纳米粒子介导p38α丝裂原活化蛋白激酶反义寡核苷酸转染对哮喘动物模型干预研究
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摘要
支气管哮喘(简称哮喘),是严重危害儿童身体健康的气道慢性炎症性疾病,其发病机制目前仍不十分明确。激素治疗仍然是首选的治疗方案,但仍有部分患儿症状得不到有效控制,故需探索新的治疗途径。丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)是近年证实的细胞外信号传入细胞内的关键性途径,在炎症反应、细胞周期和应激中起着重要的作用。本研究旨在通过反义寡核苷酸(antisense oligonucleotides, ASO)与人血清白蛋白和硫酸鱼精蛋白构成的生物性纳米粒子(nanoparticle, NP)呼吸道转染p38α-ASO,探讨p38α-ASO对哮喘动物模型的影响。采用电镜、逆转录聚合酶链反应、酶联免疫反应、蛋白质免疫印记和组织形态学等技术研究了NP理化性质;p38α-ASO-NP在体外T细胞中的转染效率、反义效应和特异性;p38α- ASO-NP对哮喘模型肺部炎症和支气管肺泡灌洗液中炎性细胞、TH1/TH2型细胞因子及转录因子GATA-3和气道重塑等方面的影响。本研究证实NP可有效提高无修饰的p38α-ASO转染效率,特异性地抑制p38αMAPK异常表达;减轻肺组织的炎症、嗜酸性粒细胞的浸润和气道粘液的高分泌性;首次提出p38α-ASO可能通过降低GATA-3表达抑制TH2型细胞因子的过度生成,纠正TH1/TH2细胞的平衡;通过抑制转化生长因子β1(transforming growth factor-β1,TGF-β1)的表达,减轻肺组织胶原沉积。证实呼吸道转染p38α-ASO-NP是控制哮喘肺部炎症和减轻气道重构的有效途径。该研究具有一定的临床意义,为今后的临床研究和药物研发奠定了一定的基础。
Allergic asthma is a chronic disease of the lungs characterized by chronic eosinophilic airway inflammation, airway remodelling, mucus hypersecretion,reversible airway obstruction and airway hyperresponsiveness to bronchoconstrictor, which is one of the commonest diseases in children and whose highest rate of increase was reported in children under 5 years old. This represents a profound public health care problem, and the annual medical care cost of asthma was huge, $6.2 billion for medical care just in the U.S. In China, the number of Chinese diagnosed with asthma was over 30 million, so placing a considerable burden to every family and society. Most patients with asthma respond well to current therapies with glucocorticoid and leukotriene receptor antagonist, however, a small percentage (10%) fail to respond well and cost more than 50% of the total asthma health care costs. So we must find other new ways to resolve it. With understanding the pathogenesis of asthma, the balance of Type 1 T-helper (TH1)/TH2 was regarded as the most important characteristic of asthma, especially TH2 lymphocytes playing an crucial role in the initiation, progression and persistence of asthma by secreting associated IL-4, IL-13, and IL-5 etc. So blocking TH2-derived cytokines or increasing the function of counteracting TH1 cells had been an area of intense research and development. However, treatment with antibody of IL-12, soluble IL-4 receptor and IL-5 have been rather disappointing to date, with only partial and transient reduction of eosinophil numbers and limited improvement of lung function. So far, people have found that just blocking a single mediator is unlikely to be very effective in this complex disease and mediator antagonists have not proved to be very effective compared with drugs that have a broad spectrum of anti-inflammatory effects, such as glucocorticoids., because over one hundred mediators have now been implicated in inflammatory diseases of the airway inflammation, including multiple cytokines, chemokines and growth factors, which interact broadly. And chronic inflammation was also involved with airway structural cells, such as airway epithelial cell, airway smooth muscle cell and fibroblast. Then people wanted to look for a new upstream approach mediating multiple inflammatory cells and mediators involved in asthma. Recently, the mitogen-activated protein kinase (MAPK) has been shown to be pivotal in the activation of various immune cells and inflammatory cells, which is ubiquitous in all tissues. It is activated by a conservative three-tiered sequential phosphorylation of MAPK kinase kinase, MAPK kinase and MAPK. There are three major groups of MAPK in mammalian cells, including extracellular signal-regulated protein kinase, p38 MAPK, and c-Jun NH2-terminal kinase. Some studies showed that p38 MAPK activity in the lungs of asthmatic mice was significantly higher as compared with normal mice and activated on ligation of T cell receptor in T cells, B cell receptor in B cells, and FcεRⅠin mast cells, leading to proliferation, differentiation, cytokine production, and degranulation. It has also been shown that the p38 MAPK pathway is critically important for the activation of the IL-5 receptor and eotaxin receptor in eosinophils, resulting in survival, activation, degranulation, and chemotaxis of eosinophils. A recent study revealed that the p38MAPK inhibitor SB239063 markedly reduced pulmonary eosinophilia in animal models of asthma, suggesting that the p38 MAPK pathway can be a pharmacologic target for the treatment of asthma. However, SB239063 is not selective among p38 MAPK isoforms, because p38 MAPK exists in four distinct isoforms , involved in many physiological processes. So inhibiting all p38 MAPK isoforms unselectively will produce many unwanted side effects. In addition, in that study, the inhibitor was given systemically, not topically, and airway eosinophilia was the only endpoint reported. In the present study, we have designed and synthesized a potent and selective p38α-ASO and investigated its anti-inflammatory effects and roles in airway remodeling in a mouse asthma model.
Antisense oligonucleotides (ASO) represent a novel therapeutic approach to the treatment of asthma. ASO are usually short strands of nucleic acid that are complementary to the target mRNA, resulting in specific inhibition of target gene expression by various mechanism and reducing the level of translation of the target mRNA species. To circumvent systemic delivery of ASO, we adopted pulmonary topical application of ASO, because the lung is considered an excellent tissue for local ASO administration. But native unmodified phosphodiester oligonucleotides (PS-ONs) are highly susceptible towards nuclease degradation. To overcome this problem, chemical modifications of DNA must be applied, but it will reduce specific inhibition and enhance cell toxicity of ASO. In this present study, we firstly prepared a biodegradable, non-toxic ternary system of albumin- protamine-oligonuleotide nanoparticle (AlPrO-NP), first developed by Vogel, with p38α-ASO, sulfate protamine and human serum albumine preventing native ONs to be degradated. The physical properties of p38α-AlPrO-NP were investigated by transmission electron microscopy and laser scattering particle size analyzer. Then using the p38α-AlPrO-NP, we investigated the effects of p38α-ASO on the in vitro cultured T cells from mouse asthma model sensitized and challenged with OVA, including inhibition of p38α/βmRNA and p38α/βprotein, the changes of TH1/TH2 cytokines in supernatant. For investigating the effects of p38α-AlPrO-NP on animal model, we sensitized and challenged mice with OVA,and 72 Balb/c mice were divided into 9 groups depending on different treatment and p38α-ASO dosage. Next, in varied groups, we investigated the effects of p38α-AlPrO-NP on the pulmonary eosinophilia, the expression of p38αmRNA/protein, and the changes of cytokines in bronchoalveolar lavage fluid (BALF) by using histologic examination, RT-PCR and ELISA respectively. To investigate the pathogenesis of the changes of cytokines in BALF, we measured the TH2 specific transcription factor GATA-3 which is pivotal for the generation of TH2 cytokines on the levels of mRNA. Last, we investigated the effects of p38α-AlPrO-NP on the animal model airway remodelling by histologic examination stained with hematoxylin and eosin for examining inflammatory cells infiltration, with periodic acid-Schiff stain for measuring mucus production and goblet cell metaplasia, with masson for collagen hyperplasia in pulmonary tissue from different groups. To probe into the possible pathogenesis of p38αMAPK involved in airway remodelling, we investigated the differece of transforming growth factor-β1(TGF-β1) expression in pulmonary tissue from groups using western-blot and RT-PCR .
Reslults: we prepared the ternary system of p38α-AlPrO-NP successfully, which was assembled in isotonic media with a dh in a range of 240- 350nm for most preparations investigated by transmission electron microscopy and laser scattering particle size analyzer. The chemical composition of the NP was investigated by ultraviolet spectrophotometer. Approximately, 85- 93% (w/w) of the ONs, 7-10% (w/w) of the HAS, and 68-75% (w/w) of the PS in preparation solution were bound to the NP. We tested p38α-AlPrO-NP on in vitro cultured T cells from asthmatic animal model and found that p38α-AlPrO-NP substantially increased the cellular uptake and antisense effect of p38α-ASO compared to phosphorothioate modified oligonuleotides (PS-ONs) and native unmodified ASO. p38α-AlPrO-NP inhalation can inhibit the expression of p38αprotein/mRNA specially, without effect on the expression of p38β, indicating the specificity of the p38α-ASO for p38α. These results described a potent, isoform- specific p38α-ASO useful for mouse asthma model study. OVA inhalation significantly increased total cell, eosinophil, macrophage, and lymphocyte counts as compared with saline control. Inhalation of p38α-AlPrO-NP (over 3.33μg/kg on p38α-ASO) substantially reduced the total cell number, which was mainly caused by a significant reduction in eosinophil count. However, inhalation of PS-ONs (just over 33.3μg/kg on p38α-ASO) partly reduced those mentioned above. The numbers of neutrophils, macrophages, and lymphocytes were not affected by the p38α-AlPrO-NP. Inhalation of p38α-AlPrO-NP (over 3.33μg/kg on p38α-ASO) significantly attenuated the pulmonary eosinophilic inflammation as compared with asthmatic control. OVA-challenged developed marked goblet cell hyperplasia and mucus hypersecretion within the bronchi in the lung. The OVA-induced mucus secretion and goblet cell hyperplasia were substantially reduced by p38α- ASO(over 3.33μg/kg) likewise compared with asthmatic control. To determine the levels of cytokines in BALF, we measured the BALF 2h after the last OVA challenge. OVA inhalation induced substantial TH2 cytokine release into BALF, compared with normal control, however, the overexpression was reduced by inhalation of p38α-ASO in a dose-related manner. To testified the inhibition of p38αby inhalation of p38α-AlPrO-NP, we measured the expression of p38αin pulmonary tissue from different groups on the g levels of gene and protein. The datum indicated that inhalation of p38α-AlPrO-NP may inhibit the expression of p38αon the levels of protein and mRNA. To explore the pathogenesis of airway inflammation of asthma deeply, we investigated the TH2-specific transcription factor GATA-3, which was pivotal in the production of TH2 cytokines. The result indicated the p38α-ASO reduced the expression of TH2 cytokines likely from downregulation of GATA-3 in inflammatory cells. For studying the role of p38αin airway remodeling, we tested the level of collagen in mice pulmonary tissue, and the result showed that overexpression of collagen was significantly observed in mice challenged with OVA as compared with mice treated with saline. After inhalation of p38α-AlPrO-NP (over 3.33μg/kg on p38α-ASO), collagen expression was substantially reduced, in good agree with the histologic determination of lung tissue stained with masson. To study the pathogenesis of asthmatic airway remodeling, the level of transforming growth factorβ1(TGF-β1) was observed and the gene and protein expression of TGF-β1 were reduced by p38α-AlPrO-NP inhalation in a dose-dependent manner, so indicating that p38αmay be implicated in airway remodeling through reducing TGF-β1 expression.
