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
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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。
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 microspheres
Objective 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 mechanism
Objective 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.
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。
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 microspheres
Objective 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 mechanism
Objective 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.
引文
[1]Carr DJ, Blalock JE. A molecular basis for intersystem communication between the immune and neuroendocrine systems. Int Rev Immunol.1989. 4(3):213-28.
[2]Nockher WA, Renz H. Neurotrophins and asthma:novel insight into neuroimmune interaction. J Allergy Clin Immunol.2006.117(1):67-71.
[3]Nassenstein C, Kutschker J, Tumes D, Braun A. Neuro-immune interaction in allergic asthma:role of neurotrophins. Biochem Soc Trans.2006.34(Pt 4): 591-3.
[4]Feng JT, Hu CP. Dysfunction of releasing adrenaline in asthma by nerve growth factor. Med Hypotheses.2005.65(6):1043-6.
[5]汪俊,胡成平,冯俊涛,Jun W, Cheng-ping HU, Jun-tao F.神经生长因子对支气管哮喘大鼠肾上腺素释放障碍的机制研究.中华结核和呼吸杂志.2006.29(12)：812-815.
[6]汪俊,胡成平,梁伟军,冯俊涛.哮喘大鼠肾上腺髓质嗜铬细胞表型转化与神经生长因子表达水平初探.中国实用内科杂志.2007.27(4)：270-272.
[7]汪俊,胡成平,冯俊涛等.新生小牛肾上腺髓质嗜铬细胞的原代培养及其儿茶酚胺的分泌.中国应用生理学杂志.2008.24(2)：138-140.
[8]朱锦琪,冯俊涛,胡成平等.神经生长因子、白血病抑制因子调控支气管哮喘大鼠神经源性炎症机制的研究.中华结核和呼吸杂志.2006.29(6)：376-380.
[9]陆彬.药物新剂型与新技术.1998.人民卫生出版社.165-249.
[10]Boisdron-Celle M, Menei P, Benoit JP. Preparation and characterization of 5-fluorouracil-loaded microparticles as biodegradable anticancer drug carriers. J Pharm Pharmacol.1995.47(2):108-14.
[11]der Lubben IM v, Verhoef JC, Borchard G, Junginger HE. Chitosan and its derivatives in mucosal drug and vaccine delivery. Eur J Pharm Sci.2001. 14(3):201-7.
[12]Konturek SJ, Pawlik W, Mysh W, et al. Comparison of organ uptake and disappearance half-time of human epidermal growth factor and insulin. Regul Pept.1990.30(2):137-48.
[13]Boisdron-Celle M, Menei P, Benoit JP. Preparation and characterization of 5-fluorouracil-loaded microparticles as biodegradable anticancer drug carriers. J Pharm Pharmacol.1995.47(2):108-14.
[14]Conti B, Pavanetto F, Genta I. Use of polylactic acid for the preparation of microparticulate drug delivery systems. J Microencapsul.1992.9(2):153-66.
[15]陈庆华,瞿文,Qinghua C, Wen Q.多肽、蛋白质类药物缓释剂型的研究进展.中国药学杂志.2000.35(3)：147-150.
[16]艾国,程远国,Guo AI, Yuan-guo C.蛋白质多肽长效微球注射剂的体内药动学及分析方法研究进展.中国新药杂志.2007.16(7)：501-506.
[17]杨彤,周云赞,王娜,徐力,吴晓霞,张学忠.细胞粘附位点RGD三肽脂质体的制备及表征.吉林大学学报(理学版).2003.41(3)：356-360.
[18]谷海刚，龙大宏,李晓滨等.异硫氰酸荧光素标记的神经生长因子缓释微球的制备及评价.中华生物医学工程杂志.2007.13(6)：393-396.
[19]Cohen S, Yoshioka T, Lucarelli M, Hwang LH, Langer R. Controlled delivery systems for proteins based on poly(lactic/glycolic acid) microspheres. Pharm Res.1991.8(6):713-20.
[20]Menei P, Montero-Menei C, Venier MC, Benoit JP. Drug delivery into the brain using poly(lactide-co-glycolide) microspheres. Expert Opin Drug Deliv. 2005.2(2):363-76.
[21]Jiang W, Gupta RK, Deshpande MC, Schwendeman SP. Biodegradable poly(lactic-co-glycolic acid) microparticles for injectable delivery of vaccine antigens. Adv Drug Deliv Rev.2005.57(3):391-410.
[22]Haller MF, Saltzman WM. Nerve growth factor delivery systems. J Control Release.1998.53(1-3):1-6.
[23]Haller MF, Saltzman WM. Localized delivery of proteins in the brain:can transport be customized. Pharm Res.1998.15(3):377-85.
[24]Obeidat WM, Price JC. Viscosity of polymer solution phase and other factors controlling the dissolution of theophylline microspheres prepared by the emulsion solvent evaporation method. J Microencapsul.2003.20(1):57-65.
[25]Ruan G, Ng JK, Feng SS. Effects of polymer, organic solvent and mixing strength on integrity of proteins and liposomes encapsulated in polymeric microspheres fabricated by the double emulsion process. J Microencapsul. 2004.21(4):399-412.
[26]刘世芸,徐超,冯乙巳等.白蛋白聚乳酸缓释微球的制备及体外释放研究.合肥工业大学学报(自然科学版).2006.29(12)：1584-1587.
[27]Zhu G, Mallery SR, Schwendeman SP. Stabilization of proteins encapsulated in injectable poly (lactide-co-glycolide). Nat Biotechnol.2000.18(1):52-7.
[28]Kang F, Jiang G, Hinderliter A, DeLuca PP, Singh J. Lysozyme stability in primary emulsion for PLGA microsphere preparation:effect of recovery methods and stabilizing excipients. Pharm Res.2002.19(5):629-33.
