【作者】 胡海洋；

【导师】 李强；

【作者基本信息】 第二军医大学 ， 内科学， 2009， 博士

【摘要】 支气管哮喘是呼吸系疾病中常见病和多发病之一。近20年来,全世界支气管哮喘的患病率和死亡率均呈逐渐上升趋势,这已经引起全球的广泛重视。既往支气管哮喘的研究过多地关注哮喘炎症反应的控制,然而对气道重塑及气道平滑肌的作用研究相对很少。由于哮喘的发病机制异常复杂,所以炎症的控制也异常困难,这方面的研究已经多年没有突破性进展。所以很多学者开始不断地调整哮喘研究主攻方向,寻找新的研究领域,哮喘气道重塑正是在这样的背景下开始引起学者的广泛关注,并逐渐成为支气管哮喘研究的热点之一。气道重塑作为慢性哮喘的核心病理改变,是影响慢性哮喘患者生活质量和劳动能力的最重要因素,它主要导致气流阻塞的可逆性减小,呼气期为主的通气功能障碍加重,肺内残气量增加,进而导致不同程度的阻塞性肺气肿,甚至肺源性心脏病。如果能有效地控制气道重塑就可以最大限度地保护患者的肺功能,但是由于长期以来对其重视不足,导致具体体制至今不明,而且始终缺乏有效的治疗手段,所以控制气道重塑已经成为哮喘预防和治疗的当务之急。慢性哮喘气道重塑是全气道复杂改变的复合体,包括哮喘患者气道不同于正常气道的全部组织学改变,主要有气道平滑肌增生及肥大、细胞外基质沉积、上皮下纤维化、管腔狭窄、黏膜肥厚、气管壁增厚、炎症细胞浸润等。气道平滑肌是气道重塑过程中核心的病理改变,气道重塑的主要变化都是围绕着气道平滑肌细胞展开的。学者已经认识到哮喘的治疗离不开气道平滑肌的治疗,所以针对气道平滑肌的治疗也已经成为哮喘治疗的重要突破口。平滑肌祖细胞是近年来血管重构领域研究的重大发现。传统的观念曾认为在血管损伤部位的平滑肌细胞,是由受损部位的中膜迁移到新生内膜的。但是现在的观点认为血管损伤部位的平滑肌细胞更有可能或至少有相当一部分来源于其外周血中的前体细胞--平滑肌祖细胞(SPCs)。目前平滑肌祖细胞的研究工作还是处于起步阶段。现有研究认为SPCs作为平滑肌细胞的前体细胞可能是一种或者一大类细胞;最有可能的来源就是骨髓中的多功能干细胞;现今仍未能找到SPC区别于其他细胞的特异性分子标记;因为SPCs同时具有平滑肌细胞和干细胞部分特征,所以目前研究SPC所使用的细胞标记大多都是干细胞、内皮祖细胞、平滑肌细胞的标记物。平滑肌祖细胞与慢性哮喘气道重塑之间的关系尚未见文献报道,但是平滑肌祖细胞在血管重构中所发挥的作用已经给我们一个全新的启示。因为在既往哮喘研究中很多学者都发现气道平滑肌细胞存在独特的迁移、肥大和增殖等现象,但对其具体机制不甚清楚;再有在哮喘相关研究中还发现①哮喘动物外周血和肺组织中的CD34+细胞出现增加;②PDGF-BB参与哮喘气道重塑的相关机制的调控,但是学者并没有对这两个现象产生的机制及意义给出非常令人信服的答案。然而PDGF-BB和CD34+均与平滑肌祖细胞密切相关,前者与平滑肌祖细胞的分化诱导相关,后者是平滑肌祖细胞的重要特征标志之一。将上述信息整合后,我们就自然联想到平滑肌祖细胞存在参与哮喘致病过程中的可能性,即平滑肌祖细胞在特定哮喘炎性因子的作用下出现趋化、定植和分化,并参与了慢性哮喘的气道重塑。为求证上面的假说我们设计了本研究课题,首先通过对比检测哮喘大鼠与正常大鼠在外周血单个核细胞中平滑肌祖细胞含量上的差别,初步分析平滑肌祖细胞含量变化与哮喘状态的是否有潜在联系;再利用性别差异Y染色体的特征基因—SRY(sex-determining region on the Y chromosome)作为外源平滑肌祖细胞示踪标记物,通过原位杂交检测SRY基因表达的方法观察雄性大鼠来源SPCs在雌性哮喘大鼠气道平滑肌层内定植和分化情况,判断SPCs是否参与了哮喘气道重塑;最后再通过使用可能影响SPCs作用的药物西司莫罗,观察药物作用后SPCs在哮喘气道重塑中作用的变化。力求阐明慢性哮喘大鼠外周血中的平滑肌祖细胞可以参与慢性哮喘的气道重塑;来源于SPCs的平滑肌细胞在气道重塑中占有重要比例;抑制平滑肌祖细胞分化、增殖可以明显改善哮喘的气道重塑,为今后慢性哮喘气道重塑的预防和治疗开拓了新的领域。第一部分OVA雾化吸入对哮喘大鼠的外周血中平滑肌祖细胞含量的影响目的:研究OVA雾化吸入对哮喘大鼠的外周血中平滑肌祖细胞含量的影响方法:OVA致敏、雾化激发制作哮喘大鼠模型,常规观察、HE染色和肺泡灌洗液细胞计数鉴定评价模型哮喘模型;取正常大鼠和哮喘大鼠的外周血,Ficoll法分离外周血单个核细胞,通过流式细胞仪检测其中SM-MHC和CD34阳性的表达情况,以此了解外周血单个核细胞中平滑肌祖细胞含量。结果:哮喘组大鼠支气管肺泡灌洗液中巨噬细胞、淋巴细胞、中性粒细胞和嗜酸性细胞数量分别为(1.34±0.06)×10~6、(0.54±0.07)×10~6、(3.20±0.79)×10~6和(5.85±0.86)×10~6肤,均明显高于正常对照组大鼠肺泡灌洗液相应细胞含量(0.96±0.11)×10~6、(0.04±0.02)×10~6、(0.43±0.16)×10~6和(0.21±0.11)×10~6;哮喘组大鼠外周血单个核细胞中CD34+为1.10±0.17%,MHC+为12.02±1.80%,CD34/MHC均阳性的比例为0.95±0.09%,亦明显高于对照组的比例:0.22±0.07%、1.65±0.11%和0.21±0.05%(P <0.05)。结论:哮喘组大鼠的外周血单个核细胞中平滑肌祖细胞含量要高于正常对照组大鼠。在哮喘状态下平滑肌祖细胞可能受到特定炎性因子刺激后,数量上升并参与到哮喘的致病过程中。第二部分大鼠骨髓间充质干细胞分离、鉴定和平滑肌祖细胞鉴定目的:分离大鼠骨髓间充质干细胞并诱导其分化为平滑肌祖细胞方法:取正常雄性Wistar大鼠四肢长骨,无菌状态用无血清培养基冲洗骨髓腔,取骨髓细胞悬液接种至培养瓶内,使用全贴壁法培养骨髓间充质干细胞,培养传代鉴定;观察细胞的生长状态,流式仪检测其细胞表型特点;观察其在体外条件下分化为成骨和脂肪细胞的能力。再取P4骨髓间充质干细胞用含PDGF-BB的培养液中诱导2周,检测其中α-SMA和CD34阳性的表达情况,了解其分化为平滑肌祖细胞情况。结果:全骨髓贴壁法培养骨髓间充质干细胞是一种确实可行的办法,培养获得的骨髓间充质干细胞符合MSC的典型特点。在体外条件下,骨髓间充质干细胞可分化为成骨和脂肪细胞。骨髓间充质干细胞在含PDGF-BB的培养液作用下,细胞性质发生明显地改变,可以同时具有干细胞和平滑肌细胞的特点,CD34阳性上升至32%,α-SMA表达阳性率为62%。结论:体外培养的骨髓间充质干细胞可以在被诱导分化为平滑肌祖细胞,可以作为研究平滑肌祖细胞的重要工具。第三部分平滑肌祖细胞在慢性哮喘气道重塑中的作用目的:观察平滑肌祖细胞在慢性哮喘气道重塑中的作用方法:采用正常雌性Wistar大鼠复制慢性哮喘气道模型,在哮喘模型雾化激发的第4周开始将雄性来源的平滑肌祖细胞(10~6个)经鼠尾静脉注射入,每周1次,连续4周。第12周分别取动物的气道组织标本制作切片,并同时分离气管进行气道平滑肌细胞进行原代培养,通过原位杂交检测SRY阳性细胞气道组织标本制作切片中表达情况;再通过原位杂交检测SRY阳性细胞在纯化后的气道平滑肌细胞中表达情况;观察平滑肌祖细胞在慢性哮喘气道重塑中的定位和分化情况。