应用RNA干扰技术对哮喘小鼠白介素15表达影响的研究
摘要
本实验的目的是探讨IL-15在哮喘中的作用以及干预IL-15后对哮喘的影响。首先利用Ambion软件根据siRNA的设计原则,设计并合成21nt碱基序列IL-15-siRNA,利用pSIREN-RetroQ-ZsGreen构建合成了重组质粒pSIREN-RetroQ-ZsGreen-IL-15-siRNA(以下简称为pSRZsi-IL-15),应用阳离子脂质体Lipofectamine 2000做为转染载体,特异性的阻断哮喘小鼠模型肺组织中IL-15 mRNA的表达水平,采用逆转录聚合酶链反应、酶联免疫反应、共聚焦显微镜、组织形态学和免疫组织化学等技术;研究了pSRZsi-IL-15-Lip对体外T细胞中的基因沉默效应和特异性;进而根据体外转染的数据,进行呼吸道体内转染pSRZsi-IL-15-Lip;对哮喘模型肺部炎症和支气管肺泡灌洗液中嗜酸粒细胞、中性粒细胞计数、血清中Th1/Th2型细胞因子以及IL-15、IL-4mRNA表达水平的影响进行了研究。本研究证实:在哮喘小鼠T细胞的体外转染中,p SRZsi-IL-15与Lipofectamine2000达到最佳基因沉默效果比例为1:2,此种比例的pSRZsi-IL-15-Lip特异性地抑制哮喘小鼠T细胞IL-15mRNA的表达,达到了特异性基因沉默效应。基于此种比例我们成功地进行了pSRZsi-IL-15-Lip在哮喘小鼠模型呼吸道转染;pSRZsi-IL-15可有效地抑制肺组织IL-15mRNA的表达,减轻肺部Eos为主的炎性;可以部分抑制IL-4 mRNA的表达以及血清IgE水平,一定程度上减轻肺部炎症;哮喘模型肺部中性粒细胞的迁移和浸润部分依赖IL-15的调节,而Eos的迁移、浸润与IL-15关系密切,二者成正相关; pSRZsi-IL-15- Lip减轻气道炎症;对气道炎症的影响可能是通过促进嗜酸性粒细胞凋亡而发挥作用的。在pSRZsi-IL-15进行呼吸道体内转染过程中,其发挥效果高峰时间可能为转染后48-72小时;证实呼吸道转染pSRZsi-IL-15可能成为控制哮喘肺部炎症的新途径。
IL15comes from various tissues. Most of tissues and cells could expressIL15,and monocaryon/macrophage is main resource. The main functional ofmonocaryon/macrophage is that could present the antigenic message to T cell,and promoted it secreted cytokines and mediator of inflammation precurosor.Well, IL15could induce T cell expressed cykines and theirs receptor,participate inflammation which make T cell activated and generation earlier,promote B cell secreted IG, enhanced NK cell cytotoxity and producedcytokines, modulated monocaryon/macrophage produce anteroinflammationcytokine. It is cleared that IL15could induce neutrophil expressed NFκB,and inhibited apoptosis of neutrophil and leukomonocyte. During inflammation,there had a certain degree neutrophilic leukocytosis, and released leukotriene,prostaglandin and platelet activating factor, participated moludatedinflammation of respiratory passage, but It is not identify the relationshipbetween IL15and asthma which is still on the stage of exploration.
RNAi is a new gene blockage technology by introducted doublestrandedRNA which is high performance, definite, satefy and operated. It could highefficiency and specific to block the special gene expression, promotedegradation of target of mRNA, and lure the absence of expression of the definite gene, got the aim of post transcriptional gene silencing. We utilizedRNA interference to research gene function, signal transduction, relationship ofeach other and antivirus, antineoplaston. Although it was applicated in multudefields and clinical research, it is still on the stage of vitro cell line research inrespiratory system disease.
Our aim is to discuse the effect of IL15on asthma and the influence afterinterfered it. In our experiment we design 21nt base sequence according toprinciple of design of RNAi. To utilize pSIRENRetroQZsGreenconstructionrecombinant plasmid of pSIRENRetroQZsGreenIL15siRNA（pSRZsiIL15）which can block the expression of IL15mRNA in the lung.,and discuss the effection of IL15siRNAin the lung tissue on an asthmamouse model.
Method
1. To establish the sequence of IL15siRNAthat is only one, amply andsequence identity which has synthetical expression plasmid pSRZsiIL15.
2. To establish an asthma mouse model in accordance with reference.Oberserve the change of ethology on mouse, to determine the level ofIL4,INFγand IgE in sera, the change of eosinophils and neutrophil cellcounts in BALF, and the change of histopathology.
3. Isolated culture spleen T lymphocyte of asthma mouse, and delivereddifference ration of pSRZsiIL15and Lipofectamine 2000 to T lymphocyte.We collected the cells after 72 hours, observed the results of transfected of Tlymphocyte by fluorescent microscope, adopted RTPCRto test the expressionof IL4mRNAand IL15mRNA,used ELISA to determine the change ofcytokines in lymphocyte culture supernatant such as IL4and IFNγ,compared the effects of different ration of pSRZsiIL15and Lipofectamine 2000.
4. Based on the asthma mouse model, the mice were divided into normalcontrol group, asthma control group, hormonal therepy group, vetor group andRNAi group. To establish an asthma mouse model in accordance withreference. Among those: 3days before sensitized and 1 day before stimulatedwas delivered the ration of pSIRENRetroQZsGreenIL15siRNAandLipofectamine 2000 1/2. Killed the mice after transfected 24,48 and 72 hourspartly. At meantime, randomly get a part of fresh lung tissue and made frozensection to observe the respiratory transfected by confocal microscopy andIL15siRNAeffected on correlation factors in lung tissue. We determined thelevel of IL4,IL15,IFNγ,IgE in each group, detected the expression ofIL4mRNAand IL15mRNAby RTPCR,determined the expression of IL15in lung tissue by immunohistochemical method, observed the changes ofhistopathology in lung tissue and change of eosinophils and neutrophil cellscounts in BALF.
Results
1.Identity construced the pSRZsiIL15was our need by amplification andsequencing.
2. We observed the ethology change of mouse, the level of IL4on asthmamouse model was higer than that of normal control group, the level of INFγwas lower than that of normal control group, at the same time, the level of IgEalso increased obviously, the eosinophils cell counts in BALF higher comparedto normal control group. Change of lung histopathology was typical that loss ofbronchial mucosa epithelium and was hurt. Eosinophils, leukomonocyte andlymphomonocyte infiltrated, mucous formed. That established an mouse model successfully.
3. Interfered on T cell on asthma mouse model in vitro
①After 48 hours observed that there had some green fluorescence proteinon asthma mouse T cell that indicate the succed in transfecting.
②After transfected to T cell, the expression of IL15is significant lowerin RNAi group than that of in asthma control group. It suggested thatpSRZsiIL15was transfected into T cell successfully, and specific blocked thehe expression of IL15got the gene silencing.
③The level of IL4was lower in RNAi group than that of asthma controlgroup in lymphocyte culture supernatant, so does in the expression of IL4mRNA. In lymphocyte culture supernatant, no change on level of IFNγof Th1cytokine which indicated that IL15has no regulated or micro modulated.
④It could get the best specificity gene silence when the ration ofpSRZsiIL15andLipofectamine 2000 is 1:2 which had advangtage of theretion of 1/1 and 1/3.
4. pSRZsiIL15Lipinterfered on asthma mouse model in vivo afterrespiratory transfection.
①After transfected 24 hours, we could observed that there has some greenfluorescent material on bronchial mucosa epithelialis and around lung tissueunder the confocal microscopy. And that suggested that respiratory transfectedsuccesfuly.
②In asthma group eosinophils and neutrophils cell counts, as well as thelevel of IL15was significant heighten. After delivered pSRZsiIL15,it coulddescend eosinophils and neutrophils cell counts in RNAi group, especially aftertransfected 4872hours which could get a stable level, and the level of IL15is positive correlation with eosinophils and neutrophils cell counts.
③The level of IgE in RNAi group is lower than asthma control group. Atmeantime, that of 48 hours and 72 hours transgected was significant lower than24 hours transfected. In the experiment the change of 48 hours transfected wassimilar to that of 72 hours, that was pSRZsiIL15Lipeffected on the level ofIgE got a stable level after respiratory transfected in vivo.
④There is no change on levels of INFγin sera in RNAi group. level ofIL4in RNAi group is lower than asthma control group. At meantime, that of48 hours and 72 hours transgected was significant lower than 24 hourstransfected. In the experiment the change of 48 hours transfected was similar tothat 72 hours, that was pSRZsiIL15Lipeffected on the level of IL4got astable level after respiratory transfected in vivo.
⑤Level of IL15in RNAi group is lower than asthma control group. Atmeantime, that of 48 hours and 72 hours transfected was significant lower than24 hours transfected. In the experiment the change of 48 hours transfected wassimilar to that 72 hours, that was pSRZsiIL15Lipeffected on IL15got astable gene silencing level after respiratory transfected in vivo.
⑥The positive staining cell counts was lower than asthma control group,The positive staining cell counts was similar to normal control group aftertransfected 48 hours and 72 hours. pSRZsiIL15Lipcould lessen eosinophilsleak out in BALF and inflammation cell infiltrating in lung tissue.