[29]Estey T, Kang J, Schwendeman SP, Carpenter JF. BSA degradation under acidic conditions:a model for protein instability during release from PLGA delivery systems. J Pharm Sci.2006.95(7): 1626-39.
[30]de Weert M v, Hennink WE, Jiskoot W. Protein instability in poly(lactic-co-glycolic acid) microparticles. Pharm Res.2000.17(10): 1159-67.
[31]Carrascosa C, Espejo L, Torrado S, Torrado JJ. Effect of gamma-sterilization process on PLGA microspheres loaded with insulin-like growth factor-Ⅰ (IGF-Ⅰ). J Biomater Appl.2003.18(2):95-108.
[32]Meinel L, Illi OE, Zapf J, Malfanti M, Peter MH, Gander B. Stabilizing insulin-like growth factor-Ⅰ in poly(D,L-lactide-co-glycolide) microspheres. J Control Release.2001.70(1-2):193-202.
[33]Yuksel E, Weinfeld AB, Cleek R, et al. Increased free fat-graft survival with the long-term, local delivery of insulin, insulin-like growth factor-Ⅰ, and basic fibroblast growth factor by PLGA/PEG microspheres. Plast Reconstr Surg. 2000.105(5):1712-20.
[34]汤渝玲,胡成平,冯俊涛等.神经生长因子调控哮喘神经源性炎症Ras-MAPK信号转导通路.中南大学学报(医学版).2006.31(3)：319-325.
[35]Menei P, Pean JM, Nerriere-Daguin V, Jollivet C, Brachet P, Benoit JP. Intracerebral implantation of NGF-releasing biodegradable microspheres protects striatum against excitotoxic damage. Exp Neurol.2000.161(1): 259-72.
[36]Pean JM, Menei P, Morel O, Montero-Menei CN, Benoit JP. Intraseptal implantation of NGF-releasing microspheres promote the survival of axotomized cholinergic neurons. Biomaterials.2000.21(20):2097-101.
[37]Mahoney MJ, Saltzman WM. Controlled release of proteins to tissue transplants for the treatment of neurodegenerative disorders. J Pharm Sci. 1996.85(12):1276-81.
[38]Gertz HJ, Cervos-Navarro J, Ewald V. The septo-hippocampal pathway in patients suffering from senile dementia of Alzheimer's type. Evidence for neuronal plasticity. Neurosci Lett.1987.76(2):228-32.
[39]Blanco-Prieto MJ, Leo E, Delie F, Gulik A, Couvreur P, Fattal E. Study of the influence of several stabilizing agents on the entrapment and in vitro release of pBC 264 from poly(lactide-co-glycolide) microspheres prepared by a W/O/W solvent evaporation method. Pharm Res.1996.13(7):1127-9.
[40]Takada S, Yamagata Y, Misaki M, Taira K, Kurokawa T. Sustained release of human growth hormone from microcapsules prepared by a solvent evaporation technique. J Control Release.2003.88(2):229-42.
[41]Pean JM, Boury F, Venier-Julienne MC, Menei P, Proust JE, Benoit JP. Why does PEG 400 co-encapsulation improve NGF stability and release from PLGA biodegradable microspheres. Pharm Res.1999.16(8):1294-9.
[42]Sun SW, Jeong YI, Jung SW, Kim SH. Characterization of FITC-albumin encapsulated poly(DL-lactide-co-glycolide) microspheres and its release characteristics. J Microencapsul.2003.20(4):479-88.
[43]Yang YY, Chung TS, Ng NP. Morphology,'drug distribution, and in vitro release profiles of biodegradable polymeric microspheres containing protein fabricated by double-emulsion solvent extraction/evaporation method. Biomaterials.2001.22(3):231-41.
[44]Bodmeier R, McGinity JW. The preparation and evaluation of drug-containing poly(dl-lactide) microspheres formed by the solvent evaporation method. Pharm Res.1987.4(6):465-71.
[45]de Weert M v, Hennink WE, Jiskoot W. Protein instability in poly(lactic-co-glycolic acid) microparticles. Pharm Res.2000.17(10): 1159-67.
[46]Perez C, Castellanos IJ, Costantino HR, Al-Azzam W, Griebenow K. Recent trends in stabilizing protein structure upon encapsulation and release from bioerodible polymers. J Pharm Pharmacol.2002.54(3):301-13.
[47]习明,胡卫列,Ming XI, Wei-lie HU.肾上腺解剖学的研究进展.中国临床解剖学杂志.2008.26(2)：227-228.
[48]汪俊,胡成平,冯俊涛,Jun W, Cheng-ping HU, Jun-tao F.神经生长因子对支气管哮喘大鼠肾上腺素释放障碍的机制研究.中华结核和呼吸杂志.2006.29(12)：812-815.
[49]林敏娟,胡成平,吴鄂生,潘频华.支气管哮喘大鼠肺组织中白血病抑制因子的表达变化.中华结核和呼吸杂志.2003.26(11)：727-728.
[50]Baumgarten CR, Witzel A, Schierhorn K, Kunkel G. Neuro-immuno regulatory mechanisms of asthma. Acta Microbiol Immunol Hung.1998.45(1): 31-42.
[51]Ball TM, Anderson D, Minto J, Halonen M. Cortisol circadian rhythms and stress responses in infants at risk of allergic disease. J Allergy Clin Immunol. 2006.117(2):306-11.
[52]Wright RJ. Stress and atopic disorders. J Allergy Clin Immunol.2005.116(6): 1301-6.
