【作者】 宋颖芳；

【导师】 戚好文；

【作者基本信息】 第四军医大学 ， 内科学， 2008， 博士

【摘要】 哮喘是全球最常见的慢性疾病之一,发病率逐年增高并造成巨大的经济及社会负担,因此研究哮喘的发生、发展及防治,已成为当前人口与健康领域的前沿课题。近年来气道重塑,又称气道重建或重构,被认为是哮喘发病机制中的一个重要环节,它在哮喘发病机制和治疗中的作用越来越受到关注。气道重塑是以气道壁的细胞和分子的组成、数量和结构的变化为特征的一系列慢性损伤和修复过程,主要表现为细胞外基质(extracellular matrix,ECM)沉积、基底膜增厚、气道平滑肌增生和肥大等。作为哮喘的特征性病理变化,气道重塑是哮喘慢性化、持续化、严重化的病理基础,是哮喘中存在的不可逆性气道阻塞及气道高反应性的病理基础,也是激素抵抗性哮喘的病理基础。治疗哮喘必须与防治气道重塑相结合,才是真正意义上的控制。1,25-二羟维生素D3(1,25-dihydroxyvitamin D3,1,25-(OH)2D3)是体内维生素D3最重要的活性代谢产物,主要通过与维生素D受体(vitaminD receptor, VDR)的结合发挥生物学效应。随着研究的深入,目前发现1,25-(OH)2D3除外经典的调节体内骨稳态及钙磷代谢平衡的生理功能,还具有更为广泛的生物学调节作用。哮喘是一种由遗传和环境因素共同作用的复杂的多基因遗传病,新近研究明确VDR基因是哮喘的易感基因,其多态性参与了哮喘的发生及发展,提示1,25-(OH)2D3可能在某种程度上通过与VDR的配体-受体效应来调节哮喘。对1,25-(OH)2D3功能的进一步研究发现,它在哮喘的免疫应答及慢性气道炎症发面均发挥复杂的调控作用,揭示了1,25-(OH)2D3在哮喘治疗中的潜在应用前景。但目前国内外尚无其调节哮喘气道重塑的相关研究报道。本课题从体内及体外两方面,研究1,25-(OH)2D3对哮喘气道重塑的调节作用并初步探讨相关作用机制,旨在加深对1,25-(OH)2D3参与哮喘发病机制的理解,为扩展其在哮喘治疗中的临床运用提供有价值的理论依据。本项研究共分三部分:第一部分1,25-二羟维生素D3对哮喘小鼠模型气道重塑及MMP-9表达的影响目的:观察1,25-二羟维生素D3对哮喘小鼠气道重塑及肺组织中基质金属蛋白酶-9(matrix metalloprotease-9,MMP-9)表达的影响,探讨它在哮喘治疗中的作用。方法:BALB/c小鼠随机分为对照组、哮喘组、VD组及DEX (dexamethasone,地塞米松)组。卵白蛋白(ovalbumin,OVA)致敏和激发建立哮喘小鼠气道重塑模型;HE染色观察气道结构改变;计算机图像分析系统测定气道形态学指标;明胶酶谱法检测各组小鼠肺组织中MMP-9的活性;同时采用逆转录-聚合酶链反应检测各组MMP-9的mRNA表达水平。结果:(1)VD组能部分逆转哮喘气道重塑的特征性病理改变;(2)VD组的支气管壁厚度、支气管壁平滑肌层厚度及支气管壁平滑肌细胞核数量均显著低于哮喘组(P<0.05),但仍高于对照组(P<0.05);(3)VD组肺组织MMP-9活性及其mRNA表达水平均明显低于哮喘组(P<0.05),但仍高于对照组(P<0.05)。结论:1,25-(OH)2D3的干预可显著减轻哮喘气道重塑的病理改变,有效抑制气道平滑肌细胞(airway smooth muscle cells,ASMCs)的增生;并可通过部分抑制肺内MMP-9的表达来延缓哮喘气道重塑的进程。第二部分1,25-二羟维生素D3对被动致敏人气道平滑肌细胞增殖及表达MMP-9、ADAM33的影响目的:检测1,25-二羟维生素D3对被动致敏人气道平滑肌细胞(human airway smooth muscle cells,HASMCs)的增殖及表达MMP-9、解整合素-金属蛋白酶33(a disintegrin and metalloprotease 33,ADAM33)的影响,从细胞水平探讨1,25-(OH)2D3对哮喘气道重塑的作用机制。方法:原代培养并被动致敏HASMCs,以1,25-(OH)2D3作为干预因素。MTT法检测10–10~10–7M的1,25- (OH)2D3对HASMCs增殖的影响并确定其最佳作用浓度;而后用最佳作用浓度的1,25-(OH)2D3预处理HASMCs,MTT法测定细胞增殖活力,流式细胞仪测定细胞周期,免疫细胞化学染色法检测细胞核增殖抗原(proliferating cell nuclear antigen,PCNA)的表达;同时用实时荧光定量PCR及蛋白免疫印记法分别检测细胞中MMP-9及ADAM33的表达情况。结果:(1)1,25-(OH)2D3在生理浓度(10–10M)下并不影响被动致敏HASMCs的增殖(P>0.05),但其在10–9、10–8、10–7M浓度下均能显著抑制被动致敏HASMCs的增殖,且表现为浓度依赖性(P<0.05);当浓度达到10–7M时,1,25-(OH)2D3抑制作用最强,故把此浓度作为实验的最佳浓度运用于之后的所有细胞实验;(2)10–7M的1,25-(OH)2D3对被动致敏HASMCs的抗增殖作用呈现时间依赖性;(3)1,25- (OH)2D3显著抑制被动致敏HASMCs中PCNA阳性表达率,并抑制细胞周期中G1/S的转化;(4)1,25-(OH)2D3明显下调被动致敏HASMCs中MMP-9与ADAM33的mRNA及蛋白表达水平。结论:1,25-(OH)2D3能直接抑制被动致敏HASMCs的增殖并下调其MMP-9、ADAM33表达,这种多重抑制作用可能是1,25-二羟维生素D3调节哮喘气道重塑的作用机制之一。第三部分NF-κB信号通路在1,25-二羟维生素D3调节被动致敏HASMCs过程中的作用研究目的:探讨核转录因子-κB (nuclear factor-κB,NF-κB)在1,25-二羟维生素D3调节被动致敏HASMCs过程中的信号传导作用。方法:原代培养HASMCs并随机分为三组:对照组、哮喘组及VD组,EMSA法检测NF-κB的DNA结合活性;间接免疫荧光细胞化学染色技术观察NF-κB亚基p65的核易位情况;实时荧光定量PCR技术检测VDR、CYP24及IκBα的mRNA表达水平;并用蛋白免疫印迹法检测各组IκBα的蛋白表达水平。结果:(1)正常HASMCs表达VDR,哮喘血清的刺激并不影响其表达,而1,25-(OH)2D3却显著上调VDR及CYP24的mRNA表达水平;(2)VD组NF-κB的DNA结合活性较哮喘组明显减弱(P<0.01),但仍强于对照组(P<0.01);(3)1,25-(OH)2D3显著削弱被动致敏HASMCs胞核NF-κBp65的荧光强度(P<0.01),即显著抑制了NF-κB p65的核易位;(4)1,25-(OH)2D3显著上调被动致敏HASMCs中IκBα的mRNA及蛋白表达水平。结论:1,25-二羟维生素D3能上调HASMCs中VDR的表达并诱发其功能性反应,增强抑制蛋白IκBα的表达,进而抑制哮喘血清对NF-κB的活化。而NF-κB的过度活化广泛地参与了哮喘状态下HASMCs的功能失调,因此1,25-(OH)2D3对NF-κB信号通路的抑制可能是其调控被动致敏HASMCs的重要作用机制。更多还原