These findings demonstrate NP is a promising potent carrier for ASO drugs administration. Combining with NP technique, the p38α-AlPrO-NP increases significantly the antisense effect. And our findings support the use of inhaled ASO as a highly effective pharmacologic approach for asthma treatment, and suggest that aerosolized p38α-AlPrO-NP may have therapeutic potential for allergic airway inflammation and remodelling.
Allergic asthma is a chronic disease of the lungs characterized by chronic eosinophilic airway inflammation, airway remodelling, mucus hypersecretion,reversible airway obstruction and airway hyperresponsiveness to bronchoconstrictor, which is one of the commonest diseases in children and whose highest rate of increase was reported in children under 5 years old. This represents a profound public health care problem, and the annual medical care cost of asthma was huge, $6.2 billion for medical care just in the U.S. In China, the number of Chinese diagnosed with asthma was over 30 million, so placing a considerable burden to every family and society. Most patients with asthma respond well to current therapies with glucocorticoid and leukotriene receptor antagonist, however, a small percentage (10%) fail to respond well and cost more than 50% of the total asthma health care costs. So we must find other new ways to resolve it. With understanding the pathogenesis of asthma, the balance of Type 1 T-helper (TH1)/TH2 was regarded as the most important characteristic of asthma, especially TH2 lymphocytes playing an crucial role in the initiation, progression and persistence of asthma by secreting associated IL-4, IL-13, and IL-5 etc. So blocking TH2-derived cytokines or increasing the function of counteracting TH1 cells had been an area of intense research and development. However, treatment with antibody of IL-12, soluble IL-4 receptor and IL-5 have been rather disappointing to date, with only partial and transient reduction of eosinophil numbers and limited improvement of lung function. So far, people have found that just blocking a single mediator is unlikely to be very effective in this complex disease and mediator antagonists have not proved to be very effective compared with drugs that have a broad spectrum of anti-inflammatory effects, such as glucocorticoids., because over one hundred mediators have now been implicated in inflammatory diseases of the airway inflammation, including multiple cytokines, chemokines and growth factors, which interact broadly. And chronic inflammation was also involved with airway structural cells, such as airway epithelial cell, airway smooth muscle cell and fibroblast. Then people wanted to look for a new upstream approach mediating multiple inflammatory cells and mediators involved in asthma. Recently, the mitogen-activated protein kinase (MAPK) has been shown to be pivotal in the activation of various immune cells and inflammatory cells, which is ubiquitous in all tissues. It is activated by a conservative three-tiered sequential phosphorylation of MAPK kinase kinase, MAPK kinase and MAPK. There are three major groups of MAPK in mammalian cells, including extracellular signal-regulated protein kinase, p38 MAPK, and c-Jun NH2-terminal kinase. Some studies showed that p38 MAPK activity in the lungs of asthmatic mice was significantly higher as compared with normal mice and activated on ligation of T cell receptor in T cells, B cell receptor in B cells, and FcεRⅠin mast cells, leading to proliferation, differentiation, cytokine production, and degranulation. It has also been shown that the p38 MAPK pathway is critically important for the activation of the IL-5 receptor and eotaxin receptor in eosinophils, resulting in survival, activation, degranulation, and chemotaxis of eosinophils. A recent study revealed that the p38MAPK inhibitor SB239063 markedly reduced pulmonary eosinophilia in animal models of asthma, suggesting that the p38 MAPK pathway can be a pharmacologic target for the treatment of asthma. However, SB239063 is not selective among p38 MAPK isoforms, because p38 MAPK exists in four distinct isoforms , involved in many physiological processes. So inhibiting all p38 MAPK isoforms unselectively will produce many unwanted side effects. In addition, in that study, the inhibitor was given systemically, not topically, and airway eosinophilia was the only endpoint reported. In the present study, we have designed and synthesized a potent and selective p38α-ASO and investigated its anti-inflammatory effects and roles in airway remodeling in a mouse asthma model.
Antisense oligonucleotides (ASO) represent a novel therapeutic approach to the treatment of asthma. ASO are usually short strands of nucleic acid that are complementary to the target mRNA, resulting in specific inhibition of target gene expression by various mechanism and reducing the level of translation of the target mRNA species. To circumvent systemic delivery of ASO, we adopted pulmonary topical application of ASO, because the lung is considered an excellent tissue for local ASO administration. But native unmodified phosphodiester oligonucleotides (PS-ONs) are highly susceptible towards nuclease degradation. To overcome this problem, chemical modifications of DNA must be applied, but it will reduce specific inhibition and enhance cell toxicity of ASO. In this present study, we firstly prepared a biodegradable, non-toxic ternary system of albumin- protamine-oligonuleotide nanoparticle (AlPrO-NP), first developed by Vogel, with p38α-ASO, sulfate protamine and human serum albumine preventing native ONs to be degradated. The physical properties of p38α-AlPrO-NP were investigated by transmission electron microscopy and laser scattering particle size analyzer. Then using the p38α-AlPrO-NP, we investigated the effects of p38α-ASO on the in vitro cultured T cells from mouse asthma model sensitized and challenged with OVA, including inhibition of p38α/βmRNA and p38α/βprotein, the changes of TH1/TH2 cytokines in supernatant. For investigating the effects of p38α-AlPrO-NP on animal model, we sensitized and challenged mice with OVA,and 72 Balb/c mice were divided into 9 groups depending on different treatment and p38α-ASO dosage. Next, in varied groups, we investigated the effects of p38α-AlPrO-NP on the pulmonary eosinophilia, the expression of p38αmRNA/protein, and the changes of cytokines in bronchoalveolar lavage fluid (BALF) by using histologic examination, RT-PCR and ELISA respectively. To investigate the pathogenesis of the changes of cytokines in BALF, we measured the TH2 specific transcription factor GATA-3 which is pivotal for the generation of TH2 cytokines on the levels of mRNA. Last, we investigated the effects of p38α-AlPrO-NP on the animal model airway remodelling by histologic examination stained with hematoxylin and eosin for examining inflammatory cells infiltration, with periodic acid-Schiff stain for measuring mucus production and goblet cell metaplasia, with masson for collagen hyperplasia in pulmonary tissue from different groups. To probe into the possible pathogenesis of p38αMAPK involved in airway remodelling, we investigated the differece of transforming growth factor-β1(TGF-β1) expression in pulmonary tissue from groups using western-blot and RT-PCR .
Reslults: we prepared the ternary system of p38α-AlPrO-NP successfully, which was assembled in isotonic media with a dh in a range of 240- 350nm for most preparations investigated by transmission electron microscopy and laser scattering particle size analyzer. The chemical composition of the NP was investigated by ultraviolet spectrophotometer. Approximately, 85- 93% (w/w) of the ONs, 7-10% (w/w) of the HAS, and 68-75% (w/w) of the PS in preparation solution were bound to the NP. We tested p38α-AlPrO-NP on in vitro cultured T cells from asthmatic animal model and found that p38α-AlPrO-NP substantially increased the cellular uptake and antisense effect of p38α-ASO compared to phosphorothioate modified oligonuleotides (PS-ONs) and native unmodified ASO. p38α-AlPrO-NP inhalation can inhibit the expression of p38αprotein/mRNA specially, without effect on the expression of p38β, indicating the specificity of the p38α-ASO for p38α. These results described a potent, isoform- specific p38α-ASO useful for mouse asthma model study. OVA inhalation significantly increased total cell, eosinophil, macrophage, and lymphocyte counts as compared with saline control. Inhalation of p38α-AlPrO-NP (over 3.33μg/kg on p38α-ASO) substantially reduced the total cell number, which was mainly caused by a significant reduction in eosinophil count. However, inhalation of PS-ONs (just over 33.3μg/kg on p38α-ASO) partly reduced those mentioned above. The numbers of neutrophils, macrophages, and lymphocytes were not affected by the p38α-AlPrO-NP. Inhalation of p38α-AlPrO-NP (over 3.33μg/kg on p38α-ASO) significantly attenuated the pulmonary eosinophilic inflammation as compared with asthmatic control. OVA-challenged developed marked goblet cell hyperplasia and mucus hypersecretion within the bronchi in the lung. The OVA-induced mucus secretion and goblet cell hyperplasia were substantially reduced by p38α- ASO(over 3.33μg/kg) likewise compared with asthmatic control. To determine the levels of cytokines in BALF, we measured the BALF 2h after the last OVA challenge. OVA inhalation induced substantial TH2 cytokine release into BALF, compared with normal control, however, the overexpression was reduced by inhalation of p38α-ASO in a dose-related manner. To testified the inhibition of p38αby inhalation of p38α-AlPrO-NP, we measured the expression of p38αin pulmonary tissue from different groups on the g levels of gene and protein. The datum indicated that inhalation of p38α-AlPrO-NP may inhibit the expression of p38αon the levels of protein and mRNA. To explore the pathogenesis of airway inflammation of asthma deeply, we investigated the TH2-specific transcription factor GATA-3, which was pivotal in the production of TH2 cytokines. The result indicated the p38α-ASO reduced the expression of TH2 cytokines likely from downregulation of GATA-3 in inflammatory cells. For studying the role of p38αin airway remodeling, we tested the level of collagen in mice pulmonary tissue, and the result showed that overexpression of collagen was significantly observed in mice challenged with OVA as compared with mice treated with saline. After inhalation of p38α-AlPrO-NP (over 3.33μg/kg on p38α-ASO), collagen expression was substantially reduced, in good agree with the histologic determination of lung tissue stained with masson. To study the pathogenesis of asthmatic airway remodeling, the level of transforming growth factorβ1(TGF-β1) was observed and the gene and protein expression of TGF-β1 were reduced by p38α-AlPrO-NP inhalation in a dose-dependent manner, so indicating that p38αmay be implicated in airway remodeling through reducing TGF-β1 expression.
These findings demonstrate NP is a promising potent carrier for ASO drugs administration. Combining with NP technique, the p38α-AlPrO-NP increases significantly the antisense effect. And our findings support the use of inhaled ASO as a highly effective pharmacologic approach for asthma treatment, and suggest that aerosolized p38α-AlPrO-NP may have therapeutic potential for allergic airway inflammation and remodelling.