结果:雌性哮喘大鼠在尾静脉注射雄性来源的SPCs后平滑气道平滑肌层出现表达SRY阳性的平滑肌细胞,而雌性正常对照组动物的气道中未见SRY阳性平滑肌细胞定植;在分离培养的同组动物气道平滑肌细胞中也发现SRY阳性的平滑肌细胞。结论:平滑肌祖细胞在慢性哮喘病程中可以定植到气道并分化为气道平滑肌参与哮喘的气道重塑。第四部分西罗莫司对哮喘气道重塑中平滑肌祖细胞作用的影响目的:观察西罗莫司对哮喘气道重塑中平滑肌祖细胞作用的影响方法:将SPC分别放在DM、0.1ng/ml、1 ng/ml、10 ng/ml和100 ng/ml不同药物浓度的培养基中作用48小时后,通过观察MTT吸光度值和计数变化,了解西罗莫司对平滑肌祖细胞增殖的影响。在含PDGF-BB的培养液中加入不同浓度的西罗莫司(对照组、0.1ng/ml和1 ng/ml),分别对MSC作用10天后,观察α-SMA阳性表达情况,了解西罗莫司对平滑肌祖细胞分化的影响。取正常雌性Wistar大鼠复制慢性哮喘气道模型,在模型制作的过程中将完成诱导的雄性来源的平滑肌祖细胞经鼠尾静脉注射入,在模型制作的后4周每天雾化激发后予以西罗莫司雾化吸入治疗,于12周分别取动物的气道组织标本制作切片,通过原位杂交检测SRY阳性细胞表达情况,观察西罗莫司雾化吸入治疗对平滑肌祖细胞在慢性哮喘气道重塑中作用的影响。结果:西罗莫司不同药物浓度组在作用48小时后,细胞计数和MTT法检测吸光度值也明显低于对照组,DM、0.1ng/ml、1ng/ml、10ng/ml和100ng/ml各药物浓度组中作用48小时后,各药物浓度组细胞的吸光度值分别为0.75±0.06、0.44±0.04、0.33±0.02、0.20±0.01和0.18±0.01(P <0.05)。西罗莫司不同药物浓度组(对照组、0.1ng/ml和1ng/ml)在作用10天后,细胞的α-SMA阳性表达率分别为71%、24%和4%(P <0.05)。OVA哮喘组和西罗莫司哮喘治疗组的气道平滑肌层中均出现SRY阳性表达的细胞,但西罗莫司治疗哮喘组大鼠气道组织中SRY阳性细胞数量要明显少于OVA哮喘组,PBS雾化对照组大鼠气道内未见SRY阳性细胞表达。结论:西罗莫司可以显著抑制平滑肌祖细胞的分化和增殖,其抑制作用与药物浓度及作用时间存在相关性;西罗莫司雾化吸入可以通过抑制平滑肌祖细胞的作用,改善慢性哮喘的气道重塑,为哮喘气道重塑的治疗开辟新的靶点。更多还原

【Abstract】 Background:Asthma is a chronic inflammatory disease characterized by reversible airflow limitation and airway hyperresponsiveness. It remains a severe health problem since current therapies are directed to suppressing, rather than preventing or reversing, the primary disease process.Persistent inflammation in airway tissues may lead to structural changes known as airway remodeling and consequently airway obstruction that is not fully reversible and progressive loss of lung function over time. Airway remodeling in asthma is a complex process that involves structural changes in virtually all tissues of the airway wall. Airway remodeling, a central feature of asthma, is characterized by airway smooth muscle hypertrophy and hyperplasia, collagen deposition to sub-epithelial basement membrane, hyperplasia of goblet cells, thickening of airway mucosa and an increase in vascularity. Current treatments may indirectly control airway remodeling through a reduction of inflammation but such a kind of approach is only in part successful. Airway smooth muscle plays a multifaceted role in the pathogenesis of airway remodeling in asthma,including airway myocytes to airway inflammation, airway wall remodeling, and airflow obstruction in this prevalent disease syndrome. Together, these roles make airway smooth muscle an attractive target for asthma therapy.Recent animal data suggest that vascular smooth muscle cells within the neointima of the vessel wall may originate from bone marrow, providing indirect evidence for circulating smooth muscle progenitor cells （SPCs）.The location, mobilization, and function of such progenitors is still poorly understood, but may include reparative or alternatively proliferative consequences for vascular injury development and progression. These observations provide a platform for re-evaluation of smooth muscle cell heterogeneity and progenitor biology in asthma, with implications for therapy of airway remodeling in asthma.Previous studies have suggested that the number of CD34+ progenitor cells is