⑦pSRZsiIL15Lipcould cut down the expression of IL4mRNAin lungtissue after airway drop in it. But there was no difference in every time intervalthat paralleled to the change of sera. At meantime, pSRZsiIL15Lipcould cutdown the expression of IL15mRNAin lung tissue especially after transfected 48 hours and 72 hours. That could got a stable the gene silencing level aftertranfected 4872hours.
Conclusion
1 Succeeded in preparation in asthma mouse model by OVA sensitizedand stimulated.
2 Succeeded in identification on amplification and sequencing ofpSRZsiIL15.
3 It could get the best specificity gene silence when the ration ofpSRZsiIL15andLipofectamine 2000 is 1:2
4 During the course of asthma IL15main effects is promoted theflammation.
5 After respiratory transfection, pSRZsiIL15Lipcould get a stable thegene silencing level after tranfected 4872hours.
6 pSRZsiIL15Lipcould lessen eosinophils inflammation and correctthe disbalance of Th1/Th2 on asthma mouse in lung tissue.
7 Thus airway inhaled pSRZsiIL15Lipcould be one of effectivemethods to cure asthma.
IL15comes from various tissues. Most of tissues and cells could expressIL15,and monocaryon/macrophage is main resource. The main functional ofmonocaryon/macrophage is that could present the antigenic message to T cell,and promoted it secreted cytokines and mediator of inflammation precurosor.Well, IL15could induce T cell expressed cykines and theirs receptor,participate inflammation which make T cell activated and generation earlier,promote B cell secreted IG, enhanced NK cell cytotoxity and producedcytokines, modulated monocaryon/macrophage produce anteroinflammationcytokine. It is cleared that IL15could induce neutrophil expressed NFκB,and inhibited apoptosis of neutrophil and leukomonocyte. During inflammation,there had a certain degree neutrophilic leukocytosis, and released leukotriene,prostaglandin and platelet activating factor, participated moludatedinflammation of respiratory passage, but It is not identify the relationshipbetween IL15and asthma which is still on the stage of exploration.
RNAi is a new gene blockage technology by introducted doublestrandedRNA which is high performance, definite, satefy and operated. It could highefficiency and specific to block the special gene expression, promotedegradation of target of mRNA, and lure the absence of expression of the definite gene, got the aim of post transcriptional gene silencing. We utilizedRNA interference to research gene function, signal transduction, relationship ofeach other and antivirus, antineoplaston. Although it was applicated in multudefields and clinical research, it is still on the stage of vitro cell line research inrespiratory system disease.
Our aim is to discuse the effect of IL15on asthma and the influence afterinterfered it. In our experiment we design 21nt base sequence according toprinciple of design of RNAi. To utilize pSIRENRetroQZsGreenconstructionrecombinant plasmid of pSIRENRetroQZsGreenIL15siRNA（pSRZsiIL15）which can block the expression of IL15mRNA in the lung.,and discuss the effection of IL15siRNAin the lung tissue on an asthmamouse model.
Method
1. To establish the sequence of IL15siRNAthat is only one, amply andsequence identity which has synthetical expression plasmid pSRZsiIL15.
2. To establish an asthma mouse model in accordance with reference.Oberserve the change of ethology on mouse, to determine the level ofIL4,INFγand IgE in sera, the change of eosinophils and neutrophil cellcounts in BALF, and the change of histopathology.
3. Isolated culture spleen T lymphocyte of asthma mouse, and delivereddifference ration of pSRZsiIL15and Lipofectamine 2000 to T lymphocyte.We collected the cells after 72 hours, observed the results of transfected of Tlymphocyte by fluorescent microscope, adopted RTPCRto test the expressionof IL4mRNAand IL15mRNA,used ELISA to determine the change ofcytokines in lymphocyte culture supernatant such as IL4and IFNγ,compared the effects of different ration of pSRZsiIL15and Lipofectamine 2000.
4. Based on the asthma mouse model, the mice were divided into normalcontrol group, asthma control group, hormonal therepy group, vetor group andRNAi group. To establish an asthma mouse model in accordance withreference. Among those: 3days before sensitized and 1 day before stimulatedwas delivered the ration of pSIRENRetroQZsGreenIL15siRNAandLipofectamine 2000 1/2. Killed the mice after transfected 24,48 and 72 hourspartly. At meantime, randomly get a part of fresh lung tissue and made frozensection to observe the respiratory transfected by confocal microscopy andIL15siRNAeffected on correlation factors in lung tissue. We determined thelevel of IL4,IL15,IFNγ,IgE in each group, detected the expression ofIL4mRNAand IL15mRNAby RTPCR,determined the expression of IL15in lung tissue by immunohistochemical method, observed the changes ofhistopathology in lung tissue and change of eosinophils and neutrophil cellscounts in BALF.
Results
1.Identity construced the pSRZsiIL15was our need by amplification andsequencing.
2. We observed the ethology change of mouse, the level of IL4on asthmamouse model was higer than that of normal control group, the level of INFγwas lower than that of normal control group, at the same time, the level of IgEalso increased obviously, the eosinophils cell counts in BALF higher comparedto normal control group. Change of lung histopathology was typical that loss ofbronchial mucosa epithelium and was hurt. Eosinophils, leukomonocyte andlymphomonocyte infiltrated, mucous formed. That established an mouse model successfully.
3. Interfered on T cell on asthma mouse model in vitro
①After 48 hours observed that there had some green fluorescence proteinon asthma mouse T cell that indicate the succed in transfecting.
②After transfected to T cell, the expression of IL15is significant lowerin RNAi group than that of in asthma control group. It suggested thatpSRZsiIL15was transfected into T cell successfully, and specific blocked thehe expression of IL15got the gene silencing.
③The level of IL4was lower in RNAi group than that of asthma controlgroup in lymphocyte culture supernatant, so does in the expression of IL4mRNA. In lymphocyte culture supernatant, no change on level of IFNγof Th1cytokine which indicated that IL15has no regulated or micro modulated.
④It could get the best specificity gene silence when the ration ofpSRZsiIL15andLipofectamine 2000 is 1:2 which had advangtage of theretion of 1/1 and 1/3.
4. pSRZsiIL15Lipinterfered on asthma mouse model in vivo afterrespiratory transfection.
①After transfected 24 hours, we could observed that there has some greenfluorescent material on bronchial mucosa epithelialis and around lung tissueunder the confocal microscopy. And that suggested that respiratory transfectedsuccesfuly.
②In asthma group eosinophils and neutrophils cell counts, as well as thelevel of IL15was significant heighten. After delivered pSRZsiIL15,it coulddescend eosinophils and neutrophils cell counts in RNAi group, especially aftertransfected 4872hours which could get a stable level, and the level of IL15is positive correlation with eosinophils and neutrophils cell counts.
③The level of IgE in RNAi group is lower than asthma control group. Atmeantime, that of 48 hours and 72 hours transgected was significant lower than24 hours transfected. In the experiment the change of 48 hours transfected wassimilar to that of 72 hours, that was pSRZsiIL15Lipeffected on the level ofIgE got a stable level after respiratory transfected in vivo.
④There is no change on levels of INFγin sera in RNAi group. level ofIL4in RNAi group is lower than asthma control group. At meantime, that of48 hours and 72 hours transgected was significant lower than 24 hourstransfected. In the experiment the change of 48 hours transfected was similar tothat 72 hours, that was pSRZsiIL15Lipeffected on the level of IL4got astable level after respiratory transfected in vivo.
⑤Level of IL15in RNAi group is lower than asthma control group. Atmeantime, that of 48 hours and 72 hours transfected was significant lower than24 hours transfected. In the experiment the change of 48 hours transfected wassimilar to that 72 hours, that was pSRZsiIL15Lipeffected on IL15got astable gene silencing level after respiratory transfected in vivo.
⑥The positive staining cell counts was lower than asthma control group,The positive staining cell counts was similar to normal control group aftertransfected 48 hours and 72 hours. pSRZsiIL15Lipcould lessen eosinophilsleak out in BALF and inflammation cell infiltrating in lung tissue.
⑦pSRZsiIL15Lipcould cut down the expression of IL4mRNAin lungtissue after airway drop in it. But there was no difference in every time intervalthat paralleled to the change of sera. At meantime, pSRZsiIL15Lipcould cutdown the expression of IL15mRNAin lung tissue especially after transfected 48 hours and 72 hours. That could got a stable the gene silencing level aftertranfected 4872hours.
Conclusion
1 Succeeded in preparation in asthma mouse model by OVA sensitizedand stimulated.
2 Succeeded in identification on amplification and sequencing ofpSRZsiIL15.
3 It could get the best specificity gene silence when the ration ofpSRZsiIL15andLipofectamine 2000 is 1:2
4 During the course of asthma IL15main effects is promoted theflammation.
5 After respiratory transfection, pSRZsiIL15Lipcould get a stable thegene silencing level after tranfected 4872hours.
6 pSRZsiIL15Lipcould lessen eosinophils inflammation and correctthe disbalance of Th1/Th2 on asthma mouse in lung tissue.