引文
1. 全国儿科哮喘防治协作组.中国城区儿童哮喘患病率调查.中华儿科杂志,2003,41(2):123-127.
2. Adcock IM, Chung KF, Caramori G, et al. Kinase inhibitors and airway inflammation. European Journal of Pharmacology, 2006, 533(1-3):118-132.
3. Shirai T, Suxuki K, Inui N, et al. Th1/Th2 profile in peripheral blood in atopic cough and atopic asthma. Clin Exp Allergy, 2003, 33(1):84-89.
4. Maizels RM, Yazdanbakhsh M. Immune regulation by helminth parasites: cellular and molecular mechanisms. Nat Rev Immunol, 2003, 3(9):733-744.
5. Irene HH, Antoon JM. Targeting T cells for asthma. Current Opinion in Pharmacology, 2005, 5(3):227-231.
6. Barnes PJ. New drugs for asthma. Nat Rev Drug Discov, 2004, 3(10):831-844.
7. Barnes PJ. Anti-leukotrienes: here to say? Curr Opin Pharmacol, 2003, 3(3):257-263.
8. Barnes PJ, Chung KF, Page CP, et al. Inflammatory mediators of asthma: an update. Pharmacol Rev, 1998,50(4):515-596.
9. Zhou M, Ouyang W. The function role of GATA-3 in TH1 and TH2 differentiation. Immunol Res, 2003, 28(1):25-37.
10. Catley MC, Cambridge IM, Nasuhara Y, et al. Inhibitors of protein kinase C prevent activated transcription: role of events downstream of NF-κB DNA binding. J Biol Chem, 279(18):18457-18466.
11. Barnes PJ, Hansel TT. Prospects for new drugs for chronic obstructive pulmonary disease. Lancet, 2004,364(9438):985-996.
12. Barnes PJ. Novel signal transduction modulators for the treatment of airway diseases. Pharmacology & Therapeutics, 2006, 109(1-2):238-245.
13. Manning G, Whyte DB, Martinez R, et al. The protein kinase complement of the human genome. Science,2002,298(5600): 1912-1934.
14. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinase. Science, 2002,298(5600):1911-1912
15. 黄翠萍,张珍祥,徐永健.p38 丝裂原活化蛋白激酶信号传导通路和支气管哮喘气道炎症.国外医学呼吸系统分册,2005,25(9):656-658.
16. Saklatvala J. The p38 MAP kinase pathway as a therapeutic target in inflammatory disease. Curr Opin Pharmacol, 2004, 4(4):372-377.
17. Dodeller F, Skapenko A, Kaldan JR, et al. The p38 mitogen activated protein kinase regulates effector functions of primary human CD4 T cells. Eur J Immunol, 2005, 35(12):3631-3642.
18. Kankaanranta H, Giembycz MA, Barnes P, et al. SB203580, an inhibitor of p38 mitogen-activated protein kinase, enhances constitutive apoptosis of cytokine-deprived human eosinophils. J Pharmacol Exp Ther, 1999, 290(2):621-628.
19. Ali S, Leonard SA, Kukoly CA, et al. Absorption, distribution, metabolism, and excretion of a respirable antisense oligonucleotide for asthma. Am J Respir Crit Care Med, 2001, 163(4): 989-993.
20. Popescu FD. Antisense- and RNA interference-based therapeutic strategies in allergy. J Cell Mol Med, 2005, 9(4):840-853.
21. Zarubin T, Han JH. Activation and signaling of the p38 MAP kinase pathway. Cell Research, 2005, 15(1):11-18.
22. Rouse J, Cohen P, Trigon S, et al. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell, 1994, 78(6):1027-1037.
23. Han J, Lee JD, Bibbs L, et al. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science, 1994, 256(5173):808-811.
24. Hanks SK, Hunter T. The cukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J, 1999, 9(8):576-596.
25. Jiang Y, Li Z, Schwarz EM, et al. Structure-function studies of p38 mitogen-activated protein kinase. Loop-12 influence substrate specifity and autophosphorylation, but not upstream kinase selection. J Biol Chem, 1997, 272(17):11096-11102.
26. Dong C, Davis RJ, Flavell RA. MAP kinases in the immune response. Annu Rev Immunol, 2002, 20(4):55-72.
27. Parker CG, Hunt J, Diener K, et al. Identification of stathmin as a novel substrate for p38 delta. Biophys Res Commun, 1998, 249(3): 791-796.
28. Jiang Y, Gram H, Zhao M, et al. Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinase, p38delta. J Biol Chem, 1997,272(48):30122-30128.
29. Salvador JM, Mittelstadt PR, Guszczynsli T, et al. Alternative p38 activation pathway mediated by T cell receptor-proximal tyrosine kinases. Nat Immunol, 2005, 6(4):390-395.
30. Rudd CE. MAPK p38: alternative and nonstressful in T cells. Nat Immunol, 2005, 6(4):368-370.
31. Camps M, Nichols A, Gillieron C, et al. Catalytic activation of the phosphatases MKP-3 by ERK2 mitogen-activated protein kinase. Science, 1998, 280(5367): 1262-1265.
32. Yee AS, Paulson EK, McDevitt MA, et al. The HBPI1 transcriptional repressor and the p38 MAP kinase: unlikely partner in G1 regulation and tumor suppression. Gene, 2004, 336(1): 1-13.
33. Pereira RC, Delany AM, Canalis E. CCAAT/enhancer binding protein homologous protein (DDTT3) induces osteoblastic cell differentiation. Endocrinology, 2004, 145(4):1952-1960.
34. McLaughlin MM, Kumar S, McDonnell PC, et al. Identification of mitogen-activated protein (MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38 MAP kinase. J Biol Chem, 1996, 271(14): 8444-8492.
35. New L, Jiang Y, Zhao M, et al. PRAK, a novel protein kinase regulated by the p38 MAP kinase. EMBO J, 1998, 17(12): 3372-3384.
36. New L, Zhao M, Li Y, et al. Cloning and characterization of RLPK, a novel RSK-related protein kinase. J Biol Chem, 1999, 274(2): 1026-1032.
37. Sweeney SE, Firestein GS. Signal transduction in rheumatoid arthritis. Curr Opin Rheumatol, 2004, 16(3): 231-237.
38. Menzies-GA, Robinson DS.Eosinophils cytokines (interleukin-5), and antieosinophilic therapy in asthma. Curr Opin Med, 2002, 8(1):33-38.
39. Wong CK, Wang CB, Ip WK, et al. Role of p38 MAPK and NF-κB for chemokine release in coculture of human eosinophils and bronchial epithelial cells. Clinical and Experimental Immunology, 2005, 139(1):90-100.
40. Adachi T, Stafford S, Kayaba H, et al. Myosin light chain kinase mediates eosinophil chemotaxis in a mitogen-activated protein kinase-dependent manner. J Allergy Clin Immunol, 2003, 111(1):113-116.
41. Kampen GT, Stafford S, Adachi T, et al. Eotaxin induces degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases. Blood, 2000, 95(6):1911-1917.
42. Cuvelier SL, Paul S, Shariat N, et al. Eosinophil adhesion under flow conditions activates mechanosensitive signaling pathways in human endothelial cells. J Exp Med, 2005, 202(6):865-876.
43. Maa SH, Wang CH, Liu CY, et al. Endogenous nitric oxide downregulates theBcl-2 expression of eosinophils through mitogen-activated protein kinase in bronchial asthma. J Allergy Clin Immunol, 2003, 112(4):761-767.
44. Zhang JP, Wong CK, Lam CW. Role of caspases in dexamethne AS-ONs- induced apoptosis and activation of c-Jun Nh2-terminal kinase and p38 mitogen-activated protein kinase in human eosinophils. Clin Exp Immunol, 2000, 122(1): 20-27.
45. Yu JJ, Tripp CS, Russell JH. Regulation and phenotype of an innate Th1 cell: role of cytokines and the p38 MAP kinase pathway. J Immunol, 2003, 171(11):6112-6118.
46. Zhang J, Salojin KV, Gao JX, et al. p38 mitogen-activated protein kinase mediates signal integration of TCR/CD28 costimulation in primary murine T cells. J Immunol, 1999, 162(7):3819-3829.
47. Doceller F, Skapenko A, Kalden JR, et al. The p38 mitogen-activated protein kinase regulates effector functions of primary human CD4 T cells. Eur J Immunol, 2005, 35(12): 3631-3642.
48. Labuda T, Sundatedt A, Dohlsten M, et al. Selective induction of p38 mitogen-activated protein kinase activity following A6H costimulation in primary human CD4+T cells. Int Immunol, 2000, 12(3):253-261.
49. Chen CH, Zhang DH, LaPorte JM, et al. Cyclic AMP activates p38 mitogen-activated protein kinase in Th2 cells: phosphorylation of GATA-3 and stimulation of Th2 cytokine gene expression. J Immunol, 2000, 165(10):5597-5605.
50. Harlin H, Pocack E, Boothby M, et al. TCR-independent CD30 signaling selectively induces IL-13 production via a TNF receptor-associated factor/p38 mitogen-activated protein kinase-dependent mechanism. J Immunol, 2002, 169(5);2451-2459.
51. Schafer PH, Wadsworth SA, Wang L, et al. p38 alpha mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+CD45RO+ T cells and Th2 effector cells. J Immunol, 1999, 162(12):7110-7119.
52. Ishizuka T, Okajima F, Ishiwara M, et al. Sensitized mast cells migrate toward the antigen: a response regulated by p38 mitogen-activated protein kinase and Rho-associated coiled-coil-forming protein kinase. J Immunol, 2000, 167(4):2298-2304.
53. Masuda A, Yoshikai Y, Aiba K, et al. Th2 cytokine production from mast cellsis directly induced by lipopolysaccharide and distinctly regulated by c-Jun N-terminal kinase and p38 pathways. J Immunol, 2002, 169(7):3801-3810.
54. Hamilton LM, Puddicombe SM, Dearman RJ, et al. Altered protein tyrosine phosphorylation in asthmatic bronchial epithelium. Eur Respir J, 2005, 25(6):978-985.
55. Tsitoura DC, Rothman PB. Enhancement of MEK/ERK signaling promotes glucocorticoid resistance in CD4 + T cells. J Clin Invest, 2004, 113(4):619-627.
56. Pargellis C, Tong L, Churchill L, et al. Inhibition of p38 MAP kinase by utilizing a novel allosteric binding site. Nat Struct Biol, 2002, 9(4):268-272.
