. Shiozaki T etal. Low oxygen extraction despite high oxygen delivery causes low oxygen consumption in patients with burns recovering slowly from operative hypothermia. Surgery,1995;118(1);44-48
31. Albert S,et al.The NFkappa b and kappa b proteins:new discoveries and insights.Annu Rev Immunl,1996,14:649-683(Review).
32. Chen ZJ,Parent L,Maniatis T,et al.Sitespecified phosphorylation of Ikappa balpha by a novel biquitinationdependent protein kinase activity.Cell, 1996,84(6):853-862.
33. Ryseck RP, Bull P,et al.A new Rel family transcription activator that can interact with p50-NF-kappa B. Mol Cell Biol, 1992;12:674-684
34. Bogdan,C,and Nathan C.Modulation of macrophage function by transforming growth factor b,interleukin-4 and interleukin-10,Ann.NY Acad.Sci.1993,685:713-739.
35. Collart MA,Baeuerle PA,et al.Vassalli P:Regulation of tumor necrosis factor alpha transcription in macrophager:Involvement of four kB-like motifs and inducible forms of NF-kB.Mol Cell Biol 1990,10:1498-1506.
36. Morishita R,Higaki J,Tomita N,et al.Application of transcription factor "decoy" strategy as means of gene therapy and study of gene expression in cardiovascular disease.Circ Res,1998，82(10):1023-1028.
37. Kupatt C,Habazettl H,Goedecke A,et al.Tumor necrosis factor-alpha contributes to ischemia- and reperfusion-induced endothelial activation in isolated hearts.Circ Res,1999，84(4):392-396.
38. Hosoya T,Takeuchi H,et al.Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides. Federation of European Biochemical Societies.1999,461:136-140.
Lim CS,Fontes JD,et al.Sequence-independent inhibition of RNA transcription by DNA dumbbells and other decoys.Nucleic Acids
39. Research,1997,vol.25.No.3:575-581.
40. Hart SL,Arancibia-Czrcamo CV,et al.Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector[J].Hum Gene Ther,1998, 9(4):575-585.
41. Ropert C,et al.Liposomes as a gene dilivery system[J].Braz J Med Biol Res,1999,32(2):163-169.
42. Blezinger P,Freimark BD,Matar M,et al.Intratracheal administration of interleukin 12 plasmidcationic lipid complexes inhibits murine lung metastases[J].Hum Gene Ther,1999,10(5):723-731.
43. Lee ER,Marshall J,Siegel CS,et al.Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung[J]. Hum Gene Ther,1996,7(14):1701-1717.
44. Gao X,Huang L.Cationic liposome-mediated gene transfer[J].Gene Ther,1995,2(10):710-722.
45. Spector MS,Schnur JM,et al.DNA ordering on a lipid membrane[J]. Science,1997,275(5301):791-792.
46. Nily D,et al.Multilamellar structures of DNA complexes with cationic liposomes[J].Biophys H,1997,73(4):1842-1846.
47. Gershon H,Ghirlando R,Guttman SB,et al.Mode of formation and structural features of DNA-cationic liposome complexes used for transfection[J]. Biochemistry,1993,32(28):7143-7151.
48. Sternberg B,Sorgi FL,Huang L,et al.New structures in complex formation between DNA and cationic liposomes visualized by freeze-fracture electron microscopy[J].FEBS Lett,1994,356(2/3):361-366.
49. Radler JO,Koltover I,SaldittT,et al.Structure of DNA-cationic liposome complexes:DNA intercalation in multilamellar membranes in distinct interhelical packing regimes[J].Science,1997,275(5301):810-814.
Baeuerle,P., and V.R.Baichwal.NF-kB as a frequent target for
50. immunosuppressive and anti-intlammatory molecules.Adv. Immunol. 1997, 65:111-137.
51. Kuhlmann,M.K.,E.Horsch,G.Burkhardt,M.Wagner,and H.Kohler.Reduction of cisplatin toxicity in cultured renal tubular cells by the bioflavonoid quercetiin.Arch.1998,Toxicol.72:536-540.
52. NersekmC.,K.Vogt,C.Platzer,F.Randow,C.Liebenthal,and H.D.Volk.Differential regualtion of monocytic tumour necrosis factor-a and interleukin-10 expression.Eur.J.Immunol.1996,26:1580-1586.
