【作者】 李园园；

【导师】 胡成平；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 研究背景支气管哮喘是一种以可逆性气流受阻、气道炎症和气道高反应性为特征的疾病,随着分子生物学、分子遗传学以及分子免疫学的众学科在哮喘研究中的不断发展,目前证实其发生发展牵涉神经、内分泌、免疫等众多机制。神经生长因子(NGF)是一种对神经生长、发育和分化起到重要作用的神经肽类,可由肾上腺髓质细胞合成并分泌。哮喘究竟是一种全身性疾病还是单独的气道性疾病?目前尚无定论。我们的前期研究发现,支气管哮喘大鼠存在肾上腺素合成和释放障碍。神经生长因子可能通过启动哮喘大鼠肾上腺髓质嗜铬细胞冗余性致肾上腺素释放障碍而参与哮喘的发生和发展。用NGF抗体经14天腹腔给药,对哮喘大鼠进行干预,在降低体内NGF表达的同时可以使气道炎症得以缓解。然而将NGF抗体(抗-NGF)成功应用于哮喘患者还存在一些困难：如外源性生长因子及其抗体的生物半衰期短,给药后使用很快被稀释和代谢,故需要多次、大剂量给药；又如抗-NGF全身应用的毒副反应尚不明确,并能拮抗机体内源性NGF,降低其正常生理学效应。用何办法能简单、有效的抑制肾上腺素减少从而减轻或抑制哮喘发作?近年来,微球和纳米粒子作为药物运载系统引起越来越多人们的兴趣,微球是一种以适宜高分子材料为载体包裹或吸附药物而制成的球形或类球形的微粒,缓释微球则是以生物可降解聚合物为载体材料制成的微球,通过微球内部的孔道以及高分子材料的融蚀降解来调控蛋白质在体内外的释放。蛋白微球的制备最常用的方法是采用水／油／水(W1／O／W2)的双乳化技术。在生物可降解聚合物中,聚乳酸(PLA)和聚(乳酸-羟基乙酸)共聚物(PLGA)由于其无毒且能在体内缓慢降解的特性而得到广泛的应用,并且是已经被美国FDA批准为可用于人体的载体材料。包载药物的缓释微球具有粒径小(1—250μm)、长周期缓慢释放药物、对生物体无毒等优点。基于此,本课题以NGF、抗-NGF为包被药物,采用W1／O／W2的双乳化-溶剂挥发技术,成功制备粒径约15μmm,释放15天以上的PLGA微球,并分别将抗-NGF缓释微球和NGF缓释微球直接注入哮喘大鼠肾上腺实现局部干预,检测对肺功能、肺组织结构、血清学指标等影响,探讨抗-NGF缓释微球局部干预对哮喘大鼠的干预作用及其机制,寻找可能的哮喘防治新方法。第一章NGF、抗NGF微球的制备及其评价目的制备粒径均匀(15μm左右)能够持续释放神经生长因子(抗神经生长因子)达15天以上,有良好的体外缓释效果的NGF-(抗-NGF)PLGA缓释微球,为后续实验以提供干预剂。方法运用水／油／水(W1／O／W2)的双乳化技术,以牛血清白蛋白(BSA)为载体,制备NGF(抗-NGF)-PLGA缓释微球,进行其外观、粒径、载药量、包封率、体外释放情况的检测与评价。结果神经生长因子缓释微球(BSA/PLGA 15/100,NGF、抗-NGF/BSA 1/2000)表面光滑,形态圆整,粒径(15.5±5.2)μm,载药量8.45%,包封率55.7%。在体外释放的第3、6、9、12、15天分别有32.14%、44.5%、50.99%、63.51%、79.53%的NGF从PLGA微球中释放。结论水／油／水(W1／O/W2)的双乳化技术可以成功制备包载神经生长因子的微球,其成球性佳,粒径均匀(15μm左右),重现性好。蛋白／多聚物比率越高,蛋白包封率则越低,但载药量也越高。神经生长因子缓释微球能够持续释放神经生长因子达15天以上,有良好的体外缓释效果。第二章抗神经生长因子微球局部植入对哮喘大鼠的干预作用及其机制目的利用抗-NGF缓释微球和NGF缓释微球直接注入哮喘大鼠肾上腺实现局部干预,探讨抗NGF对哮喘的治疗作用,寻找可能的哮喘治疗新方法。方法雄性SD大鼠32只,随机分为正常对照组、哮喘模型组、NGF微球干预组、抗-NGF微球干预组,分别干预各组大鼠。进行大鼠行为学观察和肺功能检测、光镜下观察肺组织切片、电镜下观察肾上腺髓质细胞超微结构的变化、免疫组织化学检测肾上腺组织中NGF和苯乙醇胺N-甲基转移酶(PNMT)阳性表达、ELISA法检测血清中NGF、皮质醇、皮质酮、肾上腺素、去甲肾上腺素的浓度变化情况。结果大鼠行为学显示哮喘组大鼠均有不同程度喷嚏、流涕、喘鸣、搔抓头面部、烦躁钻洞、大小便失禁、攻击性增强等行为,抗-NGF组则上述症状相对较轻,动物表现较安静。肺功能检测提示哮喘组、抗-NGF组、NGF组大鼠均存在明显的气道高反应,同时抗NGF组与哮喘组相比,气道阻力值减低,动态肺顺应性值升高。肺组织切片HE染色显示：哮喘组大鼠存在明显支气管收缩征象及其周围炎症细胞浸润,小血管周围、肺泡腔、肺间质大量炎细胞浸润,可见支气管上皮细胞脱落,抗-NGF组支气管收缩征象不明显,炎性细胞浸润现象较哮喘组轻。气管上皮较完整,腔内无明显炎性分泌物渗出现象。电镜观察可见：哮喘组、NGF组肾上腺组髓质细胞均出现空泡样改变,嗜铬颗粒分布不均匀,数量和浓度较正常组明显降低；NGF组肾上腺髓质细胞膜似见杵状和绒毛状突起；抗NGF组无明显空泡样改变,嗜铬颗粒数量及浓度均基本接近正常组。图像分析结果显示抗NGF组PNMT和NGF免疫组化结果平均灰度值分别为(193.39±0.95)、(173.78±3.23),与哮喘组比较均有明显差异(P分别<0.05、0.01)；ELISA结果显示：(1)各组间肾上腺素平均浓度水平依次为对照组>抗NGF组>哮喘组>NGF组；抗NGF组与哮喘组、哮喘组与正常组、NGF组与哮喘组对比均有明显差异(P均<0.01)。(2)各组间去甲肾上腺素平均浓度水平依次为NGF组>哮喘组>抗NGF组>对照组；抗NGF组与哮喘组、哮喘组与正常组、NGF组与哮喘组对比均有明显差异(P均<0.01)。(3)各组间皮质醇浓度水平差异无显著性(P均>0.05)。(4)各组皮质酮平均浓度水平依次为对照组>抗NGF组>哮喘组>NGF组；抗NGF组与哮喘组、哮喘组与对照组、NGF组与哮喘组对比均有明显差异(P<0.01、0.01、0.05)。结论局部植入抗-NGF微球能减轻哮喘大鼠肺组织炎性浸润、改善肺功能。其作用机制可能为抗-NGF将NGF拮抗后逆转了其启动肾上腺髓质细胞转分化过程。更多还原

【Abstract】 Background Bronchial asthma is a disease characterized by reversible airflow obstruction, airway inflammation and airway hyperresponsiveness. In recent years, as the development of molecular biology, molecular genetics and molecular immunology in the asthma research, it is confirmed that the exacerbation of asthma involves the nervous, endocrine, immune and many other mechanisms. Nerve growth factor (NGF) is a kind of neuropeptide. It plays an important role in neural development and differentiation. NGF is synthesized and secreted by the adrenal medullary cell. Whether asthma is a systemic disease or a separate airway disease? There is no conclusion. Our previous study found that the adrenaline was decreased due to the dysfunction of synthesis and release in asthmatic rats. NGF may initiate dysfunction of releasing adrenaline in asthmatic rats due to functional redundancy of adrenal medulla chromaffin cells to participate in the occurrence and development of asthma. After 14 days intervention with NGF antibody intraperitoneally injection in asthmatic rats we found that it could reduce the NGF expression in vivo while airway inflammation were also