57. Stelmach JE, Liu L, Patel SB, et al. Design and synthesis of potent, orally bioavailable dihydroquinazolinone inhibitors of p38 MAP kinase. Bioorg Med Chem, Lett, 2003, 13(2):277-280
58. Fijen JW, Tulleken JE, Kobold AC, et al. Inhibition of p38 mitogen-activated protein kinase: dose-dependent suppression of leukocyte and endothelial response after endotoxin challerge in humans. Crit Care Med, 2002, 30(4):841-845
59. Kuma Y, Sabio G, Bain J, et al. BIRB796 inhibits all p38 MAPK isoforms in vitro and in vivo. J Biol Chem, 2005, 280(20):19472-19479.
60. Kurreck J. Antisense technologies. Improvement through novel chemical modification. Eur J Biochem, 2003, 270(8):1628-1644.
61. 陈援越,吕时铭. 反义核酸技术的现状.国外医学临床生物化学与检验学分册,2001,22(1):20-21.
62. Crooke ST. Molecular mechanisms of action of antisense drugs. Biochim Biophys Acta, 1999, 1489(1):31-44.
63. 张冰,龚磊,刘贤锡. 肿瘤反义治疗研究进展.国外医学肿瘤学分册,2004,31(9):674-677.
64. Zhao Q, Yu D, Agrawal S, et al. Site of chemical modification in CpG containing phosphorothioate oligodeoxynucleotide modulates its immunostimulatory activity. J Bioorg Med Chem Lett, 1999, 9(24):3453-3458.
65. Nielsen PE, Egholm M, Berg RH, et al. Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science, 1991, 254(5037): 1497-1500.
66. Suqimoto N, Yamamoto K, Satoh N. Positional effect of single bulgenucleotide on PNA (peptide nucleic acid)/DNA hybrid stability. J Nucleic Acids Symp Ser, 1999, 42:95-96.
67. Ball HA, Van MR, Robinson CB. Sense and antisense: therapeutic potential of oligonucleotides and interference RNA in asthma and allergic disorders. Clin Rev Allergy Immunol, 2004, 27(3):207-217.
68. Nyce JT. Respirable antisense oligonucleotides: a new, third drug class targeting respiratory disease. Curr Opin Allergy Clin Immunol, 2002, 2 (6):533-536.
69. Crosby JR, Tung D, Guba M, et al. Inhaled TNF-α antisense oligonucleotides inhibits lung inflammation and airway hyper-responsiveness in a mouse model of chronic asthma (poster 311). ATS 2005 San Diego, Abstract Page: A905.
70. Turner AJ, Murphy LJ. Molecular pharmacology of endothelin converting enzyme. Biochem Pharmaco, 1996, 51(2): 91-102.
71. 文忠成,徐军,钟南山. 反义寡核苷酸对气道平滑肌细胞内皮素转换酶及内皮素-1 表达的影响. 中华结核和呼吸杂志,1999,22(5):279-282.
72. Kelly AE, Melo ME, Smith E, et al. Complex role of the IL-4 receptor in a murine model of airway inflammation: expression of the IL-4 receptor on nonlymphoid cells of bone marrow origin contributes to severity of inflammation. J Immunol, 2004, 172(7): 4545-4555.
73. 谢华,孙滨. 粘附分子与哮喘气道炎症细胞的迁移.细胞与分子免疫学杂志,1997,13(2):45-48.
74. Borchers MT, Crosby J, Farmer S, et al. Blockade of CD49d inhibits allergic airway pathologies independent of effects on leukocyte recruitment. Am J Physicol Lung Cell Mol Physicol, 2001, 280(4):L813-521.
75. Lofthouse SA, Crosby JR, Tung D, et al. Aerosol delivery of VLA-4 specific antisense oligonucleotides inhibites airway inflammation and hyperresponsiveness in mice. Respirology, 2005, 10:A11.
76. Fiset PO, Soussi-Gounni A, Christodoulopoulos P, et al. Modulation of allergic response in nasal mucosa by antisense oligodeoxynucleotides for IL-4. J Allergy Clin Immunol, 2003, 111(3):580-586.
77. Mousavi T, Mazer B, Tebianian M. Inhibition of IL-13 by antisense oligonucleotides changes immunoglobulin isotype profile in cultured B-lymphocytes. Iran Biomed J, 2004, 8:185-191.
78. Lach TE, McKay RA, Monia BP, et al. In vitro and in vivo inhibition ofinterleukin IL-5-mediated eosinopoiesis by murine IL-5Rα antisense oligonucleotides. Am J Respir Cell Mol Biol, 2001, 24(2):116-122.
79. Hanai K, Kurokawa T, Minakuchi Y, et al. Potential of atelocollagen- mediated systemic antisense therapeutics for inflammatory disease. Hum Gene Ther, 2004, 15(3):263-272.
80. 林科雄,王长征,钱桂生. 雷公腾对哮喘豚鼠 IL-5、GM-CSF 基因转录的影响及作用机制.中国免疫学杂志,2000,16(6):325-328.
81. Allakhverdi Z, Allam M, Renzi PM. Inhibition of antigen-induced eosinophilia and airway hyperresponsiveness by antisense oligonucleotides directed against the common β chain of IL-3,IL-5,GM-CSF receptors in a rat model of allergic asthma. Am J Respir Crit Care Med, 2002, 165(7):1015-1021.
82. Finotto S, De Sanctis GT, Lehr HA, et al. Treatment of allergic inflammation and hyperresponsiveness by antisense-induced local blockade of GATA-3 expression. J Exp Med, 2001, 193(11):1247-1260.
83. Peng Q, Matsuda T, Hirst SJ, et al. Signaling pathways regulating interleukin- 13-stimulated chemokine release from airway smooth muscle. Am J Respir Crit Care Med, 2004, 169(5):596-603.
84. Choi IW, Kim DK, Ko HM, et al. Administration of antisense phosphorothiate oligonucleotide to the p65 subunit of NF-κB inhibits established asthmatic reaction in mice. Int Immunopharmacol, 2004, 4(14):1817-1828.
85. Stafford S, Lowell C, Sur S, et al. Lyn tyrosine kinase is important for IL-5-stimulated eosinophil differentiation. J Immunol, 2002, 168 (4): 1978 - 1983.
86. Ulanova M, Puttagunta L, Kim MK, et al. Antisense oligonucleotides to Syk kinase: a novel therapeutic approach for respiratory disorders. Curr Opin Investig Drugs, 2003, 4(5):552-555.
87. Duan W, Chan JH, McKay K, et al. Inhaled p38αmitogen-activated protein kinase antisense oligonucleotide attenuates asthma in mice. Am J Respir Crit Care Med, 2005, 171(6):571-578.
88. 伍志坚,陈诗书,彭朝晖.逆转录病毒载体用于基因治疗的安全性. 国外医学分子生物学分册,1995,17(2):71.
89. 许海燕. 纳米技术在医学领域的应用研究. 中华医学杂志,2006,86(8):505-506.
90. Lockman PR, Mumper RJ, Khan MA, et al. Nanoparticle technology for drug delivery across the blood-brain barrier.Drug Dev Ind Pharm, 2002,28(1):1-13.
91. Borchard G., Junginger HE. Modern drug delivery applications of chitosan. Adv Drug Deliver Rev, 2001, 52(2):103.
92. Mumper RJ, Wang J, Klaspell SL, et al. Protective interactive noncondensing (PINC) polymers for enhanced plasmid distribution and expression in rat skeletal muscle. J Control Release, 1998, 52(1-2):191-203.
93. Illum L, Jabbal-Gill I, Hinchcliffe M, et al.Chitosan as a novel nasal delivery system for vaccines. Adv Drug Deliver Rev, 2001, 51(1-3):81.
94. Erbacher P,Zou S,Bettinger T,et al. Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability. Pharm Res, 1998, 15(9):1332.
95. Nakanishi M, Noguchi A. Confocal and probe microscopy to study gene transfection mediated by cationic liposomes with a cationic cholesterol derivative. Adv Drug Deliver Rev, 2001, 52(3):197
96. Kiang T, Wen J, Lim H W, e1 al. The effect of the degree of chitosan deacetylation on the efficiency of gene transfection. Biomalerials, 2004, 25( 22):5293.
97. 黄伟.壳聚糖纳米粒基因递送系统的研究.北京.中国人民解放军军事医学科学院.2002.
98. Richardson SC, Kolbe HV, Duncan R. Potential of low molecular mass chitosan as a DNA delivery system: biocompatibility, body distribution and ability to complex and protect DNA. Int J Pharm, 1999, 178 (2):231-243.
99. Lee KY, Kwon IC, Kim YH, et al. Preparation of chitosan self-aggregates as a gene delivery system. Macromolecules, 1998, 51(2):213-220.
100. Park YK, Park YH, Shin BA, et al.Galactosylated chitosan-graft-dextran as hepatocyte-targeting DNA carrier. J Controlled Release, 2000, 69(1):97.
101. Murata J, Kitamoto T, Ohya Y, et al. Effect of dimerization of the D-glucose analogue of muramyl dipeptide on stimulation of macrophage-like cells. Carbohydr Res, 1997, 297(2):127-133.
102. Kim TH, Ihm JE, Choi YJ, et al. Efficient gene delivery by urocanic acid-modified chitosan. J Controlled Release, 2003, 93(3):389.
103. Junghans M, Kreuter J, Zimmer A. Antisense delivery using protamine- oligonucleotide particles. Nucleic Acids Res, 2000, 28(10):E45.
104. Lochmann D, Vogel V, Weyermann J, et al. Physicochemical characterization of protamine-phosphorothioate nanoparticles. J Microencapsulation, 2004,21(6):625-641.
105. Vogel V, Lochmann D, Weyermann J, et al. Oligonucleotide– protamine- albumin nanoparticles: preparation, physical properties, and intracellular distribution. J Controlled Rel, 2005, 103 (1):99-111.
106. Lochmann D, Weyermann J, Georgens C, et al. Albumin- protamine- oligonucleotide-nanoparticles as a new antisense delivery system. Part 1: Physicochemical characterization. European Journal of Pharmaceutics and Biopharmaceutics, 2005, 59(3):419-429.
107. Arnedo A, Irache JM, Merodio M, et al. Albumin nanoparticles improved the stability, nuclear accumulation and anticytomegaloviral activity of a phosphodiester oligonuceotide. Journal of Controlled Release, 2004, 94 (1):217-227.
108. Mayer G, Vogel V, Weyermann J, et al. Oligonucleotide- protamine- albumin nanoparticles: Protamine sulfate causes drastic size reduction. Journal of Controlled Release, 2006, 106 (1-2):181-187.
109. 陈美英,王士斌. 可生物降解聚合物作为药物载体的应用进展.国际生物医学杂志,2006,29(5):300-303.
110. Wang J, Wang BM, Schwendeman SP. Characterization of the initial burst release of a model peptide from poly (D.L-lactide-co-glycolide) microspheres. J Contr Rel, 2002, 82(2-3):289-307.