53. Nemeth Z H.Hasko G, et al.Pyrrolidine dithiocarbamate augments IL-10, inhibits TNF-a,MIP-1a,IL-12,and nitric oxide production and protects from the lethal effect of endotoxin. 1998,Shock, vol.10,No.1,pp. 49-53.
54. Bondeson J,Browne KA, et al. Selective Regulation of Cytokine Induction by Adenoviral Gene Transfer of IkBa into Human Macrophager: Lipopolysaccharide-Induced,But Not Zymosan-Induced,Proinflammatory Cytokines Are Inhibited,But IL-10 is Nuclear Factor-kB Independent.The Journal of Immunology,1999,162:2939-2945.
55. Wang P, Wu P, Siegel MI, Egan RW, Billah MM. Interleukin (IL)-10 inhibits nuclear factor kappa B (NF-kappa B) activation in human monocytes. IL-10 and IL-4 suppress cytokine synthesis by different mechanisms. J Biol Chem, 1995;270:9558-9563.
56. Seng Song,Hsiang L H,Roebuck K A,et al.Interleukin10 inhibits interferongammainduced intercellular adhesion molecule1 gene transcription in human monocytes.blood,1997,89(12):4461-4469.
57. J.Barsig,et al.Lipopolysaccharide-induced interleukin-10 in mice:role of endogenous tumor necrosis factor-α.Eur J Immunol.1995,25:2888-2893
Butler,D.M.,R.N.Maini.M.Williams,M.Feidmann.B.M.rennan. Modulation of proinflammatory cytokine release I rheumaoid synovial membrane cell cultures:comparison of monoclonal anti-TNFa annbody with the IL-1
58. receptor antagonist.Eur.Cytokine Nerwork.1995,6:225.
59. Brennan.F.M.D.Chantry,A.Jackson.R.Maini,and M.Feldmann. Inhibitory effect of TNF a antibodies on synovial cell interieukin-1 production in rheumatoid arthritis.Lancet.1989,2:244.
60. Haworth.C.F.M.Brennan,D.Chantry.M.Tumer.R.N.Maini,and M.Feldmann. Expression of granulocyte-macyophage colony-stimulating factor in rheumaroid arthritis:regulation by tumor necrosis ractor a.Eur. J.Immunol. 1991,21:2575.
61. Platzer C., C.Meisel.K.Vogt,Mplarzer.and H.D.Volk. Up-reguiation of monocvtic IL-10 by tumor necrosis ractor-a and cAMP cievating drugs.Int.lmmunol.1995,7:517.
62. Foey A D,.Parry S L,.Williams L M,.Feidmann M..Foxwell B.N.J,and Brennan F N. Regulation of monocyte IL-10production by endogenous IL-1 and TNF:role of the p38 and p42/44MAP kinases. J.bounol. 1998,160:920.
63. Parry.S.L.N.Sebbag,F.M.Brennan.and Feldmann M. Contact with T cells modulates monocyte IL-10 production by TNFα-dependent mechanisms. J.Immunol. 1997,158:3673.
64. Katsikis.P.C.Q.Chu.F.M.Brennan.R.N.Maini.and M.Feldmann. Immunoregulatory role of interieukin 10(IL-10)in rheumarord arthritis.J.Exp.Med.1994,179:1517.
2. Abraham E,Wunderink R,Silverman H,et al.Efficacy sand safety of monoclonal antibody to human necrosis factor-alpha in patients with sepsis syndrome:a randomized,controlled,doubleblind,multic enter clinical trial.JAMA,1995,273-934.
3. Perenboom R M,Beckers P,Van-Der-Meer J W,et al.Proinflammatory cytokines in lung and blood during steroid-induced pneumocystis carinii pneumonia in rats.J Leukoc Biol,1996,60:710-715.
4. Marik P E,Zaloga G P,et al.Hypothermia and cytokines in septic shock.Norasept study investigators.North American study of the safety and efficacy of murine monoclonal antibody to tumor necrosis factor for the treatment of septic shock.Intensive Care Med,2000,26:716-721.