7 Thus airway inhaled pSRZsiIL15Lipcould be one of effectivemethods to cure asthma.
引文
1. Grabatein KH, Eisenman K, Shanebeck K, et al. Cloning of T cell growthfactor that interacts with the beta chain of the interleukin2receptor.Science, 1994, 264: 965-968
2. Musso T, Calosso L, Zucca M, et al. Human monocytes constitutivelyexpress membranebound,biologically active, and interferongammaupregulatedinterleukin15.Blood, 1999, 93: 3531-3539
3. Burton JD, Bamford RN, Peters C, et al. A lymphokine, provisionallydesignated interleukin T and produced by a human adult Tcellleukemialine, stimulates Tcellproliferation and the induction of lymphokineactivatedkiller cells. Proc Natl Acad Sci USA,1994,91:4935-4939
4. Krause H, Jandrig B, Wernicke C, et al. Genomic structure and chromosomallocalization of the human interleukin 15 gene (IL15).Cytokine,1996,8:667-674
5. 高巍综述，韩守信审校.白介素15与支气管哮喘.国外医学呼吸系统分册,2004，24（1）：15-17
6. Perera LP, Goldman CK, Waldmann TA. IL15induces the expression ofchemokines and their receptors in T lymphocytes .J Immunol,1999,162:2606-2612
7. Liu K, Catalfamo M, Li Y, et al. IL15mimics T cell receptor cross linkingin the induction of cellular proliferation, gene expression, and cytotoxicityin CD+8memory T cells. Proc Natl Acad Sci USA,2002,99:6192-6197
8. Fehniger TA, Shah MH, Turner MJ, et al. Differential cytokine geneexpression by human NK cell following activation with IL8or IL15incombination with IL12:implications for the innate immune response.J Immunol,1999,162(8):4511-4520
9. Neely GG, Robbins SM, Amankwah EK, et al. Lipopolysaccharidestimulated or granulocytemacrophagecolonystimulatingfactorstimulatedmonocytes rapidly express biologically active IL15on their cell surfaceindependent of new protein synthesis. J Immunol, 2001,167:5011-5017
10. Alleva DG, Kaser SB, Monroy MA, et al. IL15functions as a potentautocrine regulator of macrophage proinflammatory cytokine production:evidence for differential receptor subunit utilization associated withstimulation or inhibition. J Immunol, 1997, 159:2941-2951
11. Avice MN, Demeure CE, Delespesse G, et al.IL15promotes IL12production by human monocytes via T celldependentcontact and maycontribute to IL12mediatedIFNgammasecretion by CD4+ T cell in theabcence of TCR ligation.J Immunol,1998;161:3408-3415
12. Hoontrakoon R, Chu HW, Gardai SJ, et al. Interlukin15inhibitsspontaneous apoptosis in human eosinophils via autocrine production ofgranulocyte macrophage colonystimulating factor and nuclear factorkBactivation. Am J Respir Cell Mol Biol, 2002, 26: 404-412
13. 高巍，韩守信，陈复辉，等.支气管哮喘患者外周血 IL15的测定及意义.哈尔滨医科大学学报,2004,38（1）: 70-72
14. Pelletier M, Ratthe C, Girard D. Mechanisms involved ininterleukin15inducedsuppression of human neutrophil apoptosis:role ofthe antiapoptoticMcl1protein and several kinases including Januskinase2,p38 mitogenactivatedprotein kinase and extracellular signalregulatedkinases1/2.FEBS Lett,2002,532:164-170
15. McDonald PP, Russo MP, Ferrini S,et al.Interleukin15(IL15)inducesNFkappaB activation and IL8production in human neutrophils.Blood,1998, 92:4828-4835
16. Girard D, Paquet ME, Paquin R, et al. Differential effects of interleukin15(IL15)and IL2on human neutrophils: modulation of phagocytosis,cytoskeleton rearrangement, gene expression, and apoptosis by IL15[J].Blood,1996,88(8):3176-3184.
17. Lai YG, Gelfanov V, Gelfanova V, et al. IL15promotes survival but noteffector function differentiation of CD8+ TCR alphabeta+ intestinalintraepithelial lymphocytes[J].J Immunol,1999,163 (11):5843-5850.
18. KomaiKomaM, McKay A, Thomson L, et al. Immunoregulatorycytokines in asthma:IL15and IL13in induced sputum. Clin ExpAllergy,2001, 31:1441-1448
19. Grunig G, Warnock M, Wokil AE, et al. Requirement for IL13independently of IL4in experimental asthma. Science, 1998, 28（2）:2261-2263
20. Ishimitsu R, Nishimura H, Yajima T, et al. Overexpression of IL15in vivoenhances Tc1 response, which inhibits allergic inflammation in a murinemodel of asthma. J Immunol,2001,166:1991-2001
21. Rene′ Ru¨ckert, Katja Brandt, Armin Braun, et al. Blocking IL15Preventsthe Induction of AllergenSpecificT Cells and Allergic Inflammation InVivo. The Journal of Immunology, 2005, 174: 5507–5515.
22. Hartmann P, Plum G. Immunological defense mechanisms in tuberculosisand MACinfection.Diagn Microbiol Infect Dis, 1999,34(2):147-152.
23. Turner J, Orme IM. The expression of early resistance to an infection withMycobacterium tuberculosis by old mice is depend on IFN typeⅡ(IFNGamma)but not IFN typeⅠ. Mech Aging Dev,2004,125 (1):19.
24. Umemura M, Nishimura H, Hirose K, et al. Overexpression of IL15invivo enhances protection agaist Mycobacterium bovis bacillus CalmetteGuerininfection via augmentation of NK and T cytotoxic responses. JImmunol,2001,167(2):946-956.
25. Di Carlo E, Meazza R, Basso R, et al. Dissimilar antitumorreactionsinduced by tumor cells engineered with the interleukin2or interleukin15gene in nude mice. J Pathol, 2000,191(2):193-201.
26. Takeuchi E, Yanagawa H, Suzuki Y, et al. Interleukin(IL)15has lessactivity than IL2to promote type 2 cytokine predominance in tumorassociated mononuclear cells from lung cancer patients.Cytokine,2001,13(2):119-123.
27. Di Carlo E, Comes A, Basso S, et al. The combined action of IL15andIL2gene transfer can induce tumor cell rejection without T and NK cellinvolvement .J Immunol,2000,165(6):3111-3118.
28. Meazza R, Lollini PL, Nanni P, et al. Gene transfer of a secretable form ofIL15in murine adenocarcinoma cells: effects on tumor igenicity,metastatic potential and immune response. Int J Cancer,2000,87(4):574-581.
29. Guo S, Ke mpheus KJ. Par1,a gene required for establishing polarity in C.elegans embryos, encodes a putative Ser/Thr kinase that is asymmetricallydistributed[J]. Cell,1995,81:611-620.
30. Fire A, Xu S, Montgomery MK, et al. Potent and specific geneticinterference by doublestrandRNA in Caenorhabditis elegans [J]. Nature,1998,391(6669) :744-745.
31. Wianny F, ZernickaGoetzM. Specific interference with gene function bydoublestrandedRNA in early mouse development. Nat Cell Biol,2000,2(2):70~75
32. Elbashir S M, Harborth J, Lendeckel W, et al. Duplexes of 21nucleotideRNAs mediate RNA interference in cultured mammalian cells.Nature,2001,411: 494~498
33. Nykanen A, Haley B, Zamore PD. ATP requirements and small interferenceRNA structure in the RNA interference pathway [J]. Cell, 2001,107(3) :309-3021.
34. Tuschil T. Expanding small RNA interference [ J ]. Nat Biotechnol,2002,20 (5) : 446-468.
35. 郑宁宁,于艳秋,宋晓宇,等.RNA 干扰抑制小鼠脾淋巴细胞分泌 IL1α.中国病理生理杂志,2007,23（3）:435-437
36. 康洁,刘福林.RNAi 的抗病毒作用及其机制[J].现代免疫学,2004,24(5):439-441.