111. Wu LB, Ding JD. In vitro degradation of three-dimensional porous poly (D.L-lactide-co-glycolide) scaffolda for tissue engineering. Biomaterials, 2004, 25(27):5821-5830.
112. Sinha VR, Trchan A. Biodegradable microspheres for protein delivery. J Contr Rel, 2003, 90(3):261-280.
113. Zamecnik PC, Stephenson ML. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Pro, Natl Acad. USA 75(1978): 280-284.
114. Lebedeva I, Benimetskaya L, Stein CA, et al. Cellular delivery of antisense oligonucleotides. Eur J Pharm Biopharm, 2000, 50(1):101-119.
115. 杨远,贾文祥,祁欣,等.可生物降解材料作为基因载体的研究.生物医学工程学杂志,2006,23(3):573-577.
116. Duzgunes N, De Ilarduya CT, Simoes S, et al. Cationic liposomes for gene delivery: novel cationic lipids and enhancement by proteins and peptides. Curr Med Chem, 2003, 10 (14):1213-1220.
117. Weyermann J, Lochmann D, Georgens C, et al. Albumin- protamine- oligonucleotide-nanoparticles as a new antisense delivery system. Part 2: cellular uptake and effect. European Journal of Pharmaceutics and Biopharmaceutics, 2005, 59(3):431-438.
118. Wartlick B, Schmitt BS, Strebhardt K, et al. Tumour cell delivery of antisense oligonuleotides by human serum albumin nanoparticles. J Control Release, 2004, 96 (3):483-495.
119. Welsh DJ, Scott PH, Peacock AJ. p38 MAP kinase isoform activity and cell cycle regulators in the proliferative response of pulmonary and systemic artery fibroblasts to acute hypoxia. Pulmonary Pharmacology Therapeutics,2006,19(2):128-138.
120. Rogers DF. Airway mucus hypersecretion in asthma: an undervalued pathology? Current Opinion in Pharmacology, 2004, 4(3):241-250.
121. Irusen E, Matthews JG, Takahashi A, et al. p38 Mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces its activity: role in steroid-insensitive asthma. J Allergy Clin Immunol, 2002 ,109(4):649-657.
122. Schreiber F, Matthias P, Müller MM, et al. Rapid detection of octamer binding protein with “mini-extracts”, prepared from a small number of cells. Nucleic Acids Res, 1989, 17(15):6419.
123. 杜宏举, 汤宁. 丝裂原活化蛋白激酶信号通路及其生物学功能. 国外医学卫生学分册,2005,32(4):197-201.
124. Underwood DC, Obsorn RR, Kotzer CJ, et al. SB239063, a potent p38 MAP kinase inhibitor, reduces inflammatory cytokine production, airways eosinophil infiltration, and persistence. J Pharmacol Exp Ther, 2000, 293(1):281-288.
125. Acosta R, Montanez C, Gomez P, et al. Delivery of antisense oligonuleotides to PC12 cells. Neurosci Res, 2002, 43(1): 81-86.
126. 吴杰,刘克良. 反义寡核苷酸类化合物的体内转运研究进展. 中国新药杂志,2003,12(5):329-333.
127. Gavett SH, Chen X, Finkelmann F, et al. Depletion of murine CD4+ T lymphocytes prevents antigen induced airway hyperreactivity and pulmonary eosinophilia. Am J Respir Cell Mol Biol, 1994, 10(6): 587-593.
128. Herrick CA, Bottomly K. To respond or not to respond:T cells in allergic asthma. Nat Rev Immunol, 2003, 3(5):1-8.
129. Elias JA, Lee CG, Zheng T, et al. New insights into the pathogenesis of asthma. J Clin Invest, 2003, 111(3): 291-297.
130. Taube C, Nick JA, Sieqmund B, et al. Inhibition of early airway neutrophilia does not affect development of airway hyperresponsiveness. Am J Respir Cell Mol Biol, 2004, 30(6):837-843.
131. Nel AE. T-cell activation through the antigen receptor. Part 1: signaling component, signaling pathways, and signal integration at the T-cell antigen receptor synapse. J Allergy Clin Immunol,2002,109(6):758-770
132. Myou S, Leff SM, Boetticher J,et al. Blockade of inflammation and airway hyperresponsiveness in immune-sensitized mice by dominant - negative phosphoinositide 3-kinase-TAT. J Exp Med, 2003,198(10):1573.
133. 刘传合,薛全福,陈育智,等. 支气管哮喘动物模型的研究状况. 中华结核和呼吸杂志,2000,23(11):647-649.
134. 商艳,李强,黄怡,等.不同剂量致敏原对小鼠过敏性哮喘模型的影响. 第二军医大学学报,2002,23(1):69-71.
135. 张延焘. 哮喘儿童吸入糖皮质激素的全身不良反应. 临床儿科杂志,2004,22(10):703.
136. Barnes PJ. Corticosteroid resistance in airway disease. Proc Am Thorac Soc, 2004,1(3):264-268.
137. Nyce JW, Metzger WJ. DNA antisense therapy for asthma in an animal model. Nature, 1997, 385(6618): 721-725.
138. 艾萍,金义光,王林.药物传送中的纳米混悬液. 国外医学药学分册,2005,32(1):61-63.
139. 郑俊杰,张英鸽,毛军文,等.纳米科技在生物医学中的应用.军事医学科学院院刊,2004,28(1):88-91.
140. Koo GC, Shah K, Ding GJF, et al. A small molecule very late antigen-4 antagonist can inhibit ovalbumin-induced lung inflammation. Am J Respir Crit Care Med, 2003, 167(10):1400-1409.
141. Chialda L, Zhang MX, Brune K, et al. Inhibitors of mitogen-activated protein kinases differentially regulate costimulated T cell cytokine production and mouse airway eosinophilia. Respiratory Research, 2005, 6(1):36.
142. Peachell P. Targeting the mast cell in asthma. Current Opinion in Pharmacology, 2005, 5(3): 1-6.
143. Khan LN, Kon OM, Macfarlane AJ, et al. Attenuation of the allergen-induced late asthmatic reaction by cyclosporin A is associated with inhibition ofbronchial eosinophils, interleukin-5, granulocyte marophage colony- stimulating factor, and eotaxin. Am J Respir Crit Care Med, 2000, 162(4 Pt1):1377-1382.
144. 刘新,王梦华.TH2 型细胞因子在支气管哮喘发作过程中的表达. 微生物学杂志,2004,24(1):54-56.
145. Dodeller F, Schulze-Koops H. The p38 mitogen-activated protein kinase signaling cascade in CD4 T cells. Arthritis Research ﹠ Therapy, 2006,8(2):205.
146. Fitzgerald SM, Lee SA, Hall HK, et al. Human lung fibroblasts express interleukin-6 in response to signaling after mast cell contact. Am J Respir Cell Mol Biol, 2004, 30(4):585-593.
147. Cui CH, Adachi T, Oyamada H, et al. The role of mitogen-activated protein kinases in eotaxin-induced cytokine production from bronchial epithelail cells. Am J Respir Cell Mol Biol, 2002, 27(3):329-335.
148. 黄艳,陈吉泉,修清玉. 雾化吸入干扰素 γ 阻断 GATA-3 表达防治哮喘的实验研究.免疫学杂志,2006,22(4):406-409.
149. 潘世秀,曾天舒,崔天盆,等.比格列酮对哮喘模型 TH1/TH2 细胞因子表达的影响.中华微生物学和免疫学杂志,2006,26(3):237-240.
150. Chen CH, Zhang DH, LaPorte JM, et al. Cyclic AMP activates p38 mitogen-activated protein kinase in Th2 cells: phosphorylation of GATA-3 and stimulation of Th2 cytokine gene expression. The Journal of Immunology, 2000,165(10):5597-5605.
151. Zhang Y, Cho YY, Petersen BL, et al. Evidence of STAT1 phosphorylation modulated by MAPKs, MEK1 and MSK1. Carcinogenesis, 2004, 25(7): 1165- 1175.
152. Saccani S, Pantano S, Natoli G, et al.p38-dependent marking of inflammatory genes for increased NF-κB recruitment. Nat Immunol, 2002, 3(1):69-75
153. Stewart AG.. Airway wall remodelling and hyperrespondsiveness: modelling remodelling in vitro and in vivo. Pulm Pharmacol Ther, 2001, 14(3):255-265.
154. Rook GA. The TGF-β1 paradox in asthma. Trends Immunol, 2001, 22(6): 299-300.
155. 冯志民,徐蘅,马旺扣. 动物组织中羟脯氨酸测定方法的建立及初步应用. 南京铁道医学院学报,1999,18(3):168-170.
156. Grant RA. Estimation of hydroxyproline by the autoanalyser. J Clin Path,1964,17(6):685-686.
157. 杨秀颖,杜冠华. 组织羟脯氨酸含量微量测定方法及应用. 中国药理学通报,2004,20(7):836-837.
158. Duvernelle C, Freund V, Frossard N. Transforming growth factor-beta and its role in asthma. Pulm Pharmacol Ther, 2003, 16(4):181-196.
159. Chen G, Khalil N. In vitro wounding of airway smooth muscle cell monolayers increases expression of TGF-beta receptors. Respir Physiol Neurobiol, 2002, 132(3):341-346.
160. Hara K, Hasegawa T, Ooi H, et al. Inhibitory role of eosinophils on cell surface plasmin generation by bronchial epithelial cells: inhibitory effects of transforming growth factor-β1. Lung, 2001, 179(1):9-20
161. McMillan SJ, Xanthou G, Lloyd CM. Manipulation of allergen-induced airway remodeling by treatment with anti-TGF-beta antibody: effect on the smad signaling pathway. J Immunol, 2005, 174(9):5774-5780.
162. Perng DW, Wu YC, Chang KT, et al. Leukotriene C4 induces TGF-β1 productioon in airway epithelium via p38 kinase pathway. Am J Respir Cell Mol Biol, 2006, 34(1):101-107.
163. Chen G, Khalil N. TGF-beta1 increases proliferation of airway smooth muscle cells by phosphorylation of map kinases. Respiratory Research, 2006, 7(1):2-11.
164. Carroll NG, Mutavdzic S, James AL. Increased mast cells and neutrophils in patients with asthma. Thorax, 2002, 57(8):677-682.
165. Jeffery P, Zhu J. Mucin-producing elements and inflammatory cells. Novartis Found Symp, 2002, 248:51-68.
166. Justice JP, Crosby J, Borchers MT, et al. T cell-dependent airway mucus production occurs in response to IL-5 expression in lung. Am J Physiol Lung Cell Mol Physiol, 2002, 282(5): L1066-1074.
167. Atherton HC, Jones G, Danahay H. IL-13-induced changes in the goblet cell density of human bronchial epithelial cell cultures: MAP kinase and phosphatidylinositol 3-kinase regulation. Am J Physiol Lung Cell Moll Physiol, 2003, 285(3):L730-739.