【Abstract】 Asthma is one of the most serious and common respiratory disease and its incidence has climbed markedly in the past two decades.Given that asthma results in an economic and social burden that is both substantial and increasing, the researches on the prevention and treatment of asthma have been one of the frontier fields in human health.While asthma is a disorder of the conducting airways characterized by Th2- directed inflammation,a second set of mechanisms is being increasingly recognized as fundamental to disease chronicity and severity,for which the term "remodeling" has been used.It is regarded as some structural changes in the airway walls,such as mucous gland hyperplasia,reticular basement membrane thickening,vascular proliferation,airway smooth muscle hypertrophy and hyper- plasia.As a significant pathological basis of irreversible airway obstruction and persistent airway hyperresponsiveness,airway remodeling is also a major change responsible for corticosteroid refractoriness in the treatment of asthma. Recently, airway remodeling has become one of the main goals in asthma research.1,25-dihydroxyvitaminD3(1,25-(OH)2D3),the active metabolite of vitamin D3,is a lypophilic molecule that exerts its activities mainly via the interaction with the vitamin D receptor(VDR).Although 1,25-(OH)2D3 has traditionally been associated with the regulation of calcium homeostasis, a multitude of other sites of action for this steroid hormone have been discovered.A major advance occurred to the study of genetics asthma with the identification of VDR gene as an asthma susceptibility gene,suggesting that the 1,25-(OH)2D3 and VDR system may function as a regulator of asthma. In recent years,the general knowledge regarding the regulation of asthma by 1,25-(OH)2D3 has rapidly increased.Existing data have showed its sophisticated regulation on the disturbed immune responses and chronic inflammation in asthma.All of these published observations have suggested promising effects of 1,25-(OH)2D3 on controling many responses that lead to pathological and physiological symptoms of asthma.However,to date, there are no data on the regulatory effect of 1,25-(OH)2D3 on asthmatic airway remodeling.And the idea that it may play a beneficial role in the alleviation of airway remodeling is relatively new .The studies presented in this paper were performed in vivo and in vitro to elusive the contribution of 1,25-(OH)2D3 to asthmatic airway remodeling and further the potential mechanisms underlying this effect.The addition of its involvement in the understanding of asthma pathogenesis can shed light on better control and treatment in asthma.The studies consisted of three parts.Part One Effects of 1,25-(OH)2D3 on airway remodeling and expression of MMP-9 in murine model of asthmaObjective:to investigate the effects of 1,25-(OH)2D3 