37. 林少微,王雪华,郑高哲,等.RNAi 的研究进展.中国医药导报·研究进展.2007,4（29）：79
38. Lipardi C, Wei Q, Paterson BM. RNAi as random degradative PCR:siRNAprimers convert mRNA into dsRNAs that are degraded to generate newsiRNAs [J].Cell,2001,107(3):297-307
39. 朱汝森,刘新光,梁念慈.RNAi 实现策略的进展[J].国外医学临床生物化学与检验学分册,2005,25(3):214-215
40. Zamore PD,Tuschl T, Sharp PA, et al. RNAi:doublestrandRNAdirectstheATPdependentcleavage ofmRNAat 21 to 23 nucleotide intervals.Cell,2000,101:25~33
41. Caplen NJ, Parrish S, Imani F, et al. Specific inhibition of gene expressionby small doublestrandedRNAs in invertebrate and vertebrate systems.Proc Natl Acad Sci USA,2001,98(17):9742~9747
42. Bernstein E, Caudy AA, Hammond SM, et al. Role for a bidentateribonuclease in the initiation step of RNA interference. Nature, 2001,409:363~366
43. Sijen T, Fleenor J, Simmer F, et al. On the role of RNA amplification indsRNAtriggeredgene silencing. Cell, 2001,107(4):465~476
44. 吴元明,陈苏民. RNA 干涉的最新研究进展.中国生物化学与分子生物学报,2003,19(4):411~417
45. Harborth J, Elbashir SM, Becheft K, et al. Identification of essential genesin cultured mammalian cells using small interfering RNAs [J].CellScience,2001,114(24):45-57
46. Bass BL.RNA interference.The short answer[J].Nature,2001,411 (6836):428-429
47. Brantl S. AntisenseRNAregulation and RNA interference. BiochimBiophys Acta, 2002, 1575(13):15-25
48.Irie N, Sakai N, Ueyama T, et al. Subtypeandspeciesspecificknockdownof PKC using short interfering RNA. Biochem Biophys Res Commun,2002, 298(5): 738-743
49.王继峰.RNAi 基因沉默技术.中国生物化学与分子生物学会中医药专业委员会,2005 年年会文集:29-37
50.Tuschl T, Borkhardt A. Small interfering RNAs: are volutionary tool for theanalysis of gene function and gene therapy . Mol Intervent,2002,2(3):158~167
51.Donz O, Picard D.RNA interference in mammalian cells using siRNAssynthesized with T7 RNA polymerase. Nucl AcidRes,2002,30(10):e46
52.Myers J W ,Jones J T, Meyer T, et al. Recombinant Dicer efficientlyconverts large dsRNAs into siRNAs suitable for gene silencing.Nat Biotech,2003,21:324~328
53.Yang D, Buchholz F, Huang Z D, et al. Short RNA duplexes produced byhydrolysis with Escherichiacoli RNaseIII mediated effective RNAinterference inmammalian cells. Proc Natl Acad Sci USA,2002,99(15):9942-9947
54.Miyagishi M, Taira K. U6 promoterdrivensiRNAs with foururidine3'overhangs efficiently suppress targeted gene expression in mammaliancells.Nat Biotech,2002,19:497~500
55.Lee N S, Dohjima T, Bauer G, et al. Expression of small interfering RNAstargeted against HIV1revtranscriptsin human cells. Nat Biotech,2002,19:500-505
56.Paul CP, Good PD, WinerI, et al. Effective expression of small interferingRNA in human cells.Nat Biotech,2002,19:505-508
57.Castanotto D, Li HT, Rossi JJ. Functional siRNA expression fromtransfected PCR products.RNA,2002,8:1454~1460
58.Wilson JA, Jayasena S, Khvorova A, et al. RNA interference blocks geneexpression and RNA synthsis from hepatitis C replication propagated inhuman liver cells[J].Proc Natl Acad Sci USA, 2003,100(5): 2783-2788
59.Hommel JD, Sears RM, DiLeone RJ, et al. Localgene knockdown in thebrain using viralmediatedRNA interference[J].Nat Med,2003,9(12):1539-1544
60.Minakuchi Y, Takeshita F, Kosaka N, et al. Atelocollagenmediatedsynthetic small interfering RNA delivery for effective gene silencing invitro and in vivo[J].Nucleic AcidsRes,2004, 32(13): 109
61.信吉阁，王晓洪，韩佃刚，等.RNA 干扰技术的应用研究进展.动物医学进展,2006,27(2):30-32
62.何承伟,刘 芳,刘新光(综述),梁念慈(审校). RNAi 在基因治疗中的应用及其设计.广 东 医 学 院 学 报,2006,24（1）:66-68
63.Anderson J, Banerjea A, Akkina R. Bispecific shorthairpin siRNAconstructs targeted to CD4, CXCR4, and CCR5 conferHIV1resistance[J].Oligonucleotides,2003,13(5): 303-312
64.Shen C, Buck AK, Reske SN, et al. Gene silencing by adenovirusdeliveredsiRNA [J].FEBS Lett,2003, 539(13):111-114
65.Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protectsmice from fulminant hepatitis[J]. Nat Med, 2003,9(3):347-351
66.陈竞.RNAi 的研究进展[J].川北医学院学报,2004,19(3):198
67.W ilda M, Fuchs U, Wossmann W, et al. Killing of leukemic cells with aBCR /ABL fusion gene by RNA interference(RNAi) [ J].Oncogene,2002,21(20): 5716-5724
68.Williams NS, Gaynor RB, Scoggin S, et al. Identification and validation ofgenes involved in the pathogenesis of colorectal cancer using cDNAmicroarrays and RNA interference[J]. Clin Cancer Res,2003,9(3):931-946
69.Ritter U, DammWelkC, Woessmann W, et al. Design and evalation ofchemically synthesized siRNA targeting the NPMALKfsion site inanaplastic large cell lymphoma (ALCL ) [ J]Oligonucleotides,2003,13(5):365-373
70.Lima RT, Martins LM, Vasconcelos MH, et al. Specific downregulationofbcl2and xIAP by RNAi enhances the effects of chemotherapeutic agentsinMCF7human breast cancer cells[J].Cancer GeneTher,2004,11(5):309-316
71.Regamey A, Hohl D, Liu JW, et al. The tumor suppressor CYLD interactswith TRIP and regulates negatively nuclear factor kappa B activation bytumor necrosis factor[J].J ExpMed,2003,198(12): 1959-1964
72.Wu H, Hait WN, Yang M. Small Interfering RNAinducedSuppressionofMDR1(PGlycoprotein)Restores Sensitivity to MultdrugresisiranceCancer Cells[ J].Cancer Res,2003, 63 (7):1515-1519
73.Nieth C, Priebsch A, Lage H, et al. Modulation of the classical multidrugresistance (MDR ) phenotype by RNA interference (RNAi)[J].FFBSLett,2003, 545(23):144-150
74.Kosciolek BA, Kalantidis K, Tabler M, et al. Inhibition of telomeraseactivity in human cancer cells by RNA interference[J].Mo CancerTher,2003, 2(3): 209-216
75.Zhang R, Guo Z, Lu J, et al. Inhibiting severe acute respiratorysyndromeassociatedcoronavirus by small interfering RNA. [J]·Chin Med J,2003,116(8):1262-1264
76.Qin L, Xiong B, Luo C, et al. Identification of probable genomic packagingsignal sequence from SARSCoVgenome by bioinformatics analysis.[J]Acta Pharmacol Sin, 2003,24(6):489-496
77. Ge Q, McManus MT, Nguyen T, et al. RNA interference of influenza virusproduction by directly targeting mRNA for degradation and indirectlyinhibiting all viral RNA transcription. [J]·Proc Natl Acad Sci USA,2003,100(5):2718-2723
78.Toyoda T. Target of developing the new antiinfluenzavirus reagents.[J]·Nippon Rinsho, 2003,61(11):1980-1986
79.McCown M, Diamond MS, Pekosz A. The utility of siRNA transcriptsproduced by RNA polymerase i in down regulating viral gene expressionand replication of negativeandpositivestrandRNA viruses. [J]·Virology,2003,313(2):514-524
80.Uematsu K, He B, You L, et al. Activation of the Wnt pathway in non smallcell lung cancer: evidence of dishevelled overexpression.[J]·Oncogene,2003,22(46):7218-7221
81.Liu LT, Chang HC, Chiang LC, et al. Histone deacetylase inhibitorupregulatesRECK to inhibit MMP2activation and cancer cell invasion.[J]·Cancer Res, 2003,63(12):3069-3072
82.Sundararaj KP, Wood RE, Ponnusamy S, et al. Rapid shortening of telomerelength in response to ceramide involves the inhibition of telomere bindingactivity of nuclear glyceraldehyde3phosphatedehydrogenase. [J]·J BiolChem, 2004, 279(7):6152-6162
83.Kristof AS, MarksKonczalikJ, Billings E, et al. Stimulation of signaltransducer and activator of transcription1(STAT1)dependentgenetranscription by lipopolysaccharide and interferongammais regulated bymammalian target of rapamycin. [J]·J Biol Chem, 2003,278(36): 33637-33644
84.Ma Y, Cress WD, Haura EB. Flavopiridolinducedapoptosis is mediatedthrough upregulationof E2F1 and repression of Mcl1.[J]·Mol CancerTher, 2003,2(1):73-81
85.Perl M, Chung CS, LomasNeiraJ, et al. Silencing of fas, but notcaspase8,in lung epithelial cells ameliorates pulmonary apoptosis,inflammation, and neutrophil influx after hemorrhagic shock and sepsis[J].Am J Pathol,2005,167(6):1545-1559
86.Krick S, Eul BG, Hanze J, et al. Role of hypoxiainduciblefactor1alphainhypoxiainducedapoptosis of primary alveolar epithelial type IIcells[J].Am J Respir Cell Mol Biol,2005,32(5):395-403
87.Sakai T, Larsen M, Yamada KM. Fibronectin requirement in branchingmorphogenesis. [J]·Nature ,2003, 423(6942):876-881
88.Chen MJ, Lai YL. Tachykinin dysfunction attenuates monocrotalineinducedpulmonary hypertension.·[J]·Toxicol Appl Pharmacol,2003,187(3):178-185
89.Li L, Yang Y, Stevens RL. Tachykinin dysfunction attenuatesmonocrotalineinducedpulmonary hypertension.·[J]·J Biol Chem,2003,278(7):4725-4729
90.Fire A，Xu S , Mentggomery M K , et al. Potent and specific geneticinterference by doublestrandedRNA in Caenorhabditis elegans J.Nature ,1998 , 391; 806-811.
91.Dykxhoorn DM, Lieberman J. The silent revolution: RNA interference asbasic biology, research tool, and therapeutic. Annu Rev Med,2005,56:401-423
92.吕辉，贺智敏. RNA 干扰技术的演进及其在基因功能和基因治疗研究中的应用.中南大学学报,2005,30（1）：102-105
93.Elbashir SM, Lendeckel W, Tuschl T. RNA interference ismediated by21and22nucleotideRNAs [ J].Genes Dev, 2001, 15(2):188-200.
94.Zamore PD, Tuschl T, Sharp PA, et al. RNAi: doublestranded RNA directstheATPdependentcleavage of mRNA at 21to 23nt intervals. Cell, 2000,101: 25-33
95.Vaysse L, Harbottle R, Bigger B, et al. Development of a Selfassemblingnuclear targeting vector system based on the tetracyclinerepressor protein. J Bio Chem,2004,279:5555-5564.
96.Plank C, Oberhauser B, Mechtler B, et al. The influence of endosomedisruptivepepides on gene transfer using synthetic viruslike gene transfersystems. J Biol chem,1994,269:12918-12924.
