【作者】 常健；

【导师】 鲁继荣；

【作者基本信息】 吉林大学 ， 内科学， 2007， 博士

【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。更多还原

【Abstract】 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.更多还原