5. Ohlsson K,Bjork P,Bergenfeldt M,et al.An interleukin-1 receptor antagonist reduce mortality from endotoxin shock.Nature,1990,348-550.
6. Barzilay E,Kessler D,Lesmes C,et al.Sequential plasma filter dialysis with slow,continuous hemofiltration additional treatment for sepsis induced AOSF treatment.J Crit Care,1988,3-163.
7. Sen R,baltimore D, et al.Multiple nuclear factors interact with the
immunoglobulin enhancer sequences.Cell,1986,46(5):705-716.
8. Marumo T,Schini-kerth V B,et al.Vascular endothelial growth factor activates nuclear factor-kappaB and induces monocyte chemoattractant protein-1 in bovine retinal endothelial cells.Diabetes,1999，48(5):1131-1137.
9. Baldwin A S,et al.Series introduction:the transcription factor NF-kappaB and human disease.J Clin Invest,2001,107(1):3-6.
10. Meng L,Mohan R,Kwok B H,et al.Epoxomicin,a potent and selective proteasome inhibitor,exhibits in vivo antiinflammatory activity.Proc Natl Acad Sci USA,1999，96(18):10403-10408.
11. Mattson M P,Camandola S,et al.NF-kappaB in neuronal plasticity and neurodegenerative disorders.J Clin Invest，2001，107(3):247-254.
12. Zandi E,Chen Y,Carin M,et al.Direct phosphorylation of IκB by IκKalpha and IκKbeta:discrimination between free and NF-kappaB-bound substrate. Science,1998，281:1360-1363.
13. May M J,D′ Acquisto F,Madge L A,et al.Selective inhibition of NF-κB activation by a peptide that blocks the interaction of NEMO with the IκB kinase complex.Science,2000,289(5485):1550-1554.
14. Bowie A, O'Neill L.A.J,et al.Oxidative stress and Nuclear factor-κB Activation. Biochemical pharmacology ,2000,59：13-24
15. Epstcin FH,et al. Nuclear factor-k B-A pivotal transcription factor in chronic inflammatory diseases. New Eng J Mrd, 1993;14:436-441
16. Schreck R, Rieber P, Baeuerle PA,et al. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription fact or and HIV-1.EMBO J, 1991;10:2247-2258.
17. Newton R,Adcock I M,barnes P J,et al.Superinduction of NFkappa b by actinomycin D and cycloheximide in epityelial cell.biochem biophys Res Commum,1996,218(2):518-523.
18. Christman J W,Lancaster L H,blackwell T S,et al.Nuclear factorκb:a pivotal role in the systemic inflammatory response syndrome and new target for therapy.Intensive Care Med,1998,24:1131-1138.
19. Miyajima T,Kotake Y.Spin trapping agent,phenyl Ntertbutyl nitrone, inhibits induction of nitric oxide synthase in endotoxininduced shock in mice.biochem biophys Res Commum,1995,215(1):114-121.
20. Epstein F H,et al.Nuclear factorκbA pivotal transcription factor in chronic inflammatory diseases.New eng J Med,1993,14:436-441.
21. Blackwell TS, Christman JW,et al.The role of nuclear factor-kappa B in cytokine gene regulation. Am J Respir Cell Mol Biol, 1997;17:3-9
22. Goebeler M,Gillitzer R,Kilian K,et al.Multiple signaling pathways regulate NF-kappaB-dependent transcription of the monocyte chemoattractant protein-1 gene in primary endothelial cells.Blood，2001，97(1):46-55.
23. Yamamoto Y,Gaynor R B,et al.Therapeutic potential of inhibition of the NF-kappaB pathway in the treatment of inflammation and cancer.J Clin Invest，2001，107(2):135-142.
24. Chung K C，Park J H,Kin C H,et al.Novel biphasic effect of pyrrolidine dithiocarbamate on neuronal cell viability is mediated by the differential regulation of intracellular zinc and copper ion levels,NF-kappaB,and MAP kinases.J Neurosci Res,2000，59(1):117-125.
25. Goebeler M,Gillitzer R,Kilian K,et al.Multiple signaling pathways regulate NF-kappaB-dependent transcription of the monocyte chemoattractant protein-1 gene in primary endothelial cells.Blood，2001，97(1):46-55.