97.Duan W, Chan JH, McKay K, et al. Inhaled p38αmitogenactivatedproteinkinase antisense oligonucleotide attenuates asthma in mice. Am J RespirCrit Care Med, 2005,171(6):571-57
98.Zhang X, Shan P, Jiang D, et al. Small interfering RNA targeting hemeoxygenase1enhances ischemiareperfusioninducedlung apoptosis. J BiolChem,2004,279(11):10677-10684
99.Oldham MJ，Phalen RF. Dosimetry implications of upper tracheobronchialairway anatomy in two mouse varieties. Anat Ree,2002,268（1）:59
100. Watson ML, White AM, Campbell EM, et al. Antiinflammatoryactionsof interleukin13:suppression of tumornecrosis factoralphaand antigeninduced leukocyte accumulation in the guinea pig lung. Am J Respir CellMoBiol, 1999,20(5):1007-1012.
101. Shim JJ, Dabbagh K, Takeyama K, et al. Suplatast tosilate inhibits gobletcell metaplasia of airway epithelium in sensitized mice.J Allergy ClinImmunol, 2000,105(4):739-745.
102. Zhao GD, Yokoyama A, Kohno N, et al. Effect of suplatest tosilate(IPD21151T)on a mouse model of asthma: inhibition of eosinophilicinflammation and bronchial hyperresponsiveness. Int Arch AllergyImmunol, 2000,121(2):116-122.
103. Zuhdi AM, Piazza FM, Selby DM, et al. Muc5ac mucin messenger RNAand protein expression is a marker of goblet cell metaplasia in murineairways. Am J Respir Cell Mol Biol,2000,22(3):253-260
104. 王妍，金先桥.支气管哮喘实验模型的研究进展.国际呼吸杂志，2006,26（1）:70-72
105. Hayashi T，Adachi Y，Hasegawa K，et al. Less sensitivity for late airwayinflammation in males than females in BALB/C mice. Scand J Immunol,2003,57（6）:562
106. Pabst R. Animal models for asthma: controversial aspects and unsolvedproblems. Pathobiology,20022003,70(5)：260-265
107. Schmitz N, Kurrer M,Kopfl M, et al. The IL1receptor 1 is criticial forTh2 type airway immune responses in a mild but not in a more severeasthma model. Eur J Immunol, 2003,33(4):991-1000
108. Elias JA, Lee CG, Zheng T, et al. New insights into the pathogenesis ofasthma. J Clin Invest,2003,111(3):291-297
109. Hannon GJ. RNA Interference [ J]. Nature, 2002, 418(6894): 244-251.
110. Sorensen DR, Leirdal M, Sioud M. Gene silencing by systemic delivery ofsynthetic siRNAs in adul tmice [ J]. J MolBio,l 2003, 327(4): 761-766.
111. McCaffrey AP, Nakai H, Pandey K, et al. Inhibition of hepatitis B virus inmice by RNA interference.Nat Biotechnol,2003,21(6):639-644
112. Lewis DL, Hagstrom JE, Loomis AG, et al. Efficient delivery of siRNAfor inhibition of gene expression in postnatal mice. Nat Genet,2002,32(1):107-108
113. Sorensen DR, Leirdal M, Sioud M. Gene silencing by systemic delivery ofsynthetic siRNAs in adult mice. J Mol Biol,2003,327(4):761-766
114. Gordon SP. Berezhna S. Scherfeld D, et al. Characterization of interactionbetween cationic lipidoligonucleotide complexes and cellular membranelipids using confocal imaging and fluorescence correlation spectroscopy [J].Biophys J,2005,88(1):305-316
115. Dodds E, Dunckley MG, Naujoks K, et al. Lipofection of cultured mousemuscle cells: a direct comparison of lipofectamine and DOSPER. GeneTher,1998,5(4):542-551
116. Hemelaers L, Louis R. Eotaxin: an important chemokine in asthma. RevMed Liege, 2006, 61 (4): 223-226
117. Humbles AA, Lloyd CM, McMillan SJ, et al. A critical role foreosinophils in allergic airways remodeling. Science, 2004,305: 1776-1779
118.Lee JJ, Dimina D, Macias MP, et al. Defining a link with asthma in micecongenitally deficient in eosinophils. Science, 2004,305: 1773-1776
119.Sexton DW, Walsh GM. Eosinophilepithelicalcell interactions:animportant facet of asthmatic inflammation. Clinical Experimental Allergy,2002, 32: 811-813
120. Duncan CJA, Lawrie A, Blaylock HG, et al. Reduced eosinophil apoptosisin induced sputum correlates with asthma severity. European RespiratoryJournal, 2003, 22: 484-490
121. Miranda C，Busaeker A，Balzar S，et al. Distinguishing severe asthmaphenotype: role of age at onset and eosinophilie inflammation. J AllergyClin Immunol,2004,13:101-108
122. Lukacs NW, Lamm WJ, Strieter RM, et al. Airway hyperreactivity isassociated with specific leukocyte subset infiltration in a mouse modelof allergic airway inflammation[J]. Pathobiology, 1996,64(6): 308-313.
123.Anticevich SZ, Hughes JM, Black JL, et al. Induction of hyperresponsivenessin human airway tissue by neutrophils mechanism of action，ClinExp Allergy, 1996,26(5):549-556.
124.Park HS, Uh ST, Park CS. lncreased neutrophil chemotactic activity i5noted in alminuminducedoccupational asthma[J]. Korean J Intern Med,1996,11(1):69-73
125.李峰,赵红洋,温暖,等.支气管哮喘发病机制进展. 实用全科医学,2007,5(10):922-923
126.Foresi A, Teodoro C, Leone C, et al. osinophil apoptosis in induced sputumfrom patients with seasonal allergic rhinitis and with asymptomatic andsymptomatic asthma. Ann Allergy Asthma Immunol, 2000, 84: 411-416
127.Kankaanranta H, Lindsay MA, Giembycz MA, et al. Delayed eosinophilapoptosis in asthma. J Allergy Clin Immunol,2000, 106: 77-83
128.Woolley KL, Gibson PG, Carty K, et al. Eosinophil apoptosis and theresolution of airway inflammation in asthma. Am J Respir Crit Care Med,1996, 154: 237-243
129.Walsh GM. Eosinphils apoptosis: mechanisms and clinical revelance inasthmatic and allergic inflammation. Br J Haematol, 2000,111: 61-67
130.Gounni AS, Gregory B, Nutku E, et al. Interleukin9enhancesinterleukin5receptor expression, differentiation, and survival of humaneosinophils. Blood, 2000;96: 2163-2171
131.Luttmann W, Knoechel B, FoersterM, et al. Activation of humaneosinophils by IL13.Induction of CD69 surface antigen, its relationshipto messenger RNA expression, and promotion of cellular viability. JImmunol, 1996,157: 1678-1683
132. Valerius T, Repp R, kalden JR, et al. Effects of IFN on human eosinophilsin comparison with other cytokines. A novel class of eosinophil activatorswith delayed onset of action. J Immunol, 1990, 145: 2950-2958
133.Fujihara S, Ward C, Dransfield I, et al. Inhibition of nuclear factorkappaBactivation unmasksthe ability of TNFalphato induce human eosinophilapoptosis. Eur J Immunol, 2002,32: 457-466
134.Hamada A, Watanabe N, Ohtomo H, et al. Nerve growth factor enhancessurvival and cytotoxic activity of human eosinophils. Br J Haematol,1996,93: 299-302
135.Robertson NM, Zangrilli JG, Steplewski, A et al. Differential expression ofTRAIL and TRAIL receptors in allergic asthmatics following segmentalantigen challenge: Evidence for a role of TRAIL in eosinophil surviva.l JImmunol, 2002,169: 5986-5996
136.Gardai SJ, Hoontrakoon R, Goddard CD, et al. Oxidantmediatedmitochondrial injury in eosinophil apoptosis: enhancement byglucocorticoids and inhibition by granulocyte macrophage colony stimulatingfactor. J Immunol, 2003,170: 556-566
137.Takeuchi M, Hayakawa A, TakagiK, et al. The ophyline induced apoptosisof the IL3activated eosinophils of patients with bronchial asthma.Apoptosis, 1999, 4: 461-468
138. Nutku E, Zhuang Q, SoussiGounniA,et al. Functional expression ofIL12receptor by human eosinophils: IL12promotes eosinophilapoptosis. J Immunol ,2001,167: 1039-1046