elevated in the peripheral blood of asthmatic patients and PDGF has a potential pathological role in asthmatic airway remodeling. Both of them are correlated with smooth muscle progenitor cells, one is an important characteristic, another is differentiation inductor. It means smooth muscle progenitor cells probably participate in asthmatic airway remodeling. Clearly, a greater understanding of the pathogenesis of smooth muscle progenitor cell in asthmatic airway remodeling is critical to the development of better therapeutic modalities.In the present study, we tested the hypothesis that smooth muscle progenitor cells may play an important role in asthmatic airway remodeling.Part I Influence of atomization inhaled ovalbumin on smooth muscle progenitor cells in peripheral blood from rat with chronic asthma.Objective:To investigate the influence of atomization inhaled ovalbumin on smooth muscle progenitor cells in peripheral blood from rat with chronic asthma.Methods and materials: Ten Wistar rats were randomly divided into two groups. OVA sensitization was performed by concurrent subcutaneous injection of OVA adsorbed to aluminum hydroxide.Fouteen days later, the animals were challenged by aerosol with an OVA solution or with phosphate-buffered saline for 8 weeks. All animals were killed 24hours after antigen challenge. On killing, lungs of the animals were rinsed with saline and total and differential cell counts were performed. The peripheral blood mononuclear cells （MNCs） were isolated from the peripheral blood of rats by density gradient centrifugation with Ficoll.Examination expressions of CD34 and SM-MHC in asthma group and control group by flow cytometric analysis.Result:After8 weeks of OVA challenge, inflammatory response and structural changes were found in airway. The area of smooth muscle in the large airway in asthmatic groups was significantly increased as compared with that in control groups. Compared with those in control group, lymphocytes, neutrophils, eosinophils, goblet cells were increased. The area of airway smooth muscle was significantly increased mainly in the large airways of rats after challenged to OVA for 8 weeks. The expressions of CD34 and SM-MHC in asthma group were higher than control group. Conclusion:Atomization inhaled ovalbumin can increase smooth muscle progenitor cells in peripheral blood from rat with chronic asthma. The increased expression of smooth muscle progenitor cells suggested that they may play a important role in airway remodeling of asthma.Part II Isolation, culture and characterization of bone marrow mesenchymal stem cells, and induce mMSCs differentiate into smooth muscle progenitor cellsObjective:To explore the potential of rat bone marrow mesenchymal stem cells （MSCs） to differentiate into smooth muscle progenitor cells.Methods and materials:After the tibias and femurs were dissected from 4～6 week-old rats ,BM mononuclear cells（MNCs）were plated in the 75cm² flasks, at a concentration of 1 10⁶/cm², in 2ml DMEM-LG medium supplemented with 10%FBS, 4 mM L-glutamine, 100u/ml