2. Adcock IM, Chung KF, Caramori G, et al. Kinase inhibitors and airway inflammation. European Journal of Pharmacology, 2006, 533(1-3):118-132.
3. Shirai T, Suxuki K, Inui N, et al. Th1/Th2 profile in peripheral blood in atopic cough and atopic asthma. Clin Exp Allergy, 2003, 33(1):84-89.
4. Maizels RM, Yazdanbakhsh M. Immune regulation by helminth parasites: cellular and molecular mechanisms. Nat Rev Immunol, 2003, 3(9):733-744.
5. Irene HH, Antoon JM. Targeting T cells for asthma. Current Opinion in Pharmacology, 2005, 5(3):227-231.
6. Barnes PJ. New drugs for asthma. Nat Rev Drug Discov, 2004, 3(10):831-844.
7. Barnes PJ. Anti-leukotrienes: here to say? Curr Opin Pharmacol, 2003, 3(3):257-263.
8. Barnes PJ, Chung KF, Page CP, et al. Inflammatory mediators of asthma: an update. Pharmacol Rev, 1998,50(4):515-596.
9. Zhou M, Ouyang W. The function role of GATA-3 in TH1 and TH2 differentiation. Immunol Res, 2003, 28(1):25-37.
10. Catley MC, Cambridge IM, Nasuhara Y, et al. Inhibitors of protein kinase C prevent activated transcription: role of events downstream of NF-κB DNA binding. J Biol Chem, 279(18):18457-18466.
11. Barnes PJ, Hansel TT. Prospects for new drugs for chronic obstructive pulmonary disease. Lancet, 2004,364(9438):985-996.
12. Barnes PJ. Novel signal transduction modulators for the treatment of airway diseases. Pharmacology & Therapeutics, 2006, 109(1-2):238-245.
13. Manning G, Whyte DB, Martinez R, et al. The protein kinase complement of the human genome. Science,2002,298(5600): 1912-1934.
14. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinase. Science, 2002,298(5600):1911-1912
15. 黄翠萍,张珍祥,徐永健.p38 丝裂原活化蛋白激酶信号传导通路和支气管哮喘气道炎症.国外医学呼吸系统分册,2005,25(9):656-658.
16. Saklatvala J. The p38 MAP kinase pathway as a therapeutic target in inflammatory disease. Curr Opin Pharmacol, 2004, 4(4):372-377.
17. Dodeller F, Skapenko A, Kaldan JR, et al. The p38 mitogen activated protein kinase regulates effector functions of primary human CD4 T cells. Eur J Immunol, 2005, 35(12):3631-3642.
18. Kankaanranta H, Giembycz MA, Barnes P, et al. SB203580, an inhibitor of p38 mitogen-activated protein kinase, enhances constitutive apoptosis of cytokine-deprived human eosinophils. J Pharmacol Exp Ther, 1999, 290(2):621-628.
19. Ali S, Leonard SA, Kukoly CA, et al. Absorption, distribution, metabolism, and excretion of a respirable antisense oligonucleotide for asthma. Am J Respir Crit Care Med, 2001, 163(4): 989-993.
20. Popescu FD. Antisense- and RNA interference-based therapeutic strategies in allergy. J Cell Mol Med, 2005, 9(4):840-853.
21. Zarubin T, Han JH. Activation and signaling of the p38 MAP kinase pathway. Cell Research, 2005, 15(1):11-18.
22. Rouse J, Cohen P, Trigon S, et al. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell, 1994, 78(6):1027-1037.
23. Han J, Lee JD, Bibbs L, et al. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science, 1994, 256(5173):808-811.
24. Hanks SK, Hunter T. The cukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J, 1999, 9(8):576-596.
25. Jiang Y, Li Z, Schwarz EM, et al. Structure-function studies of p38 mitogen-activated protein kinase. Loop-12 influence substrate specifity and autophosphorylation, but not upstream kinase selection. J Biol Chem, 1997, 272(17):11096-11102.
26. Dong C, Davis RJ, Flavell RA. MAP kinases in the immune response. Annu Rev Immunol, 2002, 20(4):55-72.
27. Parker CG, Hunt J, Diener K, et al. Identification of stathmin as a novel substrate for p38 delta. Biophys Res Commun, 1998, 249(3): 791-796.
28. Jiang Y, Gram H, Zhao M, et al. Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinase, p38delta. J Biol Chem, 1997,272(48):30122-30128.
29. Salvador JM, Mittelstadt PR, Guszczynsli T, et al. Alternative p38 activation pathway mediated by T cell receptor-proximal tyrosine kinases. Nat Immunol, 2005, 6(4):390-395.
30. Rudd CE. MAPK p38: alternative and nonstressful in T cells. Nat Immunol, 2005, 6(4):368-370.
31. Camps M, Nichols A, Gillieron C, et al. Catalytic activation of the phosphatases MKP-3 by ERK2 mitogen-activated protein kinase. Science, 1998, 280(5367): 1262-1265.
32. Yee AS, Paulson EK, McDevitt MA, et al. The HBPI1 transcriptional repressor and the p38 MAP kinase: unlikely partner in G1 regulation and tumor suppression. Gene, 2004, 336(1): 1-13.
33. Pereira RC, Delany AM, Canalis E. CCAAT/enhancer binding protein homologous protein (DDTT3) induces osteoblastic cell differentiation. Endocrinology, 2004, 145(4):1952-1960.
34. McLaughlin MM, Kumar S, McDonnell PC, et al. Identification of mitogen-activated protein (MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38 MAP kinase. J Biol Chem, 1996, 271(14): 8444-8492.
35. New L, Jiang Y, Zhao M, et al. PRAK, a novel protein kinase regulated by the p38 MAP kinase. EMBO J, 1998, 17(12): 3372-3384.
36. New L, Zhao M, Li Y, et al. Cloning and characterization of RLPK, a novel RSK-related protein kinase. J Biol Chem, 1999, 274(2): 1026-1032.
37. Sweeney SE, Firestein GS. Signal transduction in rheumatoid arthritis. Curr Opin Rheumatol, 2004, 16(3): 231-237.
38. Menzies-GA, Robinson DS.Eosinophils cytokines (interleukin-5), and antieosinophilic therapy in asthma. Curr Opin Med, 2002, 8(1):33-38.
39. Wong CK, Wang CB, Ip WK, et al. Role of p38 MAPK and NF-κB for chemokine release in coculture of human eosinophils and bronchial epithelial cells. Clinical and Experimental Immunology, 2005, 139(1):90-100.
40. Adachi T, Stafford S, Kayaba H, et al. Myosin light chain kinase mediates eosinophil chemotaxis in a mitogen-activated protein kinase-dependent manner. J Allergy Clin Immunol, 2003, 111(1):113-116.
41. Kampen GT, Stafford S, Adachi T, et al. Eotaxin induces degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases. Blood, 2000, 95(6):1911-1917.
42. Cuvelier SL, Paul S, Shariat N, et al. Eosinophil adhesion under flow conditions activates mechanosensitive signaling pathways in human endothelial cells. J Exp Med, 2005, 202(6):865-876.
43. Maa SH, Wang CH, Liu CY, et al. Endogenous nitric oxide downregulates theBcl-2 expression of eosinophils through mitogen-activated protein kinase in bronchial asthma. J Allergy Clin Immunol, 2003, 112(4):761-767.
44. Zhang JP, Wong CK, Lam CW. Role of caspases in dexamethne AS-ONs- induced apoptosis and activation of c-Jun Nh2-terminal kinase and p38 mitogen-activated protein kinase in human eosinophils. Clin Exp Immunol, 2000, 122(1): 20-27.
45. Yu JJ, Tripp CS, Russell JH. Regulation and phenotype of an innate Th1 cell: role of cytokines and the p38 MAP kinase pathway. J Immunol, 2003, 171(11):6112-6118.
46. Zhang J, Salojin KV, Gao JX, et al. p38 mitogen-activated protein kinase mediates signal integration of TCR/CD28 costimulation in primary murine T cells. J Immunol, 1999, 162(7):3819-3829.
47. Doceller F, Skapenko A, Kalden JR, et al. The p38 mitogen-activated protein kinase regulates effector functions of primary human CD4 T cells. Eur J Immunol, 2005, 35(12): 3631-3642.
48. Labuda T, Sundatedt A, Dohlsten M, et al. Selective induction of p38 mitogen-activated protein kinase activity following A6H costimulation in primary human CD4+T cells. Int Immunol, 2000, 12(3):253-261.
49. Chen CH, Zhang DH, LaPorte JM, et al. Cyclic AMP activates p38 mitogen-activated protein kinase in Th2 cells: phosphorylation of GATA-3 and stimulation of Th2 cytokine gene expression. J Immunol, 2000, 165(10):5597-5605.
50. Harlin H, Pocack E, Boothby M, et al. TCR-independent CD30 signaling selectively induces IL-13 production via a TNF receptor-associated factor/p38 mitogen-activated protein kinase-dependent mechanism. J Immunol, 2002, 169(5);2451-2459.
51. Schafer PH, Wadsworth SA, Wang L, et al. p38 alpha mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+CD45RO+ T cells and Th2 effector cells. J Immunol, 1999, 162(12):7110-7119.
52. Ishizuka T, Okajima F, Ishiwara M, et al. Sensitized mast cells migrate toward the antigen: a response regulated by p38 mitogen-activated protein kinase and Rho-associated coiled-coil-forming protein kinase. J Immunol, 2000, 167(4):2298-2304.
53. Masuda A, Yoshikai Y, Aiba K, et al. Th2 cytokine production from mast cellsis directly induced by lipopolysaccharide and distinctly regulated by c-Jun N-terminal kinase and p38 pathways. J Immunol, 2002, 169(7):3801-3810.
54. Hamilton LM, Puddicombe SM, Dearman RJ, et al. Altered protein tyrosine phosphorylation in asthmatic bronchial epithelium. Eur Respir J, 2005, 25(6):978-985.
55. Tsitoura DC, Rothman PB. Enhancement of MEK/ERK signaling promotes glucocorticoid resistance in CD4 + T cells. J Clin Invest, 2004, 113(4):619-627.
56. Pargellis C, Tong L, Churchill L, et al. Inhibition of p38 MAP kinase by utilizing a novel allosteric binding site. Nat Struct Biol, 2002, 9(4):268-272.
57. Stelmach JE, Liu L, Patel SB, et al. Design and synthesis of potent, orally bioavailable dihydroquinazolinone inhibitors of p38 MAP kinase. Bioorg Med Chem, Lett, 2003, 13(2):277-280
58. Fijen JW, Tulleken JE, Kobold AC, et al. Inhibition of p38 mitogen-activated protein kinase: dose-dependent suppression of leukocyte and endothelial response after endotoxin challerge in humans. Crit Care Med, 2002, 30(4):841-845
59. Kuma Y, Sabio G, Bain J, et al. BIRB796 inhibits all p38 MAPK isoforms in vitro and in vivo. J Biol Chem, 2005, 280(20):19472-19479.