139.Peachell P. Targeting the mast cell in asthma. Current Opinion inPharmacology,2005,5(3):16
140.Elias JA, Lee CG, Zheng T, et al. New insights into the pathogenesis ofasthma. J Clin Invest,2003,111(3):291-297
141.杜静综述，梁 晓燕审校.RNA干扰的基础研究和临床应用前景.国外医学计划生育/生殖健康分册,2006,25（6）：307-310
142.Amarzguioui M, Holen T, Babaie E, et al. Tolerance for mutations andchemical modifications in a siRNA. Nucleic Acid Res,2003,31(2):589-595
143.Saxena S, Jonsson ZO, Dutta A. Small RNAs with imperfect match toendogenous mRNA repress translation. Implications for offtargetactivityof small inhibitory RNA in mammalian cells. J Biol Chem,2003,278(45):44312-44319
2. Musso T, Calosso L, Zucca M, et al. Human monocytes constitutivelyexpress membranebound,biologically active, and interferongammaupregulatedinterleukin15.Blood, 1999, 93: 3531-3539
3. Burton JD, Bamford RN, Peters C, et al. A lymphokine, provisionallydesignated interleukin T and produced by a human adult Tcellleukemialine, stimulates Tcellproliferation and the induction of lymphokineactivatedkiller cells. Proc Natl Acad Sci USA,1994,91:4935-4939
4. Krause H, Jandrig B, Wernicke C, et al. Genomic structure and chromosomallocalization of the human interleukin 15 gene (IL15).Cytokine,1996,8:667-674
5. 高巍综述，韩守信审校.白介素15与支气管哮喘.国外医学呼吸系统分册,2004，24（1）：15-17
6. Perera LP, Goldman CK, Waldmann TA. IL15induces the expression ofchemokines and their receptors in T lymphocytes .J Immunol,1999,162:2606-2612
7. Liu K, Catalfamo M, Li Y, et al. IL15mimics T cell receptor cross linkingin the induction of cellular proliferation, gene expression, and cytotoxicityin CD+8memory T cells. Proc Natl Acad Sci USA,2002,99:6192-6197
8. Fehniger TA, Shah MH, Turner MJ, et al. Differential cytokine geneexpression by human NK cell following activation with IL8or IL15incombination with IL12:implications for the innate immune response.J Immunol,1999,162(8):4511-4520
9. Neely GG, Robbins SM, Amankwah EK, et al. Lipopolysaccharidestimulated or granulocytemacrophagecolonystimulatingfactorstimulatedmonocytes rapidly express biologically active IL15on their cell surfaceindependent of new protein synthesis. J Immunol, 2001,167:5011-5017
10. Alleva DG, Kaser SB, Monroy MA, et al. IL15functions as a potentautocrine regulator of macrophage proinflammatory cytokine production:evidence for differential receptor subunit utilization associated withstimulation or inhibition. J Immunol, 1997, 159:2941-2951
11. Avice MN, Demeure CE, Delespesse G, et al.IL15promotes IL12production by human monocytes via T celldependentcontact and maycontribute to IL12mediatedIFNgammasecretion by CD4+ T cell in theabcence of TCR ligation.J Immunol,1998;161:3408-3415
12. Hoontrakoon R, Chu HW, Gardai SJ, et al. Interlukin15inhibitsspontaneous apoptosis in human eosinophils via autocrine production ofgranulocyte macrophage colonystimulating factor and nuclear factorkBactivation. Am J Respir Cell Mol Biol, 2002, 26: 404-412
13. 高巍，韩守信，陈复辉，等.支气管哮喘患者外周血 IL15的测定及意义.哈尔滨医科大学学报,2004,38（1）: 70-72
14. Pelletier M, Ratthe C, Girard D. Mechanisms involved ininterleukin15inducedsuppression of human neutrophil apoptosis:role ofthe antiapoptoticMcl1protein and several kinases including Januskinase2,p38 mitogenactivatedprotein kinase and extracellular signalregulatedkinases1/2.FEBS Lett,2002,532:164-170
15. McDonald PP, Russo MP, Ferrini S,et al.Interleukin15(IL15)inducesNFkappaB activation and IL8production in human neutrophils.Blood,1998, 92:4828-4835
16. Girard D, Paquet ME, Paquin R, et al. Differential effects of interleukin15(IL15)and IL2on human neutrophils: modulation of phagocytosis,cytoskeleton rearrangement, gene expression, and apoptosis by IL15[J].Blood,1996,88(8):3176-3184.
17. Lai YG, Gelfanov V, Gelfanova V, et al. IL15promotes survival but noteffector function differentiation of CD8+ TCR alphabeta+ intestinalintraepithelial lymphocytes[J].J Immunol,1999,163 (11):5843-5850.
18. KomaiKomaM, McKay A, Thomson L, et al. Immunoregulatorycytokines in asthma:IL15and IL13in induced sputum. Clin ExpAllergy,2001, 31:1441-1448
19. Grunig G, Warnock M, Wokil AE, et al. Requirement for IL13independently of IL4in experimental asthma. Science, 1998, 28（2）:2261-2263
20. Ishimitsu R, Nishimura H, Yajima T, et al. Overexpression of IL15in vivoenhances Tc1 response, which inhibits allergic inflammation in a murinemodel of asthma. J Immunol,2001,166:1991-2001
21. Rene′ Ru¨ckert, Katja Brandt, Armin Braun, et al. Blocking IL15Preventsthe Induction of AllergenSpecificT Cells and Allergic Inflammation InVivo. The Journal of Immunology, 2005, 174: 5507–5515.
22. Hartmann P, Plum G. Immunological defense mechanisms in tuberculosisand MACinfection.Diagn Microbiol Infect Dis, 1999,34(2):147-152.
23. Turner J, Orme IM. The expression of early resistance to an infection withMycobacterium tuberculosis by old mice is depend on IFN typeⅡ(IFNGamma)but not IFN typeⅠ. Mech Aging Dev,2004,125 (1):19.
24. Umemura M, Nishimura H, Hirose K, et al. Overexpression of IL15invivo enhances protection agaist Mycobacterium bovis bacillus CalmetteGuerininfection via augmentation of NK and T cytotoxic responses. JImmunol,2001,167(2):946-956.
25. Di Carlo E, Meazza R, Basso R, et al. Dissimilar antitumorreactionsinduced by tumor cells engineered with the interleukin2or interleukin15gene in nude mice. J Pathol, 2000,191(2):193-201.
26. Takeuchi E, Yanagawa H, Suzuki Y, et al. Interleukin(IL)15has lessactivity than IL2to promote type 2 cytokine predominance in tumorassociated mononuclear cells from lung cancer patients.Cytokine,2001,13(2):119-123.
27. Di Carlo E, Comes A, Basso S, et al. The combined action of IL15andIL2gene transfer can induce tumor cell rejection without T and NK cellinvolvement .J Immunol,2000,165(6):3111-3118.
28. Meazza R, Lollini PL, Nanni P, et al. Gene transfer of a secretable form ofIL15in murine adenocarcinoma cells: effects on tumor igenicity,metastatic potential and immune response. Int J Cancer,2000,87(4):574-581.
29. Guo S, Ke mpheus KJ. Par1,a gene required for establishing polarity in C.elegans embryos, encodes a putative Ser/Thr kinase that is asymmetricallydistributed[J]. Cell,1995,81:611-620.
30. Fire A, Xu S, Montgomery MK, et al. Potent and specific geneticinterference by doublestrandRNA in Caenorhabditis elegans [J]. Nature,1998,391(6669) :744-745.
31. Wianny F, ZernickaGoetzM. Specific interference with gene function bydoublestrandedRNA in early mouse development. Nat Cell Biol,2000,2(2):70~75
32. Elbashir S M, Harborth J, Lendeckel W, et al. Duplexes of 21nucleotideRNAs mediate RNA interference in cultured mammalian cells.Nature,2001,411: 494~498
33. Nykanen A, Haley B, Zamore PD. ATP requirements and small interferenceRNA structure in the RNA interference pathway [J]. Cell, 2001,107(3) :309-3021.
34. Tuschil T. Expanding small RNA interference [ J ]. Nat Biotechnol,2002,20 (5) : 446-468.
35. 郑宁宁,于艳秋,宋晓宇,等.RNA 干扰抑制小鼠脾淋巴细胞分泌 IL1α.中国病理生理杂志,2007,23（3）:435-437
36. 康洁,刘福林.RNAi 的抗病毒作用及其机制[J].现代免疫学,2004,24(5):439-441.