both on the regulation of airway remodeling and the expression of matrix metalloprotease-9(MMP-9) in a murine model of asthma,and to explore its potential role in the treatment of asthma.Methods:BALB/c mice were sensitized and challenged with ovalbumin to establish the asthmatic airway remodeling model.They were randomly divided into four groups:control group,asthma group, VD group and DEX (dexamethasone)group.The characteristic airway structure was detected by HE staining. Morphometric analysis of the stained sections was performed using computerized image analysis system.The expression of MMP-9 in both activity and mRNA level was detected by gelatin zymograph and RT-PCR, respectively. Results:(1)The infiltration of inflammatory cells, smooth muscle cell layer thickening and epithelial loss were decreased in VD group when compared with those in asthma group,but these changes were still more significant than those in the control group; (2)WAm/Pbm、WAi/Pbm and N/Pbm were decreased markly in VD group when compared with those in asthma group(P<0.05),but they were still higher than those in the control group (P<0.05); (3)The activity and mRNA level of MMP-9 in VD group were decreased when compared with those in asthma group(P<0.05),but it was still higher than those in the control group (P<0.05).Conclusions:Intervention with 1,25-(OH)2D3 could markly alleviate the asthmatic airway remodeling and partly restore the appropriate structure of airway wall.It could also inhibit the ASMC proliferation in vivo and lower the expression of MMP-9 in the lung tissue on asthmatic condition,thus delay the process of airway remodeling.Part Two Effects of 1,25-(OH)2D3 on passively sensitized human airway smooth muscle cellsObjective:to investigate the effects of 1,25-(OH)2D3 on passively sensitized human airway smooth muscle cells(HASMCs) proliferation and their MMP-9 and a disintegrin and metalloprotease 33(ADAM33) expressions.Methods: HASMCs were passively sensitized with 10% serum from asthmatic patients.1,25- (OH)2D3 was used as the interventor.MTT colorimetri assay was used to examine the effect of 1,25-(OH)2D3 on cell proliferation at different concentrations(10–10M、10–9M、10–8M、10–7M).By this way,its optimal concentration was determined. And then the effects of 1,25-(OH)2D3 at the optimal concentration on cell proliferation was examined by the same MTT assay;cell cycle analysis by flow cytometry and immunocytochemical staining for proliferating cell nuclear antigen (PCNA).The expressions of MMP-9 and ADAM33 in HASMCs were studied by real-time quantitative RT-PCR and Western blotting analysis. Results:(1)Inhibition of cell proliferation by 1,25-(OH)2D3 was barely detectable at 10–10M.But with the increasing concentration ranging from10–9M to 10–7M, 1,25-(OH)2D3 markly inhibited the cell proliferation concentration-dependently and reached the maximum effect at the concentration of 10–7 M.Accordingly,10–7M was chosed as the