26. Sha W C,Liou H C,Tuomanen EI,et al.Targeted disruption of the p50/p65 subunits of NF-κB leads to multifocal defects in immune responses. Cell,1995,80(2):321-330.
27. Marumo T,Schini-kerth V B,et al.Vascular endothelial growth factor activates nuclear factor-kappaB and induces monocyte chemoattractant protein-1 in bovine retinal endothelial cells.Diabetes,1999,48(5): 1131-1137.
28. Patrick DA etal.The inflammatory profile of interleukin-6, interleukin-8, and soluble intercellular adhesion molecule-1 in postinjury multiple organ failure.Am J Surg.1996;172:425-31
Van Zee KJ,etal. Effects of intravenous IL-8 administration in nonhuman
29. primates.J Immunol 1992;148:1746-52
30. Shiozaki T etal. Low oxygen extraction despite high oxygen delivery causes low oxygen consumption in patients with burns recovering slowly from operative hypothermia. Surgery,1995;118(1);44-48
31. Albert S,et al.The NFkappa b and kappa b proteins:new discoveries and insights.Annu Rev Immunl,1996,14:649-683(Review).
32. Chen ZJ,Parent L,Maniatis T,et al.Sitespecified phosphorylation of Ikappa balpha by a novel biquitinationdependent protein kinase activity.Cell, 1996,84(6):853-862.
33. Ryseck RP, Bull P,et al.A new Rel family transcription activator that can interact with p50-NF-kappa B. Mol Cell Biol, 1992;12:674-684
34. Bogdan,C,and Nathan C.Modulation of macrophage function by transforming growth factor b,interleukin-4 and interleukin-10,Ann.NY Acad.Sci.1993,685:713-739.
35. Collart MA,Baeuerle PA,et al.Vassalli P:Regulation of tumor necrosis factor alpha transcription in macrophager:Involvement of four kB-like motifs and inducible forms of NF-kB.Mol Cell Biol 1990,10:1498-1506.
36. Morishita R,Higaki J,Tomita N,et al.Application of transcription factor "decoy" strategy as means of gene therapy and study of gene expression in cardiovascular disease.Circ Res,1998，82(10):1023-1028.
37. Kupatt C,Habazettl H,Goedecke A,et al.Tumor necrosis factor-alpha contributes to ischemia- and reperfusion-induced endothelial activation in isolated hearts.Circ Res,1999，84(4):392-396.
38. Hosoya T,Takeuchi H,et al.Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides. Federation of European Biochemical Societies.1999,461:136-140.
Lim CS,Fontes JD,et al.Sequence-independent inhibition of RNA transcription by DNA dumbbells and other decoys.Nucleic Acids
39. Research,1997,vol.25.No.3:575-581.
40. Hart SL,Arancibia-Czrcamo CV,et al.Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector[J].Hum Gene Ther,1998, 9(4):575-585.
41. Ropert C,et al.Liposomes as a gene dilivery system[J].Braz J Med Biol Res,1999,32(2):163-169.
42. Blezinger P,Freimark BD,Matar M,et al.Intratracheal administration of interleukin 12 plasmidcationic lipid complexes inhibits murine lung metastases[J].Hum Gene Ther,1999,10(5):723-731.
43. Lee ER,Marshall J,Siegel CS,et al.Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung[J]. Hum Gene Ther,1996,7(14):1701-1717.
44. Gao X,Huang L.Cationic liposome-mediated gene transfer[J].Gene Ther,1995,2(10):710-722.
45. Spector MS,Schnur JM,et al.DNA ordering on a lipid membrane[J]. Science,1997,275(5301):791-792.
46. Nily D,et al.Multilamellar structures of DNA complexes with cationic liposomes[J].Biophys H,1997,73(4):1842-1846.
47. Gershon H,Ghirlando R,Guttman SB,et al.Mode of formation and structural features of DNA-cationic liposome complexes used for transfection[J]. Biochemistry,1993,32(28):7143-7151.
48. Sternberg B,Sorgi FL,Huang L,et al.New structures in complex formation between DNA and cationic liposomes visualized by freeze-fracture electron microscopy[J].FEBS Lett,1994,356(2/3):361-366.