37. 林少微,王雪华,郑高哲,等.RNAi 的研究进展.中国医药导报·研究进展.2007,4（29）：79
38. Lipardi C, Wei Q, Paterson BM. RNAi as random degradative PCR:siRNAprimers convert mRNA into dsRNAs that are degraded to generate newsiRNAs [J].Cell,2001,107(3):297-307
39. 朱汝森,刘新光,梁念慈.RNAi 实现策略的进展[J].国外医学临床生物化学与检验学分册,2005,25(3):214-215
40. Zamore PD,Tuschl T, Sharp PA, et al. RNAi:doublestrandRNAdirectstheATPdependentcleavage ofmRNAat 21 to 23 nucleotide intervals.Cell,2000,101:25~33
41. Caplen NJ, Parrish S, Imani F, et al. Specific inhibition of gene expressionby small doublestrandedRNAs in invertebrate and vertebrate systems.Proc Natl Acad Sci USA,2001,98(17):9742~9747
42. Bernstein E, Caudy AA, Hammond SM, et al. Role for a bidentateribonuclease in the initiation step of RNA interference. Nature, 2001,409:363~366
43. Sijen T, Fleenor J, Simmer F, et al. On the role of RNA amplification indsRNAtriggeredgene silencing. Cell, 2001,107(4):465~476
44. 吴元明,陈苏民. RNA 干涉的最新研究进展.中国生物化学与分子生物学报,2003,19(4):411~417
45. Harborth J, Elbashir SM, Becheft K, et al. Identification of essential genesin cultured mammalian cells using small interfering RNAs [J].CellScience,2001,114(24):45-57
46. Bass BL.RNA interference.The short answer[J].Nature,2001,411 (6836):428-429
47. Brantl S. AntisenseRNAregulation and RNA interference. BiochimBiophys Acta, 2002, 1575(13):15-25
48.Irie N, Sakai N, Ueyama T, et al. Subtypeandspeciesspecificknockdownof PKC using short interfering RNA. Biochem Biophys Res Commun,2002, 298(5): 738-743
49.王继峰.RNAi 基因沉默技术.中国生物化学与分子生物学会中医药专业委员会,2005 年年会文集:29-37
50.Tuschl T, Borkhardt A. Small interfering RNAs: are volutionary tool for theanalysis of gene function and gene therapy . Mol Intervent,2002,2(3):158~167
51.Donz O, Picard D.RNA interference in mammalian cells using siRNAssynthesized with T7 RNA polymerase. Nucl AcidRes,2002,30(10):e46
52.Myers J W ,Jones J T, Meyer T, et al. Recombinant Dicer efficientlyconverts large dsRNAs into siRNAs suitable for gene silencing.Nat Biotech,2003,21:324~328
53.Yang D, Buchholz F, Huang Z D, et al. Short RNA duplexes produced byhydrolysis with Escherichiacoli RNaseIII mediated effective RNAinterference inmammalian cells. Proc Natl Acad Sci USA,2002,99(15):9942-9947
54.Miyagishi M, Taira K. U6 promoterdrivensiRNAs with foururidine3'overhangs efficiently suppress targeted gene expression in mammaliancells.Nat Biotech,2002,19:497~500
55.Lee N S, Dohjima T, Bauer G, et al. Expression of small interfering RNAstargeted against HIV1revtranscriptsin human cells. Nat Biotech,2002,19:500-505
56.Paul CP, Good PD, WinerI, et al. Effective expression of small interferingRNA in human cells.Nat Biotech,2002,19:505-508
57.Castanotto D, Li HT, Rossi JJ. Functional siRNA expression fromtransfected PCR products.RNA,2002,8:1454~1460
58.Wilson JA, Jayasena S, Khvorova A, et al. RNA interference blocks geneexpression and RNA synthsis from hepatitis C replication propagated inhuman liver cells[J].Proc Natl Acad Sci USA, 2003,100(5): 2783-2788
59.Hommel JD, Sears RM, DiLeone RJ, et al. Localgene knockdown in thebrain using viralmediatedRNA interference[J].Nat Med,2003,9(12):1539-1544
60.Minakuchi Y, Takeshita F, Kosaka N, et al. Atelocollagenmediatedsynthetic small interfering RNA delivery for effective gene silencing invitro and in vivo[J].Nucleic AcidsRes,2004, 32(13): 109
61.信吉阁，王晓洪，韩佃刚，等.RNA 干扰技术的应用研究进展.动物医学进展,2006,27(2):30-32
62.何承伟,刘 芳,刘新光(综述),梁念慈(审校). RNAi 在基因治疗中的应用及其设计.广 东 医 学 院 学 报,2006,24（1）:66-68
63.Anderson J, Banerjea A, Akkina R. Bispecific shorthairpin siRNAconstructs targeted to CD4, CXCR4, and CCR5 conferHIV1resistance[J].Oligonucleotides,2003,13(5): 303-312
64.Shen C, Buck AK, Reske SN, et al. Gene silencing by adenovirusdeliveredsiRNA [J].FEBS Lett,2003, 539(13):111-114
65.Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protectsmice from fulminant hepatitis[J]. Nat Med, 2003,9(3):347-351
66.陈竞.RNAi 的研究进展[J].川北医学院学报,2004,19(3):198
67.W ilda M, Fuchs U, Wossmann W, et al. Killing of leukemic cells with aBCR /ABL fusion gene by RNA interference(RNAi) [ J].Oncogene,2002,21(20): 5716-5724
68.Williams NS, Gaynor RB, Scoggin S, et al. Identification and validation ofgenes involved in the pathogenesis of colorectal cancer using cDNAmicroarrays and RNA interference[J]. Clin Cancer Res,2003,9(3):931-946
69.Ritter U, DammWelkC, Woessmann W, et al. Design and evalation ofchemically synthesized siRNA targeting the NPMALKfsion site inanaplastic large cell lymphoma (ALCL ) [ J]Oligonucleotides,2003,13(5):365-373
70.Lima RT, Martins LM, Vasconcelos MH, et al. Specific downregulationofbcl2and xIAP by RNAi enhances the effects of chemotherapeutic agentsinMCF7human breast cancer cells[J].Cancer GeneTher,2004,11(5):309-316
71.Regamey A, Hohl D, Liu JW, et al. The tumor suppressor CYLD interactswith TRIP and regulates negatively nuclear factor kappa B activation bytumor necrosis factor[J].J ExpMed,2003,198(12): 1959-1964
72.Wu H, Hait WN, Yang M. Small Interfering RNAinducedSuppressionofMDR1(PGlycoprotein)Restores Sensitivity to MultdrugresisiranceCancer Cells[ J].Cancer Res,2003, 63 (7):1515-1519
73.Nieth C, Priebsch A, Lage H, et al. Modulation of the classical multidrugresistance (MDR ) phenotype by RNA interference (RNAi)[J].FFBSLett,2003, 545(23):144-150
74.Kosciolek BA, Kalantidis K, Tabler M, et al. Inhibition of telomeraseactivity in human cancer cells by RNA interference[J].Mo CancerTher,2003, 2(3): 209-216
75.Zhang R, Guo Z, Lu J, et al. Inhibiting severe acute respiratorysyndromeassociatedcoronavirus by small interfering RNA. [J]·Chin Med J,2003,116(8):1262-1264
76.Qin L, Xiong B, Luo C, et al. Identification of probable genomic packagingsignal sequence from SARSCoVgenome by bioinformatics analysis.[J]Acta Pharmacol Sin, 2003,24(6):489-496
77. Ge Q, McManus MT, Nguyen T, et al. RNA interference of influenza virusproduction by directly targeting mRNA for degradation and indirectlyinhibiting all viral RNA transcription. [J]·Proc Natl Acad Sci USA,2003,100(5):2718-2723
78.Toyoda T. Target of developing the new antiinfluenzavirus reagents.[J]·Nippon Rinsho, 2003,61(11):1980-1986
79.McCown M, Diamond MS, Pekosz A. The utility of siRNA transcriptsproduced by RNA polymerase i in down regulating viral gene expressionand replication of negativeandpositivestrandRNA viruses. [J]·Virology,2003,313(2):514-524
80.Uematsu K, He B, You L, et al. Activation of the Wnt pathway in non smallcell lung cancer: evidence of dishevelled overexpression.[J]·Oncogene,2003,22(46):7218-7221
81.Liu LT, Chang HC, Chiang LC, et al. Histone deacetylase inhibitorupregulatesRECK to inhibit MMP2activation and cancer cell invasion.[J]·Cancer Res, 2003,63(12):3069-3072
82.Sundararaj KP, Wood RE, Ponnusamy S, et al. Rapid shortening of telomerelength in response to ceramide involves the inhibition of telomere bindingactivity of nuclear glyceraldehyde3phosphatedehydrogenase. [J]·J BiolChem, 2004, 279(7):6152-6162
83.Kristof AS, MarksKonczalikJ, Billings E, et al. Stimulation of signaltransducer and activator of transcription1(STAT1)dependentgenetranscription by lipopolysaccharide and interferongammais regulated bymammalian target of rapamycin. [J]·J Biol Chem, 2003,278(36): 33637-33644
84.Ma Y, Cress WD, Haura EB. Flavopiridolinducedapoptosis is mediatedthrough upregulationof E2F1 and repression of Mcl1.[J]·Mol CancerTher, 2003,2(1):73-81
85.Perl M, Chung CS, LomasNeiraJ, et al. Silencing of fas, but notcaspase8,in lung epithelial cells ameliorates pulmonary apoptosis,inflammation, and neutrophil influx after hemorrhagic shock and sepsis[J].Am J Pathol,2005,167(6):1545-1559
86.Krick S, Eul BG, Hanze J, et al. Role of hypoxiainduciblefactor1alphainhypoxiainducedapoptosis of primary alveolar epithelial type IIcells[J].Am J Respir Cell Mol Biol,2005,32(5):395-403
87.Sakai T, Larsen M, Yamada KM. Fibronectin requirement in branchingmorphogenesis. [J]·Nature ,2003, 423(6942):876-881
88.Chen MJ, Lai YL. Tachykinin dysfunction attenuates monocrotalineinducedpulmonary hypertension.·[J]·Toxicol Appl Pharmacol,2003,187(3):178-185
89.Li L, Yang Y, Stevens RL. Tachykinin dysfunction attenuatesmonocrotalineinducedpulmonary hypertension.·[J]·J Biol Chem,2003,278(7):4725-4729
90.Fire A，Xu S , Mentggomery M K , et al. Potent and specific geneticinterference by doublestrandedRNA in Caenorhabditis elegans J.Nature ,1998 , 391; 806-811.
91.Dykxhoorn DM, Lieberman J. The silent revolution: RNA interference asbasic biology, research tool, and therapeutic. Annu Rev Med,2005,56:401-423
92.吕辉，贺智敏. RNA 干扰技术的演进及其在基因功能和基因治疗研究中的应用.中南大学学报,2005,30（1）：102-105
93.Elbashir SM, Lendeckel W, Tuschl T. RNA interference ismediated by21and22nucleotideRNAs [ J].Genes Dev, 2001, 15(2):188-200.
94.Zamore PD, Tuschl T, Sharp PA, et al. RNAi: doublestranded RNA directstheATPdependentcleavage of mRNA at 21to 23nt intervals. Cell, 2000,101: 25-33
95.Vaysse L, Harbottle R, Bigger B, et al. Development of a Selfassemblingnuclear targeting vector system based on the tetracyclinerepressor protein. J Bio Chem,2004,279:5555-5564.