[53]Theoharides TC, Kempuraj D, Tagen M, Vasiadi M, Cetrulo CL. Human umbilical cord blood-derived mast cells:a unique model for the study of neuro-immuno-endocrine interactions. Stem Cell Rev.2006.2(2):143-54.
[54]Besedovsky HO, del RA. Feed-back interactions between immunological cells and the hypothalamus-pituitary-adrenal axis. Neth J Med.1991.39(3-4): 274-80.
[55]Besedovsky HO, del RA. Interactions between immunological cells and the hypothalamus pituitary-adrenal axis:an example of neuroendocrine immunoregulation. Recenti Prog Med.1988.79(7-8):300-4.
[56]孔灵菲,Ling-fei K.最新全球哮喘防治指南解读.中国实用内科杂志.2007.27(4)：255-257.
[57]Levi-Montalcini R, Dal Toso R, della VF, Skaper SD, Leon A. Update of the NGF saga. J Neurol Sci.1995.130(2):119-27.
[58]Levi-Montalcini R, Skaper SD, Dal Toso R, Petrelli L, Leon A. Nerve growth factor:from neurotrophin to neurokine. Trends Neurosci.1996.19(11): 514-20.
[59]Aloe L, Bracci-Laudiero L, Bonini S, Manni L. The expanding role of nerve growth factor:from neurotrophic activity to immunologic diseases. Allergy. 1997.52(9):883-94.
[60]周敏,徐永健,熊盛道,赵建平,倪望,张珍祥.神经生长因子在哮喘患者诱导痰炎性细胞的表达.中华内科杂志.2003.42(11)：764-767.
[61]Lindsay RM, Harmar AJ. Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurons. Nature.1989.337(6205):362-4.
[62]Lindsay RM, Lockett C, Sternberg J, Winter J. Neuropeptide expression in cultures of adult sensory neurons:modulation of substance P and calcitonin gene-related peptide levels by nerve growth factor. Neuroscience.1989.33(1): 53-65.
[63]Undem BJ, Hunter DD, Liu M, Haak-Frendscho M, Oakragly A, Fischer A. Allergen-induced sensory neuroplasticity in airways. Int Arch Allergy Immunol.1999.118(2-4):150-3.
[64]Virchow JC, Julius P, Lommatzsch M, Luttmann W, Renz H, Braun A. Neurotrophins are increased in bronchoalveolar lavage fluid after segmental allergen provocation. Am J Respir Crit Care Med.1998.158(6):2002-5.
[65]Sanico AM, Stanisz AM, Gleeson TD, et al. Nerve growth factor expression and release in allergic inflammatory disease of the upper airways. Am J Respir Crit Care Med.2000.161(5):1631-5.
[66]Bonini S, Lambiase A, Bonini S, et al. Circulating nerve growth factor levels are increased in humans with allergic diseases and asthma. Proc Natl Acad Sci U S A.1996.93(20):10955-60.
[67]Olgart HC, Frossard N. Nerve growth factor and asthma. Pulm Pharmacol Ther.2002.15(1):51-60.
[68]Olgart C, Frossard N. Human lung fibroblasts secrete nerve growth factor: effect of inflammatory cytokines and glucocorticoids. Eur Respir J.2001. 18(1):115-21.
[69]Carr MJ, Hunter DD, Undem BJ. Neurotrophins and asthma. Curr Opin Pulm Med.2001.7(1):1-7.
[70]Fox AJ, Patel HJ, Barnes PJ, Belvisi MG. Release of nerve growth factor by human pulmonary epithelial cells:role in airway inflammatory diseases. Eur J Pharmacol.2001.424(2):159-62.
[71]Bonini S, Lambiase A, Bonini S, Levi-Schaffer F, Aloe L. Nerve growth factor: an important molecule in allergic inflammation and tissue remodelling. Int Arch Allergy Immunol.1999.118(2-4):159-62.
[72]Braun A, Quarcoo D, Schulte-Herbruggen O, Lommatzsch M, Hoyle G, Renz H. Nerve growth factor induces airway hyperresponsiveness in mice. Int Arch Allergy Immunol.2001.124(1-3):205-7.
[73]Quarcoo D, Schulte-Herbruggen O, Lommatzsch M, et al. Nerve growth factor induces increased airway inflammation via a neuropeptide-dependent mechanism in a transgenic animal model of allergic airway inflammation. Clin Exp Allergy.2004.34(7):1146-51.
[74]朱锦琪,冯俊涛,胡成平等.神经生长因子、白血病抑制因子调控支气管哮喘大鼠神经源性炎症机制的研究.中华结核和呼吸杂志.2006.29(6)：376-380.
[75]Kubota T, Koga K, Araki H, et al. [The relationships of mononuclear leukocyte beta-adrenergic receptors to aerobic capacity and exercise-induced asthma in asthmatic children]. Arerugi.2000.49(1):40-51.
[76]Counil FP, Varray A, Karila C, Hayot M, Voisin M, Prefaut C. Wingate test performance in children with asthma:aerobic or anaerobic limitation. Med Sci Sports Exerc.1997.29(4):430-5.
[77]Greene LA, Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A.1976.73(7):2424-8.
[78]吴克复,马小彤,宋玉华.细胞的功能冗余性及其意义.生物化学与生物物理进展.2004.31(12)：1054-1057.
[79]Unsicker K, Krisch B, Otten U, Thoenen H. Nerve growth factor-induced fiber outgrowth from isolated rat adrenal chromaffin cells:impairment by glucocorticoids. Proc Natl Acad Sci U S A.1978.75(7):3498-502.
[80]Unsicker K, Krisch B, Otten U, Thoenen H. [Nerve growth factor-induced axon growth and its antagonism with dexamethasone:in vitro studies on adrenal medulla cells]. Verh Anat Ges.1978. (72):371-2.