penicillin and 100,ug/ml streptomycin. Flasks were maintained in incubator with 5%CO₂ at 37.0℃. After 24h, nonadherent cells were eliminated by medium changing, and then the medium were changed every 3 days. The cells were grown for 2-3 weeks until attaching to 80% confluences, and then they were trypsinized with 0.05% trypsin-0.02% EDTA and replanted at about 10 cells/well in the 96-well flasks. When the colonies were grown up, cells were expanded at density vary from 1.5 103/cm² to 3.0 103/cm². The characters of the cell, such as morphology, cell growth curve, cell cycle and phenotype were demonstrated. Their abilities to differentiate along adipocytic and osteoblastic pathways were also investigated.The MSCs, which were obtained, cultured in DMEM medium containing 20 %FBS, 50ng/ ml Platelet-derived growth factor - BB （PDGF - BB）. The cultured cells in vitro were identified with smooth muscle cell specificαactin （α-SMA） and CD34 by immunohistochemistry and flow cytometric analysis was used to identify the smooth muscle progenitor cells level after PDGF - BB effect.Result: The MSCs population consisted of spindle- and star-shaped cells.Flow-cytometric analysis showed that they were have typical characteristics.MSCs can be differentiated into adipocyte and osteoblast cell.Smooth muscle-like cells outgrew from the culture of MSCs in presence of PDGF– BB after cultured 2 weeks. These cells were positive stain forα-SMA and CD34, butα-SMA and CD34 were not expressed in fresh isolated MSCs.Conclusion:MSCs have been isolated and purified successfully.The growing cell population consisted of spindle- and star-shaped cells with significant renewal capacity and multiple differentiaed abilities as they were culture.Smooth muscle progenitor cells can outgrow from bone MSC, This method may be useful in study of smooth muscle progenitor cell.Part III Role of smooth muscle progenitor cells in airway remodeling in asthmaObjective:To explore the role of smooth muscle progenitor cells in airway remodeling in asthma.Methods and materials:Twenty Wistar rats were randomly divided into four groups: female control+SPC group, female asthma group, female asthma+SPC group, male asthma group. OVA sensitization was performed by concurrent subcutaneous injection of OVA adsorbed to aluminum hydroxide.Fouteen days later, the animals were challenged by aerosol with an OVA solution or with phosphate-buffered saline for 8 weeks.Smooth progenitor cells（1.0×10⁶） which isolated from male rats was intravenous injection since fourth week,the injections were repeated for four times once a week. All animals were killed 24hours after antiger challenge. Airway samples were prepared for histology by formalin fixation and paraffin embedding. Each groups rats airway smooth muscle cells （ASMC） were isolated and subculture. Airway samples and ASMC were examinated Sry （sex-determining region on the Y chromosome） positive cell count by hybridization in situ（ISH）.Result:Both of airway samples and ASMC in female asthma+SPC group were examinnted Sry positive cell count by hybridization