optimal concentration of 1,25-(OH)2D3 for the following study;(2)At the concentration of 10–7M,1,25-(OH)2D3 could inhibit the cell proliferation of passively sensitized HASMCs in a time-dependent manner;(3)1,25-(OH)2D3 markly suppressed the PCNA- positive rate and hampered the G1/S transition in HASMCs passively sensitized with asthmatic serum;(4)1,25-(OH)2D3 also markly down-regulated the expressions of protein for MMP-9 and ADAM33,as well as their mRNA levels in passively sensitized HASMCs. Conclusions: 1,25- (OH)2D3 has directly inhibitory effects on passively sensitized HASMCs in vitro,including the inhibition of the cell proliferation and the expressions of MMP-9 and ADAM33,which is maybe concerned with the beneficial role for 1,25-(OH)2D3 on the prevention and therapy of asthmatic airway remodeling.Part three Potential role of NF-κB in the multiple inhibition of 1,25-(OH)2D3 in passively sensitized HASMCsObjective: to evaluate the potential role of nuclear factor-κB(NF-κB) in the regulation of 1,25-(OH)2D3 on passively sensitized HASMCs.Methods:HASMCs were primarily cultured and randomly divided into three groups: control group, asthma group and VD group. NF-κB DNA-binding activity was assayed by electrophoretic mobility shift assay(EMSA),while the expression of NF-κB p65 in cell nuclear was observed by immunofluorescence staining.Real-time quanti- tative RT-PCR was used to determine the mRNA levels of VDR,CYP24 and the inhibitor protein IκBα.Additionally,the protein level of IκBαwas also detected by western blotting.Results:(1)The expression of VDR has been detected in the normal HASMCs. And passive sensitization with asthmatic serum alone did not alter its mRNA level compared with that in the control cultures,but 1,25-(OH)2D3 pretreatment increased its mRNA level. Moreover,the mRNA encoding CYP24 was also markly up-regulated in the VD group.(2)The NF-κB DNA-binding activity of HASMCs in VD group was significantly decreased as compared with that in asthma group(P<0.01),but it was still higher than that in the control group(P<0.01);(3)1,25-(OH)2D3 markly weakened the fluorescence intensity of NF-κB p65 in the nuclear of passively sensitized HASMCs(P<0.01);namely,it markly arrested the NF-κB p65 nuclear translocation in passively sensitized HASMCs;(4)Addition of 1,25-(OH)2D3 also markly up-regulated the expressions of protein for IκBα,as well as its mRNA levels in passively sensitized HASMCs. Conclusions:HASMCs express functional VDR and 1,25-(OH)2D3 could up- regulate its mRNA levle and evoke its functional response in passively sensitized HASMCs,leading to the up-regulation of the IκBαexpression.And by this way,1,25-(OH)2D3 could decrease the NF-κB activity.Given that NF-κB signaling pathway extensively participates in HASMC dysregulation,these results indicated that the suppression of 1,25-(OH)2D3 on NF-κB activity was maybe a relevant mechanism for its negative effects on passively sensitized HASMCs更多还原