[81]Unsicker K, Krisch B, Otten U, Thoenen H. Nerve growth factor-induced fiber outgrowth from isolated rat adrenal chromaffin cells:impairment by glucocorticoids. Proc Natl Acad Sci U S A.1978.75(7):3498-502.
[1]刘玉梅,张自强.微球制剂临床应用进展.齐鲁药事.2006,25(4)：234-236
[2]韩敏,苏秀霞,李仲谨.载药微球制剂的研究进展.应用化工.2007,36(5)：493-495
[3]Longo W E, Iw ata H.Preparation of hydrophilic album in microspheres using polymeric dispersing agents. J Pharm Sci.1982,71(9):1323-1328.
[4]李凤前,陆彬,曾仁杰.地塞米松磷酸盐牛血清清蛋白微球的研制.中国医药工业杂志.1999,30(3)：113-115.
[5]丁红,刑桂琴,谢茵.阿霉素明胶微球的制备与特性研究.中国医院药学杂志.2000,20(7)：387-389.
[6]方华丰,周宜开.壳聚糖微球的研究进展.国外医药合成药生化药制剂分册.1999,20(5)：315-318.
[7]张珠.利福平壳聚糖蛋白微球的制备及性能研究.武汉理工大学学报.2001,23(1)：21-23.
[8]许天开,赵树杰.PHB在生物医学中的应用研究进展.应用与环境生物学报.1995,7(1)：85-88.
[9]王正容,陆彬,杨红.左炔诺孕酮-聚-3-羟基丁酸酯缓释微球的研究.药学学报,1 999,34(1)：57-59.
[10]刘哲鹏,潘俊,陆伟跃.生物黏附微球研究及前景.国外医药合成药生化药制剂 分册.2001,22(2)：111-114.
[11]张瑶. 缓释微球制剂的研究进展.2004,23(11)：843-844
[12]周新腾,潘卫三,张汝华.局部麻醉药生物可降解缓释微球的研究进展.中国新药杂志.2001,10(9)：658-660.
[13]董爱梅,张建强.眼用微球制剂研究进展.齐鲁药事.2005,24(1)：36-38.
[14]王红梅,江燕,王静.鼻腔给药新剂型初探.方药研究,2002,(2)：53.
[15]王晶,王勤,李静.聚乳酸微球制剂的研究概况.山东生物医学工程,1999.18(3)：50-52
[16]甘莉,鲜平.前列腺素E1脂微球制剂治疗糖尿病周围神经病变的疗效观察.临床医药实践杂志.2004,13(2)：126-27.
[17]易玉新,杨宇,屈晓冰,等.前列腺素E1脂微球制剂治疗老年脑梗塞的疗效.中日友好医院学报.2004,18(1)：20-22.
[18]顾慧华,田力,薛来英.前列腺素El脂微球载体制剂治疗慢性重型肝炎的临床研究.中国现代应用药学杂志.2004,21(1)：69-70.
[19]邵景范,罗永湘.神经生长因子的载体及其控释系统.中华显微外科杂志.1995,18(4)：303-305.
[20]蒋涛,任先军.神经营养因子及其微球制剂.中国药师.2006,9(5)：457-458
[21]黄芳,费俭,刘晓等.神经营养因子.细胞生物学杂志.1996,18：17-20
[22]Shahrooz R, Justin OH, Zhong CT, et al. Induction of apoptosis by the low-affinity NGF receptor.Science,1993,261:345-348
[23]Barrett GL, Bartlett PF. The p75 nerve growth factor receptor mediated survival or death depending on the stage of sensory neuron development.Proc Nail Acad Sci USA,1994,91:6501-6505
[24]邓小龙,陈浩凡.几种可生物降解材料在胶体微粒给药系统中的应用.中国药师,2002,5(8)：492-494
[25]Camarata PJ, Suryanarayanan R,Turner DA, et al. Sustained release of nerve growth factor from biodegradable polymer microspheres. Neurosurgery, 1992,30(3):313-319
[26]Pean JM, Venier-Julienne MC, Boury F, et al. NGF release from poly (D,L-lactide-co-glycolide) microspheres. Efect of some formulation parameters on encapsulated NGF stability. J Control Release,1998,56 (1-3):175-187
[27]Menei P,Pean JM,Neriere-Dafuin V,et al.Intracerebralimplantation of NGF-releasing biodegradable Microspheres Protects striatum against excitotoxic damage. Exp Neural,2000,161(1):259-272
[28]Aubert-Pouessel A,Venier-Julienne AC,Clavreul A,et al.In vitro study of GDNF release From Biodegradable PLGA microspheres. J Control Release,2004, 95(3):463-475
[29]Jollivet C,Aubert-Pouessel A,ClavreulA,et al. Long-term effect of intra-striatal glial cell line-derived neurotrophic factor-releasing microspheres in a partial rat model of Parkinson's disease. Neurosci Lett,2004,356(3):207-210
[30]Jolivet C, Aubert-Pouessel A, Clavreul A, et al. Striatal implantation of GDNF releasing biodegradable microspheres promotes recovery of motor function in a partial model of Parkinson's disease.Biornaterials,2004,2 5(5):933-942
[31]Maysinger D, Krieglstein K, Filipovic-Greic J, et al. Microencapsulated ciliary neurotrophic factor:physical properties and biological activities. Exp Neurol, 1996,138(2):177-188
[32]Mittal S, Cohen A, Maysinger D. In vitro effects of brain derived neurotrophic fact or released from microspheres.Neuronreport,1994,5(18):2577-2582
[33]Rellinger RJ, Mailliard RB, Barksdale EMJ.Neurobla stoma and dendritic cell function. Semin Pediatr Surg,2004,13(1):61.