60. Kurreck J. Antisense technologies. Improvement through novel chemical modification. Eur J Biochem, 2003, 270(8):1628-1644.
61. 陈援越,吕时铭. 反义核酸技术的现状.国外医学临床生物化学与检验学分册,2001,22(1):20-21.
62. Crooke ST. Molecular mechanisms of action of antisense drugs. Biochim Biophys Acta, 1999, 1489(1):31-44.
63. 张冰,龚磊,刘贤锡. 肿瘤反义治疗研究进展.国外医学肿瘤学分册,2004,31(9):674-677.
64. Zhao Q, Yu D, Agrawal S, et al. Site of chemical modification in CpG containing phosphorothioate oligodeoxynucleotide modulates its immunostimulatory activity. J Bioorg Med Chem Lett, 1999, 9(24):3453-3458.
65. Nielsen PE, Egholm M, Berg RH, et al. Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science, 1991, 254(5037): 1497-1500.
66. Suqimoto N, Yamamoto K, Satoh N. Positional effect of single bulgenucleotide on PNA (peptide nucleic acid)/DNA hybrid stability. J Nucleic Acids Symp Ser, 1999, 42:95-96.
67. Ball HA, Van MR, Robinson CB. Sense and antisense: therapeutic potential of oligonucleotides and interference RNA in asthma and allergic disorders. Clin Rev Allergy Immunol, 2004, 27(3):207-217.
68. Nyce JT. Respirable antisense oligonucleotides: a new, third drug class targeting respiratory disease. Curr Opin Allergy Clin Immunol, 2002, 2 (6):533-536.
69. Crosby JR, Tung D, Guba M, et al. Inhaled TNF-α antisense oligonucleotides inhibits lung inflammation and airway hyper-responsiveness in a mouse model of chronic asthma (poster 311). ATS 2005 San Diego, Abstract Page: A905.
70. Turner AJ, Murphy LJ. Molecular pharmacology of endothelin converting enzyme. Biochem Pharmaco, 1996, 51(2): 91-102.
71. 文忠成,徐军,钟南山. 反义寡核苷酸对气道平滑肌细胞内皮素转换酶及内皮素-1 表达的影响. 中华结核和呼吸杂志,1999,22(5):279-282.
72. Kelly AE, Melo ME, Smith E, et al. Complex role of the IL-4 receptor in a murine model of airway inflammation: expression of the IL-4 receptor on nonlymphoid cells of bone marrow origin contributes to severity of inflammation. J Immunol, 2004, 172(7): 4545-4555.
73. 谢华,孙滨. 粘附分子与哮喘气道炎症细胞的迁移.细胞与分子免疫学杂志,1997,13(2):45-48.
74. Borchers MT, Crosby J, Farmer S, et al. Blockade of CD49d inhibits allergic airway pathologies independent of effects on leukocyte recruitment. Am J Physicol Lung Cell Mol Physicol, 2001, 280(4):L813-521.
75. Lofthouse SA, Crosby JR, Tung D, et al. Aerosol delivery of VLA-4 specific antisense oligonucleotides inhibites airway inflammation and hyperresponsiveness in mice. Respirology, 2005, 10:A11.
76. Fiset PO, Soussi-Gounni A, Christodoulopoulos P, et al. Modulation of allergic response in nasal mucosa by antisense oligodeoxynucleotides for IL-4. J Allergy Clin Immunol, 2003, 111(3):580-586.
77. Mousavi T, Mazer B, Tebianian M. Inhibition of IL-13 by antisense oligonucleotides changes immunoglobulin isotype profile in cultured B-lymphocytes. Iran Biomed J, 2004, 8:185-191.
78. Lach TE, McKay RA, Monia BP, et al. In vitro and in vivo inhibition ofinterleukin IL-5-mediated eosinopoiesis by murine IL-5Rα antisense oligonucleotides. Am J Respir Cell Mol Biol, 2001, 24(2):116-122.
79. Hanai K, Kurokawa T, Minakuchi Y, et al. Potential of atelocollagen- mediated systemic antisense therapeutics for inflammatory disease. Hum Gene Ther, 2004, 15(3):263-272.
80. 林科雄,王长征,钱桂生. 雷公腾对哮喘豚鼠 IL-5、GM-CSF 基因转录的影响及作用机制.中国免疫学杂志,2000,16(6):325-328.
81. Allakhverdi Z, Allam M, Renzi PM. Inhibition of antigen-induced eosinophilia and airway hyperresponsiveness by antisense oligonucleotides directed against the common β chain of IL-3,IL-5,GM-CSF receptors in a rat model of allergic asthma. Am J Respir Crit Care Med, 2002, 165(7):1015-1021.
82. Finotto S, De Sanctis GT, Lehr HA, et al. Treatment of allergic inflammation and hyperresponsiveness by antisense-induced local blockade of GATA-3 expression. J Exp Med, 2001, 193(11):1247-1260.
83. Peng Q, Matsuda T, Hirst SJ, et al. Signaling pathways regulating interleukin- 13-stimulated chemokine release from airway smooth muscle. Am J Respir Crit Care Med, 2004, 169(5):596-603.
84. Choi IW, Kim DK, Ko HM, et al. Administration of antisense phosphorothiate oligonucleotide to the p65 subunit of NF-κB inhibits established asthmatic reaction in mice. Int Immunopharmacol, 2004, 4(14):1817-1828.
85. Stafford S, Lowell C, Sur S, et al. Lyn tyrosine kinase is important for IL-5-stimulated eosinophil differentiation. J Immunol, 2002, 168 (4): 1978 - 1983.
86. Ulanova M, Puttagunta L, Kim MK, et al. Antisense oligonucleotides to Syk kinase: a novel therapeutic approach for respiratory disorders. Curr Opin Investig Drugs, 2003, 4(5):552-555.
87. Duan W, Chan JH, McKay K, et al. Inhaled p38αmitogen-activated protein kinase antisense oligonucleotide attenuates asthma in mice. Am J Respir Crit Care Med, 2005, 171(6):571-578.
88. 伍志坚,陈诗书,彭朝晖.逆转录病毒载体用于基因治疗的安全性. 国外医学分子生物学分册,1995,17(2):71.
89. 许海燕. 纳米技术在医学领域的应用研究. 中华医学杂志,2006,86(8):505-506.
90. Lockman PR, Mumper RJ, Khan MA, et al. Nanoparticle technology for drug delivery across the blood-brain barrier.Drug Dev Ind Pharm, 2002,28(1):1-13.
91. Borchard G., Junginger HE. Modern drug delivery applications of chitosan. Adv Drug Deliver Rev, 2001, 52(2):103.
92. Mumper RJ, Wang J, Klaspell SL, et al. Protective interactive noncondensing (PINC) polymers for enhanced plasmid distribution and expression in rat skeletal muscle. J Control Release, 1998, 52(1-2):191-203.
93. Illum L, Jabbal-Gill I, Hinchcliffe M, et al.Chitosan as a novel nasal delivery system for vaccines. Adv Drug Deliver Rev, 2001, 51(1-3):81.
94. Erbacher P,Zou S,Bettinger T,et al. Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability. Pharm Res, 1998, 15(9):1332.
95. Nakanishi M, Noguchi A. Confocal and probe microscopy to study gene transfection mediated by cationic liposomes with a cationic cholesterol derivative. Adv Drug Deliver Rev, 2001, 52(3):197
96. Kiang T, Wen J, Lim H W, e1 al. The effect of the degree of chitosan deacetylation on the efficiency of gene transfection. Biomalerials, 2004, 25( 22):5293.
97. 黄伟.壳聚糖纳米粒基因递送系统的研究.北京.中国人民解放军军事医学科学院.2002.
98. Richardson SC, Kolbe HV, Duncan R. Potential of low molecular mass chitosan as a DNA delivery system: biocompatibility, body distribution and ability to complex and protect DNA. Int J Pharm, 1999, 178 (2):231-243.
99. Lee KY, Kwon IC, Kim YH, et al. Preparation of chitosan self-aggregates as a gene delivery system. Macromolecules, 1998, 51(2):213-220.
100. Park YK, Park YH, Shin BA, et al.Galactosylated chitosan-graft-dextran as hepatocyte-targeting DNA carrier. J Controlled Release, 2000, 69(1):97.
101. Murata J, Kitamoto T, Ohya Y, et al. Effect of dimerization of the D-glucose analogue of muramyl dipeptide on stimulation of macrophage-like cells. Carbohydr Res, 1997, 297(2):127-133.
102. Kim TH, Ihm JE, Choi YJ, et al. Efficient gene delivery by urocanic acid-modified chitosan. J Controlled Release, 2003, 93(3):389.
103. Junghans M, Kreuter J, Zimmer A. Antisense delivery using protamine- oligonucleotide particles. Nucleic Acids Res, 2000, 28(10):E45.
104. Lochmann D, Vogel V, Weyermann J, et al. Physicochemical characterization of protamine-phosphorothioate nanoparticles. J Microencapsulation, 2004,21(6):625-641.
105. Vogel V, Lochmann D, Weyermann J, et al. Oligonucleotide– protamine- albumin nanoparticles: preparation, physical properties, and intracellular distribution. J Controlled Rel, 2005, 103 (1):99-111.
106. Lochmann D, Weyermann J, Georgens C, et al. Albumin- protamine- oligonucleotide-nanoparticles as a new antisense delivery system. Part 1: Physicochemical characterization. European Journal of Pharmaceutics and Biopharmaceutics, 2005, 59(3):419-429.
107. Arnedo A, Irache JM, Merodio M, et al. Albumin nanoparticles improved the stability, nuclear accumulation and anticytomegaloviral activity of a phosphodiester oligonuceotide. Journal of Controlled Release, 2004, 94 (1):217-227.
108. Mayer G, Vogel V, Weyermann J, et al. Oligonucleotide- protamine- albumin nanoparticles: Protamine sulfate causes drastic size reduction. Journal of Controlled Release, 2006, 106 (1-2):181-187.
109. 陈美英,王士斌. 可生物降解聚合物作为药物载体的应用进展.国际生物医学杂志,2006,29(5):300-303.
110. Wang J, Wang BM, Schwendeman SP. Characterization of the initial burst release of a model peptide from poly (D.L-lactide-co-glycolide) microspheres. J Contr Rel, 2002, 82(2-3):289-307.
111. Wu LB, Ding JD. In vitro degradation of three-dimensional porous poly (D.L-lactide-co-glycolide) scaffolda for tissue engineering. Biomaterials, 2004, 25(27):5821-5830.