in situ（ISH）.But female control+SPC group were negative results.Conclusion:Smooth muscle progenitor cells take participate in asthmatic airway remodeling and play a critical role. Part IV Effect of atomization inhaled Sirolimus to smooth muscle progenitor cells in airway remodeling of asthma.Objective:To investigate the effect of sirolimus on proliferation and differentiation of mooth muscle progenitor cells and effect of atomization inhaled Sirolimus to smooth muscle progenitor cells in airway remodeling of asthma.Methods and materials:The bone marrow mesenchymal cells （MSCs） were isolated from the bone marrow of rats by adhering to the culture plastic.MSCs were cultured infibronectin-coated dishes in smooth muscle progenitor cells growth supplements with or without sirolimus （final concentrations: 0.1, 1, 10, 100ng/ml） for 3 days, SPCs proliferation were assayed by MTT assay for various time points （0h, 12 h, 24 h, 48 h）. After 10 days primarily cultured, attached cells were treated with sirolimus（final concentrations: 0.1, 1ng/ml） , SPCs were identified as adherent cells positive for a -SMA by indirect immunohistochemistry staining. Fifteen Wistar rats were randomly divided into three groups,female control group, female OVA+SPC group and female OVA/sirolimus+SPC group. OVA sensitization was performed by concurrent subcutaneous injection of OVA adsorbed to aluminum hydroxide.Fouteen days later, the animals were challenged by aerosol with an OVA solution or with phosphate-buffered saline for 8 weeks.The female OVA/sirolimus+SPC group also give atomization inhaled Sirolimus for 10 mins after OVA challenge. Airway samples were prepared for histology by formalin fixation and paraffin embedding. Each groups rats airway smooth muscle cells （ASMC） were isolated and subculture. Airway samples were examinated Sry positive cell count by hybridization in situ（ISH）.Result:Sirolimus also significantly inhibited the proliferative of SPC in a time and dose dependent manner.After cultured in different drug concentration of sirolimus of DM,0.1ng/ml,1ng/ml,10ng/ml and 100ng/ml for 48 hours,the optical density of each groups were 0.75±0.06,0.44±0.04,0.33±0.02,0.20±0.01 and 0.18±0.01（P<0.05）.SPC number diferentiated from MSC （final concentrations:DM, 0.1, 1ng/ml） at 10 days was significantly lower in sirolimus treated cells in a dose—dependent manner than that of vehicle treated cells.After cultured in different drug concentration of sirolimus of DM,0.1ng/ml and 1ng/ml for 10 days, the rate of a-SMA-positive SPCs from MSCs of each groups were 71%,24% and 4%（P <0.05）. Both of OVA/OVA and OVA/sirolimus groups rats’airway samples were examinnted Sry positive cells by hybridization in situ（ISH）.But Sry positive cell count in OVA/sirolimus group was lower than OVA/OVA group. OVA/PBS control+SPC group were negative results.Conclusion:Sirolimus could inhibitthe proliferation and diferentiation of smooth muscle progenitor cells,and it helpful for therapy of asthmatic airway remodeling by reduce smooth muscle progenitor cells take participate in it.更多还原