[34]李爱敏,孙洪亮,张锦华.Caspase-8沉默-神经母细胞瘤耐药新机制.国外医学-儿科学分册,2004,31(3)：165.
[35]高强,董倩,吕振华,等.转神经生长因子基因诱导神经母细胞瘤分化的研究.中华小儿外科杂志,2005,26(1)：34.
[36]陆莹,赵宝东,赵春玉.NGF对缺血损伤引起大鼠海马CA1区神经细胞凋亡的作用.锦州医学院学报,2004,3(1)：21.
[37]Hoyle, G. W., Graham, R. M., Finkelstein, J. B., Nguyen, K. P., Gozal, D.,& Friedman, M. Hyperinnervation of the airways in transgenic mice overexpressing nerve growth factor. Am J Respir Cell Mol Biol. (1998).18(2),149-157.
[38]Hunter, D. D., Myers, A. C.,& Undem, B. J. Nerve growth factorinduced phenotypic switch in guinea pig airway sensory neurons.Am J Respir Crit Care Med,(2000).161(6),1985-1990.
[39]Dinh, Q. T., Groneberg, D. A., Peiser, C., Springer, J., Joachim, R. A., Arck, P. C.,et al. Nerve growth factor-induced substance P in capsaicininsensitive vagal neurons innervating the lower mouse airway. Clin Exp Allergy.(2004).34(9), 1474-1479.
[40]Nassenstein, C., Dawbarn, D., Pollock, K., Allen, S. J., Erpenbeck, V. J., Spies, E., et al. Pulmonary distribution, regulation, and functional role of Trk receptors in a murine model of asthma. J Allergy Clin Immunol. (2006a).118(3),597-605.
[41]Micera, A., Vigneti, E., Pickholtz, D., Reich, R., Pappo, O., Bonini, S., et al.Nerve growth factor displays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair.Proc Natl Acad Sci USA.(2001).98(11),6162-6167.
[42]Kohyama, T., Liu, X., Wen, F. Q., Kobayashi, T., Abe, S., Ertl, R., et al. Nerve growth factor stimulates fibronectin-induced fibroblast migration.J Lab Clin Med. (2002).140(5),329-335.
[43]Chevet, E., Lemaitre, G., Janjic, N., Barritault, D., Bikfalvi, A.,&Katinka, M. D.Fibroblast growth factor receptors participate in the control of mitogen-activated protein kinase activity during nerve growth factor-induced neuronal differentiation of PC12 cells. J Biol Chem.(1999).274(30), 20901-20908.
[44]Cosgaya, J. M.,& Aranda, A.. Nerve growth factor regulates transforming growth factor-beta 1 gene expression by both transcriptional and posttranscriptional mechanisms in PC12 cells. J Neurochem. (1995)65(6),2484-2490.
[45]Sin, A. Z., Roche, E. M., Togias, A., Lichtenstein, L. M.,& Schroeder, J. T. Nerve growth factor or IL-3 induces more IL-13 production from basophils of allergic subjects than from basophils of nonallergic subjects.J Allergy Clin Immunol.(2001).108(3),387-393.
[46]Noga,O., Englmann, C., Hanf, G., Grutzkau, A., Guhl, S., & Kunkel, G. Activation of the specific neurotrophin receptors TrkA, TrkB and TrkC influences the function of eosinophils. Clin Exp Allergy. (2002).32(9), 1348-1354.
[47]Nithya, M., Suguna, L.,& Rose, C. The effect of nerve growth factor on the early responses during the process of wound healing. Biochim Biophys Acta.(2003).1620(1-3),25-31.
[48]Takahashi, H., Uno, S., Watanabe, Y., Arakawa, K.,& Nakagawa, S. Expression of nerve growth factor-induced type 1 plasminogen activator inhibitor (PAI-1) mRNA is inhibited by genistein and wortmannin. Neuroreport.(2000).11(5), 1111-1115.
[49]Rahbek, U. L., Dissing, S., Thomassen, C., Hansen, A. J.,& Tritsaris, K. Nerve growth factor activates aorta endothelial cells causing PI3K/Akt- and ERK-dependent migration. Pflugers Arch.(2005).450(5),355-361.
[50]Raychaudhuri, S. K., Raychaudhuri, S. P.,Weltman, H., & Farber, E.M. Effect of nerve growth factor on endothelial cell biology:proliferation and adherence molecule expression on human dermal microvascular endothelial cells. Arch Dermatol Res. (2001).293(6),291-295.
[51]Cantarella, G., Lempereur, L., Presta, M., Ribatti, D., Lombardo,G., Lazarovici, P., et al. Nerve growth factor-endothelial cell interaction leads to angiogenesis in vitro and in vivo. FASEB J. (2002).16(10):1307-1309.
[52]Moser, K. V., Reindl, M., Blasig, I.,& Humpel, C. Brain capillary endothelial cells proliferate in response to NGF, express NGF receptors and secrete NGF after inflammation. Brain Res. (2004).1017(1-2):53-60.
[53]Dolle, J. P., Rezvan, A., Allen, F. D., Lazarovici, P.,& Lelkes, P. I. Nerve growth factor-induced migration of endothelial cells. J Pharmacol Exp Ther.(2005).315(3),1220-1227.
[54]Donovan, M. J., Miranda, R. C., Kraemer, R., McCaffrey, T. A., Tessarollo, L.,Mahadeo, D., et al. Neurotrophin and neurotrophin receptors in vascular smooth muscle cells. Regulation of expression in response to injury. Am J Pathol. (1995).147(2),309-324.