alleviated. However, there are still some difficulties in application of NGF antibody in patients with asthma. For example, the exogenous growth factor needs a multiple and large administered dose due to its short biological half life period which shows the short dilution and metabolism after use. In addition, NGF in vivo may also be neutralized by NGF antibody incuding the reduction of its normal physiological effect while the toxicity of NGF antibody after its systemic application is still unknown. Which can be a simple and effective method in inhibition of dysfunction of releasing adrenaline in order to relieve or suppress the asthma attack?In recent years, as drug delivery systems, microspheres and nanoparticles caused more and more people interested in them. Microsphere is a kind of polymer material suitable for wrapping or adsorption of drug carrier made of spherical or spherical particles, releases microspheres are based on biodegradable polymers as carrier material made of microspheres through the pores of the microspheres and the polymer material to control erosion degradation of the protein in vivo and in vitro release. The protein microsphere is commonly prepared by the (water-in-oil)-in-water (W1/O/W2) emulsion and solvent evaporation technique with some modification. In biodegradable polymer, polylactic acid (PLA) and Poly (D,L-lactic-co-glycollic acid) (PLGA) is widely used because of its non-toxic and slow-degradation characteristics in vivo.While it is already used by the U.S. FDA approved carrier material in the human body. In this research, the PLGA-protein microspheres with NGF and anti-NGF as coated drug are successfully prepared by the (water-in-oil)-in-water (W1/O/W2) emulsion and solvent evaporation technique. The particle diameter of microspheres is about 15μm, and the releasing is more than 15 days. Anti-NGF and NGF microspheres are respectively and directly injected into the adrenal gland of rat model of asthma as a local embedding way so as to evaluate the effect of lung function, lung histology structure, serological indicators and etc after intervention. We also discuss the effect and mechanism of the local intervention of anti-NGF microspheres of rat model of asthma in order to find a possible new method of prevention and treatment of asthma.Chapter One The preparation and evaluation of NGF and anti-NGF microspheresObjective To prepare NGF (anti-NGF) PLGA microspheres which possessed uniform size (15μm) and continuous releasing of NGF (anti-NGF) for 15 days with good effect in vitro to provide intervention agent to follow-up experiment.Method Using bovine serum albumin (BSA) as the carrier, NGF (anti-NGF) PLGA microspheres were prepared by the (water-in-oil)-in-water (W1/O/W2) emulsion and solvent evaporation technique. Then their appearance, size, Drug-loading rate, entrapment rate, in vitro release testing were evaluated.Result NGF microspheres(BSA/PLGA 15/100,NGF、anti-NGF/ BSA 1/2000) had smooth surface and spherical morphology with (15.5±5.2)μm particle size,8.45% drug loading,55.7%encapsulation efficiency. In vitro release of the first 3,6,9,12,15 days there were 32.14%, 44.5%,50.99%,63.51%,79.53%of the NGF released from PLGA microspheres.Conclusion (water-in-oil)-in-water (W1/O/W2) emulsion and solvent evaporation technique could be successfully and reproducibly prepared package containing nerve growth factor microspheres which possess best spherical morphology and uniform particle size (15μm or so). The results showed that the higher the protein/polymer ratio, the higher the drug loading into the microspheres, and the lower the efficiency of protein encapsulation in the microspheres. NGF(anti-NGF) microspheres maintained a sustained release of NGF(anti-NGF) for at least 15 days in vitro. Chapter Two The local embedding