49. Radler JO,Koltover I,SaldittT,et al.Structure of DNA-cationic liposome complexes:DNA intercalation in multilamellar membranes in distinct interhelical packing regimes[J].Science,1997,275(5301):810-814.
Baeuerle,P., and V.R.Baichwal.NF-kB as a frequent target for
50. immunosuppressive and anti-intlammatory molecules.Adv. Immunol. 1997, 65:111-137.
51. Kuhlmann,M.K.,E.Horsch,G.Burkhardt,M.Wagner,and H.Kohler.Reduction of cisplatin toxicity in cultured renal tubular cells by the bioflavonoid quercetiin.Arch.1998,Toxicol.72:536-540.
52. NersekmC.,K.Vogt,C.Platzer,F.Randow,C.Liebenthal,and H.D.Volk.Differential regualtion of monocytic tumour necrosis factor-a and interleukin-10 expression.Eur.J.Immunol.1996,26:1580-1586.
53. Nemeth Z H.Hasko G, et al.Pyrrolidine dithiocarbamate augments IL-10, inhibits TNF-a,MIP-1a,IL-12,and nitric oxide production and protects from the lethal effect of endotoxin. 1998,Shock, vol.10,No.1,pp. 49-53.
54. Bondeson J,Browne KA, et al. Selective Regulation of Cytokine Induction by Adenoviral Gene Transfer of IkBa into Human Macrophager: Lipopolysaccharide-Induced,But Not Zymosan-Induced,Proinflammatory Cytokines Are Inhibited,But IL-10 is Nuclear Factor-kB Independent.The Journal of Immunology,1999,162:2939-2945.
55. Wang P, Wu P, Siegel MI, Egan RW, Billah MM. Interleukin (IL)-10 inhibits nuclear factor kappa B (NF-kappa B) activation in human monocytes. IL-10 and IL-4 suppress cytokine synthesis by different mechanisms. J Biol Chem, 1995;270:9558-9563.
56. Seng Song,Hsiang L H,Roebuck K A,et al.Interleukin10 inhibits interferongammainduced intercellular adhesion molecule1 gene transcription in human monocytes.blood,1997,89(12):4461-4469.
57. J.Barsig,et al.Lipopolysaccharide-induced interleukin-10 in mice:role of endogenous tumor necrosis factor-α.Eur J Immunol.1995,25:2888-2893
Butler,D.M.,R.N.Maini.M.Williams,M.Feidmann.B.M.rennan. Modulation of proinflammatory cytokine release I rheumaoid synovial membrane cell cultures:comparison of monoclonal anti-TNFa annbody with the IL-1
58. receptor antagonist.Eur.Cytokine Nerwork.1995,6:225.
59. Brennan.F.M.D.Chantry,A.Jackson.R.Maini,and M.Feldmann. Inhibitory effect of TNF a antibodies on synovial cell interieukin-1 production in rheumatoid arthritis.Lancet.1989,2:244.
60. Haworth.C.F.M.Brennan,D.Chantry.M.Tumer.R.N.Maini,and M.Feldmann. Expression of granulocyte-macyophage colony-stimulating factor in rheumaroid arthritis:regulation by tumor necrosis ractor a.Eur. J.Immunol. 1991,21:2575.
61. Platzer C., C.Meisel.K.Vogt,Mplarzer.and H.D.Volk. Up-reguiation of monocvtic IL-10 by tumor necrosis ractor-a and cAMP cievating drugs.Int.lmmunol.1995,7:517.
62. Foey A D,.Parry S L,.Williams L M,.Feidmann M..Foxwell B.N.J,and Brennan F N. Regulation of monocyte IL-10production by endogenous IL-1 and TNF:role of the p38 and p42/44MAP kinases. J.bounol. 1998,160:920.
63. Parry.S.L.N.Sebbag,F.M.Brennan.and Feldmann M. Contact with T cells modulates monocyte IL-10 production by TNFα-dependent mechanisms. J.Immunol. 1997,158:3673.
64. Katsikis.P.C.Q.Chu.F.M.Brennan.R.N.Maini.and M.Feldmann. Immunoregulatory role of interieukin 10(IL-10)in rheumarord arthritis.J.Exp.Med.1994,179:1517.