[55]Kraemer, R., Nguyen, H., March, K. L.,& Hempstead, B. NGF activates similar intracellular signaling pathways in vascular smooth muscle cells as PDGF-BB but elicits different biological responses. Arterioscler Thromb Vasc Biol. (1999).19(4),1041-1050.
[56]Calza, L., Giardino, L., Giuliani, A., Aloe, L., & Levi-Montalcini, R. Nerve growth factor control of neuronal expression of angiogenetic and vasoactive factors. Proc Natl Acad Sci U S A.(2001).98(7),4160-4165.
[57]Freund-Michel, V., Bertrand, C.,& Frossard, N. TrkA signalling pathways in human airway smooth muscle cell proliferation. Cell Signal.(2006).18(5), 621-627.
[58]Watson, J. J., Fahey, M. S., van den Worm, E., Engels, F., Nijkamp, F. P.,Stroemer, F., et al. TrkAd5:A novel therapeutic agent for treatmentof inflammatory pain and asthma. J Pharmacol Exp Ther.(2006).316(3),1122-1129.
[59]Freund-Michel V, Frossard N. The nerve growth factor and its receptors in airway inflammatory diseas.Pharmacol Ther,2008,117(1):52.
[60]唐晓梅,汪哗.神经生长因子治疗Bell's面瘫临床疗效观察.临床医学,2008,28(6)：23.
[61]李志飞.神经生长因子佐治难治性Tourette综合症疗观察.现代实用医学,2006,18(8)：548.
[62]王国华,严超英,武辉.外源性神经生长因子治疗中度新生儿缺氧缺血性脑病临床疗效观察及探讨治疗时间窗.中国妇幼保健,2008,23(10)：642.
[63]刘洪波,方树友,张丙谦等.神经生长因子治疗格林巴利综合征116例疗效观察.山东医药,2007,(10)：38.
[64]Paryaneh TA. Nerve growth factor prevents demylinon, cell death and progression of the disease in experimental allergic encephalomyelitis.Iran J Allergy Ashma Immunol,2006,5(4):177.
[65]Bella AJ, Lin G, Lin CS, et al. Nerve growth fact modulation of the cavernous nerve response to injury. J Sex Med,2009,6(Suppl3):34
[66]Mufson EJ, Counts SE, Perez SE,et al. Cholinergic System cluring the progression of Alzheimer's disease:therapeutic implications. Expert Rev Neurother,2008,8(11):1703.
[67]刘雪纯,余国正.神经生长因子治疗糖尿病周围神经病变临床观察.中国冶金工业医学杂志,2006,23(6)：661.
[68]Ieda M, Kimura K, Kanazawa H,et al. Regulation cardiac nerves:a new paradigm in the management sudelen cardiac death. Curr Med Chem, 2008,1(17):1 731.
[69]陈萍.神经生长因子对外伤性视神经视网膜挫伤的疗效观察.首都医药,2007,6(1):20.
[70]Sun W,Lin H,Chen B,et al. Collagen scaffolds loaded with collagen-binding NGF-beta accelerate ulcer healing.J Biomed Mater Res A,2009,3(12):50.
[71]黄荣道,杨杰,陈柳林.支气管镜局部用药治疗复发性肺结核27例疗效观察.微创医学,2008,3 (4):376-377
[72]崔福德等.药剂学.中国医药科技出版社.2002.153
[73]王亚秋.口腔局部用药浅析.上海医药.2001,22(12)：548-549
[2]Nockher WA, Renz H. Neurotrophins and asthma:novel insight into neuroimmune interaction. J Allergy Clin Immunol.2006.117(1):67-71.
[3]Nassenstein C, Kutschker J, Tumes D, Braun A. Neuro-immune interaction in allergic asthma:role of neurotrophins. Biochem Soc Trans.2006.34(Pt 4): 591-3.
[4]Feng JT, Hu CP. Dysfunction of releasing adrenaline in asthma by nerve growth factor. Med Hypotheses.2005.65(6):1043-6.
[5]汪俊,胡成平,冯俊涛,Jun W, Cheng-ping HU, Jun-tao F.神经生长因子对支气管哮喘大鼠肾上腺素释放障碍的机制研究.中华结核和呼吸杂志.2006.29(12)：812-815.
[6]汪俊,胡成平,梁伟军,冯俊涛.哮喘大鼠肾上腺髓质嗜铬细胞表型转化与神经生长因子表达水平初探.中国实用内科杂志.2007.27(4)：270-272.
[7]汪俊,胡成平,冯俊涛等.新生小牛肾上腺髓质嗜铬细胞的原代培养及其儿茶酚胺的分泌.中国应用生理学杂志.2008.24(2)：138-140.
[8]朱锦琪,冯俊涛,胡成平等.神经生长因子、白血病抑制因子调控支气管哮喘大鼠神经源性炎症机制的研究.中华结核和呼吸杂志.2006.29(6)：376-380.
[9]陆彬.药物新剂型与新技术.1998.人民卫生出版社.165-249.
[10]Boisdron-Celle M, Menei P, Benoit JP. Preparation and characterization of 5-fluorouracil-loaded microparticles as biodegradable anticancer drug carriers. J Pharm Pharmacol.1995.47(2):108-14.
[11]der Lubben IM v, Verhoef JC, Borchard G, Junginger HE. Chitosan and its derivatives in mucosal drug and vaccine delivery. Eur J Pharm Sci.2001. 14(3):201-7.
[12]Konturek SJ, Pawlik W, Mysh W, et al. Comparison of organ uptake and disappearance half-time of human epidermal growth factor and insulin. Regul Pept.1990.30(2):137-48.
[13]Boisdron-Celle M, Menei P, Benoit JP. Preparation and characterization of 5-fluorouracil-loaded microparticles as biodegradable anticancer drug carriers. J Pharm Pharmacol.1995.47(2):108-14.