96.Plank C, Oberhauser B, Mechtler B, et al. The influence of endosomedisruptivepepides on gene transfer using synthetic viruslike gene transfersystems. J Biol chem,1994,269:12918-12924.
97.Duan W, Chan JH, McKay K, et al. Inhaled p38αmitogenactivatedproteinkinase antisense oligonucleotide attenuates asthma in mice. Am J RespirCrit Care Med, 2005,171(6):571-57
98.Zhang X, Shan P, Jiang D, et al. Small interfering RNA targeting hemeoxygenase1enhances ischemiareperfusioninducedlung apoptosis. J BiolChem,2004,279(11):10677-10684
99.Oldham MJ，Phalen RF. Dosimetry implications of upper tracheobronchialairway anatomy in two mouse varieties. Anat Ree,2002,268（1）:59
100. Watson ML, White AM, Campbell EM, et al. Antiinflammatoryactionsof interleukin13:suppression of tumornecrosis factoralphaand antigeninduced leukocyte accumulation in the guinea pig lung. Am J Respir CellMoBiol, 1999,20(5):1007-1012.
101. Shim JJ, Dabbagh K, Takeyama K, et al. Suplatast tosilate inhibits gobletcell metaplasia of airway epithelium in sensitized mice.J Allergy ClinImmunol, 2000,105(4):739-745.
102. Zhao GD, Yokoyama A, Kohno N, et al. Effect of suplatest tosilate(IPD21151T)on a mouse model of asthma: inhibition of eosinophilicinflammation and bronchial hyperresponsiveness. Int Arch AllergyImmunol, 2000,121(2):116-122.
103. Zuhdi AM, Piazza FM, Selby DM, et al. Muc5ac mucin messenger RNAand protein expression is a marker of goblet cell metaplasia in murineairways. Am J Respir Cell Mol Biol,2000,22(3):253-260
104. 王妍，金先桥.支气管哮喘实验模型的研究进展.国际呼吸杂志，2006,26（1）:70-72
105. Hayashi T，Adachi Y，Hasegawa K，et al. Less sensitivity for late airwayinflammation in males than females in BALB/C mice. Scand J Immunol,2003,57（6）:562
106. Pabst R. Animal models for asthma: controversial aspects and unsolvedproblems. Pathobiology,20022003,70(5)：260-265
107. Schmitz N, Kurrer M,Kopfl M, et al. The IL1receptor 1 is criticial forTh2 type airway immune responses in a mild but not in a more severeasthma model. Eur J Immunol, 2003,33(4):991-1000
108. Elias JA, Lee CG, Zheng T, et al. New insights into the pathogenesis ofasthma. J Clin Invest,2003,111(3):291-297
109. Hannon GJ. RNA Interference [ J]. Nature, 2002, 418(6894): 244-251.
110. Sorensen DR, Leirdal M, Sioud M. Gene silencing by systemic delivery ofsynthetic siRNAs in adul tmice [ J]. J MolBio,l 2003, 327(4): 761-766.
111. McCaffrey AP, Nakai H, Pandey K, et al. Inhibition of hepatitis B virus inmice by RNA interference.Nat Biotechnol,2003,21(6):639-644
112. Lewis DL, Hagstrom JE, Loomis AG, et al. Efficient delivery of siRNAfor inhibition of gene expression in postnatal mice. Nat Genet,2002,32(1):107-108
113. Sorensen DR, Leirdal M, Sioud M. Gene silencing by systemic delivery ofsynthetic siRNAs in adult mice. J Mol Biol,2003,327(4):761-766
114. Gordon SP. Berezhna S. Scherfeld D, et al. Characterization of interactionbetween cationic lipidoligonucleotide complexes and cellular membranelipids using confocal imaging and fluorescence correlation spectroscopy [J].Biophys J,2005,88(1):305-316
115. Dodds E, Dunckley MG, Naujoks K, et al. Lipofection of cultured mousemuscle cells: a direct comparison of lipofectamine and DOSPER. GeneTher,1998,5(4):542-551
116. Hemelaers L, Louis R. Eotaxin: an important chemokine in asthma. RevMed Liege, 2006, 61 (4): 223-226
117. Humbles AA, Lloyd CM, McMillan SJ, et al. A critical role foreosinophils in allergic airways remodeling. Science, 2004,305: 1776-1779
118.Lee JJ, Dimina D, Macias MP, et al. Defining a link with asthma in micecongenitally deficient in eosinophils. Science, 2004,305: 1773-1776
119.Sexton DW, Walsh GM. Eosinophilepithelicalcell interactions:animportant facet of asthmatic inflammation. Clinical Experimental Allergy,2002, 32: 811-813
120. Duncan CJA, Lawrie A, Blaylock HG, et al. Reduced eosinophil apoptosisin induced sputum correlates with asthma severity. European RespiratoryJournal, 2003, 22: 484-490
121. Miranda C，Busaeker A，Balzar S，et al. Distinguishing severe asthmaphenotype: role of age at onset and eosinophilie inflammation. J AllergyClin Immunol,2004,13:101-108
122. Lukacs NW, Lamm WJ, Strieter RM, et al. Airway hyperreactivity isassociated with specific leukocyte subset infiltration in a mouse modelof allergic airway inflammation[J]. Pathobiology, 1996,64(6): 308-313.
123.Anticevich SZ, Hughes JM, Black JL, et al. Induction of hyperresponsivenessin human airway tissue by neutrophils mechanism of action，ClinExp Allergy, 1996,26(5):549-556.
124.Park HS, Uh ST, Park CS. lncreased neutrophil chemotactic activity i5noted in alminuminducedoccupational asthma[J]. Korean J Intern Med,1996,11(1):69-73
125.李峰,赵红洋,温暖,等.支气管哮喘发病机制进展. 实用全科医学,2007,5(10):922-923
126.Foresi A, Teodoro C, Leone C, et al. osinophil apoptosis in induced sputumfrom patients with seasonal allergic rhinitis and with asymptomatic andsymptomatic asthma. Ann Allergy Asthma Immunol, 2000, 84: 411-416
127.Kankaanranta H, Lindsay MA, Giembycz MA, et al. Delayed eosinophilapoptosis in asthma. J Allergy Clin Immunol,2000, 106: 77-83
128.Woolley KL, Gibson PG, Carty K, et al. Eosinophil apoptosis and theresolution of airway inflammation in asthma. Am J Respir Crit Care Med,1996, 154: 237-243
129.Walsh GM. Eosinphils apoptosis: mechanisms and clinical revelance inasthmatic and allergic inflammation. Br J Haematol, 2000,111: 61-67
130.Gounni AS, Gregory B, Nutku E, et al. Interleukin9enhancesinterleukin5receptor expression, differentiation, and survival of humaneosinophils. Blood, 2000;96: 2163-2171
131.Luttmann W, Knoechel B, FoersterM, et al. Activation of humaneosinophils by IL13.Induction of CD69 surface antigen, its relationshipto messenger RNA expression, and promotion of cellular viability. JImmunol, 1996,157: 1678-1683
132. Valerius T, Repp R, kalden JR, et al. Effects of IFN on human eosinophilsin comparison with other cytokines. A novel class of eosinophil activatorswith delayed onset of action. J Immunol, 1990, 145: 2950-2958
133.Fujihara S, Ward C, Dransfield I, et al. Inhibition of nuclear factorkappaBactivation unmasksthe ability of TNFalphato induce human eosinophilapoptosis. Eur J Immunol, 2002,32: 457-466
134.Hamada A, Watanabe N, Ohtomo H, et al. Nerve growth factor enhancessurvival and cytotoxic activity of human eosinophils. Br J Haematol,1996,93: 299-302
135.Robertson NM, Zangrilli JG, Steplewski, A et al. Differential expression ofTRAIL and TRAIL receptors in allergic asthmatics following segmentalantigen challenge: Evidence for a role of TRAIL in eosinophil surviva.l JImmunol, 2002,169: 5986-5996
136.Gardai SJ, Hoontrakoon R, Goddard CD, et al. Oxidantmediatedmitochondrial injury in eosinophil apoptosis: enhancement byglucocorticoids and inhibition by granulocyte macrophage colony stimulatingfactor. J Immunol, 2003,170: 556-566
137.Takeuchi M, Hayakawa A, TakagiK, et al. The ophyline induced apoptosisof the IL3activated eosinophils of patients with bronchial asthma.Apoptosis, 1999, 4: 461-468
138. Nutku E, Zhuang Q, SoussiGounniA,et al. Functional expression ofIL12receptor by human eosinophils: IL12promotes eosinophilapoptosis. J Immunol ,2001,167: 1039-1046
139.Peachell P. Targeting the mast cell in asthma. Current Opinion inPharmacology,2005,5(3):16
140.Elias JA, Lee CG, Zheng T, et al. New insights into the pathogenesis ofasthma. J Clin Invest,2003,111(3):291-297
141.杜静综述，梁 晓燕审校.RNA干扰的基础研究和临床应用前景.国外医学计划生育/生殖健康分册,2006,25（6）：307-310
142.Amarzguioui M, Holen T, Babaie E, et al. Tolerance for mutations andchemical modifications in a siRNA. Nucleic Acid Res,2003,31(2):589-595
143.Saxena S, Jonsson ZO, Dutta A. Small RNAs with imperfect match toendogenous mRNA repress translation. Implications for offtargetactivityof small inhibitory RNA in mammalian cells. J Biol Chem,2003,278(45):44312-44319