112. Sinha VR, Trchan A. Biodegradable microspheres for protein delivery. J Contr Rel, 2003, 90(3):261-280.
113. Zamecnik PC, Stephenson ML. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Pro, Natl Acad. USA 75(1978): 280-284.
114. Lebedeva I, Benimetskaya L, Stein CA, et al. Cellular delivery of antisense oligonucleotides. Eur J Pharm Biopharm, 2000, 50(1):101-119.
115. 杨远,贾文祥,祁欣,等.可生物降解材料作为基因载体的研究.生物医学工程学杂志,2006,23(3):573-577.
116. Duzgunes N, De Ilarduya CT, Simoes S, et al. Cationic liposomes for gene delivery: novel cationic lipids and enhancement by proteins and peptides. Curr Med Chem, 2003, 10 (14):1213-1220.
117. Weyermann J, Lochmann D, Georgens C, et al. Albumin- protamine- oligonucleotide-nanoparticles as a new antisense delivery system. Part 2: cellular uptake and effect. European Journal of Pharmaceutics and Biopharmaceutics, 2005, 59(3):431-438.
118. Wartlick B, Schmitt BS, Strebhardt K, et al. Tumour cell delivery of antisense oligonuleotides by human serum albumin nanoparticles. J Control Release, 2004, 96 (3):483-495.
119. Welsh DJ, Scott PH, Peacock AJ. p38 MAP kinase isoform activity and cell cycle regulators in the proliferative response of pulmonary and systemic artery fibroblasts to acute hypoxia. Pulmonary Pharmacology Therapeutics,2006,19(2):128-138.
120. Rogers DF. Airway mucus hypersecretion in asthma: an undervalued pathology? Current Opinion in Pharmacology, 2004, 4(3):241-250.
121. Irusen E, Matthews JG, Takahashi A, et al. p38 Mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces its activity: role in steroid-insensitive asthma. J Allergy Clin Immunol, 2002 ,109(4):649-657.
122. Schreiber F, Matthias P, Müller MM, et al. Rapid detection of octamer binding protein with “mini-extracts”, prepared from a small number of cells. Nucleic Acids Res, 1989, 17(15):6419.
123. 杜宏举, 汤宁. 丝裂原活化蛋白激酶信号通路及其生物学功能. 国外医学卫生学分册,2005,32(4):197-201.
124. Underwood DC, Obsorn RR, Kotzer CJ, et al. SB239063, a potent p38 MAP kinase inhibitor, reduces inflammatory cytokine production, airways eosinophil infiltration, and persistence. J Pharmacol Exp Ther, 2000, 293(1):281-288.
125. Acosta R, Montanez C, Gomez P, et al. Delivery of antisense oligonuleotides to PC12 cells. Neurosci Res, 2002, 43(1): 81-86.
126. 吴杰,刘克良. 反义寡核苷酸类化合物的体内转运研究进展. 中国新药杂志,2003,12(5):329-333.
127. Gavett SH, Chen X, Finkelmann F, et al. Depletion of murine CD4+ T lymphocytes prevents antigen induced airway hyperreactivity and pulmonary eosinophilia. Am J Respir Cell Mol Biol, 1994, 10(6): 587-593.
128. Herrick CA, Bottomly K. To respond or not to respond:T cells in allergic asthma. Nat Rev Immunol, 2003, 3(5):1-8.
129. Elias JA, Lee CG, Zheng T, et al. New insights into the pathogenesis of asthma. J Clin Invest, 2003, 111(3): 291-297.
130. Taube C, Nick JA, Sieqmund B, et al. Inhibition of early airway neutrophilia does not affect development of airway hyperresponsiveness. Am J Respir Cell Mol Biol, 2004, 30(6):837-843.
131. Nel AE. T-cell activation through the antigen receptor. Part 1: signaling component, signaling pathways, and signal integration at the T-cell antigen receptor synapse. J Allergy Clin Immunol,2002,109(6):758-770
132. Myou S, Leff SM, Boetticher J,et al. Blockade of inflammation and airway hyperresponsiveness in immune-sensitized mice by dominant - negative phosphoinositide 3-kinase-TAT. J Exp Med, 2003,198(10):1573.
133. 刘传合,薛全福,陈育智,等. 支气管哮喘动物模型的研究状况. 中华结核和呼吸杂志,2000,23(11):647-649.
134. 商艳,李强,黄怡,等.不同剂量致敏原对小鼠过敏性哮喘模型的影响. 第二军医大学学报,2002,23(1):69-71.
135. 张延焘. 哮喘儿童吸入糖皮质激素的全身不良反应. 临床儿科杂志,2004,22(10):703.
136. Barnes PJ. Corticosteroid resistance in airway disease. Proc Am Thorac Soc, 2004,1(3):264-268.
137. Nyce JW, Metzger WJ. DNA antisense therapy for asthma in an animal model. Nature, 1997, 385(6618): 721-725.
138. 艾萍,金义光,王林.药物传送中的纳米混悬液. 国外医学药学分册,2005,32(1):61-63.
139. 郑俊杰,张英鸽,毛军文,等.纳米科技在生物医学中的应用.军事医学科学院院刊,2004,28(1):88-91.
140. Koo GC, Shah K, Ding GJF, et al. A small molecule very late antigen-4 antagonist can inhibit ovalbumin-induced lung inflammation. Am J Respir Crit Care Med, 2003, 167(10):1400-1409.
141. Chialda L, Zhang MX, Brune K, et al. Inhibitors of mitogen-activated protein kinases differentially regulate costimulated T cell cytokine production and mouse airway eosinophilia. Respiratory Research, 2005, 6(1):36.
142. Peachell P. Targeting the mast cell in asthma. Current Opinion in Pharmacology, 2005, 5(3): 1-6.
143. Khan LN, Kon OM, Macfarlane AJ, et al. Attenuation of the allergen-induced late asthmatic reaction by cyclosporin A is associated with inhibition ofbronchial eosinophils, interleukin-5, granulocyte marophage colony- stimulating factor, and eotaxin. Am J Respir Crit Care Med, 2000, 162(4 Pt1):1377-1382.
144. 刘新,王梦华.TH2 型细胞因子在支气管哮喘发作过程中的表达. 微生物学杂志,2004,24(1):54-56.
145. Dodeller F, Schulze-Koops H. The p38 mitogen-activated protein kinase signaling cascade in CD4 T cells. Arthritis Research ﹠ Therapy, 2006,8(2):205.
146. Fitzgerald SM, Lee SA, Hall HK, et al. Human lung fibroblasts express interleukin-6 in response to signaling after mast cell contact. Am J Respir Cell Mol Biol, 2004, 30(4):585-593.
147. Cui CH, Adachi T, Oyamada H, et al. The role of mitogen-activated protein kinases in eotaxin-induced cytokine production from bronchial epithelail cells. Am J Respir Cell Mol Biol, 2002, 27(3):329-335.
148. 黄艳,陈吉泉,修清玉. 雾化吸入干扰素 γ 阻断 GATA-3 表达防治哮喘的实验研究.免疫学杂志,2006,22(4):406-409.
149. 潘世秀,曾天舒,崔天盆,等.比格列酮对哮喘模型 TH1/TH2 细胞因子表达的影响.中华微生物学和免疫学杂志,2006,26(3):237-240.
150. Chen CH, Zhang DH, LaPorte JM, et al. Cyclic AMP activates p38 mitogen-activated protein kinase in Th2 cells: phosphorylation of GATA-3 and stimulation of Th2 cytokine gene expression. The Journal of Immunology, 2000,165(10):5597-5605.
151. Zhang Y, Cho YY, Petersen BL, et al. Evidence of STAT1 phosphorylation modulated by MAPKs, MEK1 and MSK1. Carcinogenesis, 2004, 25(7): 1165- 1175.
152. Saccani S, Pantano S, Natoli G, et al.p38-dependent marking of inflammatory genes for increased NF-κB recruitment. Nat Immunol, 2002, 3(1):69-75
153. Stewart AG.. Airway wall remodelling and hyperrespondsiveness: modelling remodelling in vitro and in vivo. Pulm Pharmacol Ther, 2001, 14(3):255-265.
154. Rook GA. The TGF-β1 paradox in asthma. Trends Immunol, 2001, 22(6): 299-300.
155. 冯志民,徐蘅,马旺扣. 动物组织中羟脯氨酸测定方法的建立及初步应用. 南京铁道医学院学报,1999,18(3):168-170.
156. Grant RA. Estimation of hydroxyproline by the autoanalyser. J Clin Path,1964,17(6):685-686.
157. 杨秀颖,杜冠华. 组织羟脯氨酸含量微量测定方法及应用. 中国药理学通报,2004,20(7):836-837.
158. Duvernelle C, Freund V, Frossard N. Transforming growth factor-beta and its role in asthma. Pulm Pharmacol Ther, 2003, 16(4):181-196.
159. Chen G, Khalil N. In vitro wounding of airway smooth muscle cell monolayers increases expression of TGF-beta receptors. Respir Physiol Neurobiol, 2002, 132(3):341-346.
160. Hara K, Hasegawa T, Ooi H, et al. Inhibitory role of eosinophils on cell surface plasmin generation by bronchial epithelial cells: inhibitory effects of transforming growth factor-β1. Lung, 2001, 179(1):9-20
161. McMillan SJ, Xanthou G, Lloyd CM. Manipulation of allergen-induced airway remodeling by treatment with anti-TGF-beta antibody: effect on the smad signaling pathway. J Immunol, 2005, 174(9):5774-5780.
162. Perng DW, Wu YC, Chang KT, et al. Leukotriene C4 induces TGF-β1 productioon in airway epithelium via p38 kinase pathway. Am J Respir Cell Mol Biol, 2006, 34(1):101-107.
163. Chen G, Khalil N. TGF-beta1 increases proliferation of airway smooth muscle cells by phosphorylation of map kinases. Respiratory Research, 2006, 7(1):2-11.
164. Carroll NG, Mutavdzic S, James AL. Increased mast cells and neutrophils in patients with asthma. Thorax, 2002, 57(8):677-682.
165. Jeffery P, Zhu J. Mucin-producing elements and inflammatory cells. Novartis Found Symp, 2002, 248:51-68.
166. Justice JP, Crosby J, Borchers MT, et al. T cell-dependent airway mucus production occurs in response to IL-5 expression in lung. Am J Physiol Lung Cell Mol Physiol, 2002, 282(5): L1066-1074.
167. Atherton HC, Jones G, Danahay H. IL-13-induced changes in the goblet cell density of human bronchial epithelial cell cultures: MAP kinase and phosphatidylinositol 3-kinase regulation. Am J Physiol Lung Cell Moll Physiol, 2003, 285(3):L730-739.