[14]Conti B, Pavanetto F, Genta I. Use of polylactic acid for the preparation of microparticulate drug delivery systems. J Microencapsul.1992.9(2):153-66.
[15]陈庆华,瞿文,Qinghua C, Wen Q.多肽、蛋白质类药物缓释剂型的研究进展.中国药学杂志.2000.35(3)：147-150.
[16]艾国,程远国,Guo AI, Yuan-guo C.蛋白质多肽长效微球注射剂的体内药动学及分析方法研究进展.中国新药杂志.2007.16(7)：501-506.
[17]杨彤,周云赞,王娜,徐力,吴晓霞,张学忠.细胞粘附位点RGD三肽脂质体的制备及表征.吉林大学学报(理学版).2003.41(3)：356-360.
[18]谷海刚，龙大宏,李晓滨等.异硫氰酸荧光素标记的神经生长因子缓释微球的制备及评价.中华生物医学工程杂志.2007.13(6)：393-396.
[19]Cohen S, Yoshioka T, Lucarelli M, Hwang LH, Langer R. Controlled delivery systems for proteins based on poly(lactic/glycolic acid) microspheres. Pharm Res.1991.8(6):713-20.
[20]Menei P, Montero-Menei C, Venier MC, Benoit JP. Drug delivery into the brain using poly(lactide-co-glycolide) microspheres. Expert Opin Drug Deliv. 2005.2(2):363-76.
[21]Jiang W, Gupta RK, Deshpande MC, Schwendeman SP. Biodegradable poly(lactic-co-glycolic acid) microparticles for injectable delivery of vaccine antigens. Adv Drug Deliv Rev.2005.57(3):391-410.
[22]Haller MF, Saltzman WM. Nerve growth factor delivery systems. J Control Release.1998.53(1-3):1-6.
[23]Haller MF, Saltzman WM. Localized delivery of proteins in the brain:can transport be customized. Pharm Res.1998.15(3):377-85.
[24]Obeidat WM, Price JC. Viscosity of polymer solution phase and other factors controlling the dissolution of theophylline microspheres prepared by the emulsion solvent evaporation method. J Microencapsul.2003.20(1):57-65.
[25]Ruan G, Ng JK, Feng SS. Effects of polymer, organic solvent and mixing strength on integrity of proteins and liposomes encapsulated in polymeric microspheres fabricated by the double emulsion process. J Microencapsul. 2004.21(4):399-412.
[26]刘世芸,徐超,冯乙巳等.白蛋白聚乳酸缓释微球的制备及体外释放研究.合肥工业大学学报(自然科学版).2006.29(12)：1584-1587.
[27]Zhu G, Mallery SR, Schwendeman SP. Stabilization of proteins encapsulated in injectable poly (lactide-co-glycolide). Nat Biotechnol.2000.18(1):52-7.
[28]Kang F, Jiang G, Hinderliter A, DeLuca PP, Singh J. Lysozyme stability in primary emulsion for PLGA microsphere preparation:effect of recovery methods and stabilizing excipients. Pharm Res.2002.19(5):629-33.
[29]Estey T, Kang J, Schwendeman SP, Carpenter JF. BSA degradation under acidic conditions:a model for protein instability during release from PLGA delivery systems. J Pharm Sci.2006.95(7): 1626-39.
[30]de Weert M v, Hennink WE, Jiskoot W. Protein instability in poly(lactic-co-glycolic acid) microparticles. Pharm Res.2000.17(10): 1159-67.
[31]Carrascosa C, Espejo L, Torrado S, Torrado JJ. Effect of gamma-sterilization process on PLGA microspheres loaded with insulin-like growth factor-Ⅰ (IGF-Ⅰ). J Biomater Appl.2003.18(2):95-108.
[32]Meinel L, Illi OE, Zapf J, Malfanti M, Peter MH, Gander B. Stabilizing insulin-like growth factor-Ⅰ in poly(D,L-lactide-co-glycolide) microspheres. J Control Release.2001.70(1-2):193-202.
[33]Yuksel E, Weinfeld AB, Cleek R, et al. Increased free fat-graft survival with the long-term, local delivery of insulin, insulin-like growth factor-Ⅰ, and basic fibroblast growth factor by PLGA/PEG microspheres. Plast Reconstr Surg. 2000.105(5):1712-20.
[34]汤渝玲,胡成平,冯俊涛等.神经生长因子调控哮喘神经源性炎症Ras-MAPK信号转导通路.中南大学学报(医学版).2006.31(3)：319-325.
[35]Menei P, Pean JM, Nerriere-Daguin V, Jollivet C, Brachet P, Benoit JP. Intracerebral implantation of NGF-releasing biodegradable microspheres protects striatum against excitotoxic damage. Exp Neurol.2000.161(1): 259-72.
[36]Pean JM, Menei P, Morel O, Montero-Menei CN, Benoit JP. Intraseptal implantation of NGF-releasing microspheres promote the survival of axotomized cholinergic neurons. Biomaterials.2000.21(20):2097-101.
[37]Mahoney MJ, Saltzman WM. Controlled release of proteins to tissue transplants for the treatment of neurodegenerative disorders. J Pharm Sci. 1996.85(12):1276-81.
[38]Gertz HJ, Cervos-Navarro J, Ewald V. The septo-hippocampal pathway in patients suffering from senile dementia of Alzheimer's type. Evidence for neuronal plasticity. Neurosci Lett.1987.76(2):228-32.