intervention of anti-nerve growth factor microspheres in asthmatic rats and its mechanismObjective Using anti-NGF and NGF microspheres injected directly into the asthmatic rat adrenal gland, to achieve partial intervention of anti-NGF microsphere treatment of asthma, identify potential new method for the treatment of asthma.Method 32 male SD rats were randomly divided into normal control group, asthma group, NGF microspheres group and anti-NGF microspheres group before the rats were intervented. To investigate the behavior of rats, lung function testing, light microscopy of lung biopsy, electron microscopy of adrenal medulla cell ultrastructure changes、NGF and phenylethanolamine N-methyltransferase (PNMT) expression in the adrenal gland by immunohistochemistry method and serumal NGF, cortisol, corticosterone, epinephrine and norepinephrine concentration changes by ELISA assay.Results Behavior in asthma rats showed varying degrees of sneezing, runny nose, wheezing, scratching the head and face, irritability holes, incontinence, increased aggression and other acts, while in the rats from anti-NGF group showed relatively slighter symptoms and the rats turned to be more quiet. Pulmonary function testing prompted asthma, anti-NGF group, NGF rats showed significant airway hyperresp- onsiveness, while anti-NGF group compared with asthma, RL value is reduced, Cdyn value increased. HE staining of lung tissue sections: asthma group were obvious signs of bronchoconstriction and inflammatory cell infiltration around small vessels and alveolar spaces, interstitial lung inflammatory cell infiltration, bronchial epithelial cells can be seen off, while anti-NGF group were not as much bronchoconstriction and inflammatory cell infiltration as asthma group. And also they have more complete tracheal epithelium with no significant inflammatory exudation cavity phenomenon. Electron microscopy: asthma, NGF group adrenal medulla cells in all groups appeared vacuolated changes, uneven distribution of chromaffin granules, the quantity and concentration were significantly lower than normal; NGF group seemed on the adrenal medulla cell membrane and villous clubbing processes; anti-NGF group had no significant vacuolar changes in chromaffin granules and the concentration close to the normal group. Image analysis revealed that anti-NGF group PNMT and NGF immunohistochemistry results mean gray values were (193.39±0.95), (173.78±3.23), and the asthma group was significantly different (P< 0.05,0.01); The ELISA results showed that:(1) The average concentration levels of epinephrine in each group were the control group > anti-NGF group> asthma> NGF group.Anti-NGF group compared with asthmatic group, as well as asthma group and control group, NGF group and asthma group, were significant different (all P<0.01). (2) The average concentration levels of norepinephrine in each group were the NGF group> asthma group>anti-NGF group> control group. Anti-NGF group compared with asthmatic group, as well as asthma group and control group, NGF group and asthma group, were significant different (all P<0.01). (3) There was no significant difference among the groups of the average concentration levels of cortisol (all P> 0.05). (4) The average concentration levels of norepinephrine in each group were control group>anti-NGF group>asthma group>NGF group. Anti-NGF group compared with asthma group, as well as asthma group and control group, NGF group and asthma group, were significant different.(P＜0.01, 0.01,0.05).Conclusion Local embedding of anti-NGF microspheres can alleviate inflammatory infiltration in lung tissue and improve lung function of rat model of asthma. The mechanism may be the anti-NGF reverse NGF’s priming of the adrenal medulla cell transdifferentiation process after the antagonism of it.更多还原