[39]Blanco-Prieto MJ, Leo E, Delie F, Gulik A, Couvreur P, Fattal E. Study of the influence of several stabilizing agents on the entrapment and in vitro release of pBC 264 from poly(lactide-co-glycolide) microspheres prepared by a W/O/W solvent evaporation method. Pharm Res.1996.13(7):1127-9.
[40]Takada S, Yamagata Y, Misaki M, Taira K, Kurokawa T. Sustained release of human growth hormone from microcapsules prepared by a solvent evaporation technique. J Control Release.2003.88(2):229-42.
[41]Pean JM, Boury F, Venier-Julienne MC, Menei P, Proust JE, Benoit JP. Why does PEG 400 co-encapsulation improve NGF stability and release from PLGA biodegradable microspheres. Pharm Res.1999.16(8):1294-9.
[42]Sun SW, Jeong YI, Jung SW, Kim SH. Characterization of FITC-albumin encapsulated poly(DL-lactide-co-glycolide) microspheres and its release characteristics. J Microencapsul.2003.20(4):479-88.
[43]Yang YY, Chung TS, Ng NP. Morphology,'drug distribution, and in vitro release profiles of biodegradable polymeric microspheres containing protein fabricated by double-emulsion solvent extraction/evaporation method. Biomaterials.2001.22(3):231-41.
[44]Bodmeier R, McGinity JW. The preparation and evaluation of drug-containing poly(dl-lactide) microspheres formed by the solvent evaporation method. Pharm Res.1987.4(6):465-71.
[45]de Weert M v, Hennink WE, Jiskoot W. Protein instability in poly(lactic-co-glycolic acid) microparticles. Pharm Res.2000.17(10): 1159-67.
[46]Perez C, Castellanos IJ, Costantino HR, Al-Azzam W, Griebenow K. Recent trends in stabilizing protein structure upon encapsulation and release from bioerodible polymers. J Pharm Pharmacol.2002.54(3):301-13.
[47]习明,胡卫列,Ming XI, Wei-lie HU.肾上腺解剖学的研究进展.中国临床解剖学杂志.2008.26(2)：227-228.
[48]汪俊,胡成平,冯俊涛,Jun W, Cheng-ping HU, Jun-tao F.神经生长因子对支气管哮喘大鼠肾上腺素释放障碍的机制研究.中华结核和呼吸杂志.2006.29(12)：812-815.
[49]林敏娟,胡成平,吴鄂生,潘频华.支气管哮喘大鼠肺组织中白血病抑制因子的表达变化.中华结核和呼吸杂志.2003.26(11)：727-728.
[50]Baumgarten CR, Witzel A, Schierhorn K, Kunkel G. Neuro-immuno regulatory mechanisms of asthma. Acta Microbiol Immunol Hung.1998.45(1): 31-42.
[51]Ball TM, Anderson D, Minto J, Halonen M. Cortisol circadian rhythms and stress responses in infants at risk of allergic disease. J Allergy Clin Immunol. 2006.117(2):306-11.
[52]Wright RJ. Stress and atopic disorders. J Allergy Clin Immunol.2005.116(6): 1301-6.
[53]Theoharides TC, Kempuraj D, Tagen M, Vasiadi M, Cetrulo CL. Human umbilical cord blood-derived mast cells:a unique model for the study of neuro-immuno-endocrine interactions. Stem Cell Rev.2006.2(2):143-54.
[54]Besedovsky HO, del RA. Feed-back interactions between immunological cells and the hypothalamus-pituitary-adrenal axis. Neth J Med.1991.39(3-4): 274-80.
[55]Besedovsky HO, del RA. Interactions between immunological cells and the hypothalamus pituitary-adrenal axis:an example of neuroendocrine immunoregulation. Recenti Prog Med.1988.79(7-8):300-4.
[56]孔灵菲,Ling-fei K.最新全球哮喘防治指南解读.中国实用内科杂志.2007.27(4)：255-257.
[57]Levi-Montalcini R, Dal Toso R, della VF, Skaper SD, Leon A. Update of the NGF saga. J Neurol Sci.1995.130(2):119-27.
[58]Levi-Montalcini R, Skaper SD, Dal Toso R, Petrelli L, Leon A. Nerve growth factor:from neurotrophin to neurokine. Trends Neurosci.1996.19(11): 514-20.
[59]Aloe L, Bracci-Laudiero L, Bonini S, Manni L. The expanding role of nerve growth factor:from neurotrophic activity to immunologic diseases. Allergy. 1997.52(9):883-94.
[60]周敏,徐永健,熊盛道,赵建平,倪望,张珍祥.神经生长因子在哮喘患者诱导痰炎性细胞的表达.中华内科杂志.2003.42(11)：764-767.
[61]Lindsay RM, Harmar AJ. Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurons. Nature.1989.337(6205):362-4.
[62]Lindsay RM, Lockett C, Sternberg J, Winter J. Neuropeptide expression in cultures of adult sensory neurons:modulation of substance P and calcitonin gene-related peptide levels by nerve growth factor. Neuroscience.1989.33(1): 53-65.
[63]Undem BJ, Hunter DD, Liu M, Haak-Frendscho M, Oakragly A, Fischer A. Allergen-induced sensory neuroplasticity in airways. Int Arch Allergy Immunol.1999.118(2-4):150-3.
[64]Virchow JC, Julius P, Lommatzsch M, Luttmann W, Renz H, Braun A. Neurotrophins are increased in bronchoalveolar lavage fluid after segmental allergen provocation. Am J Respir Crit Care Med.1998.158(6):2002-5.
[65]Sanico AM, Stanisz AM, Gleeson TD, et al. Nerve growth factor expression and release in allergic inflammatory disease of the upper airways. Am J Respir Crit Care Med.2000.161(5):1631-5.
[66]Bonini S, Lambiase A, Bonini S, et al. Circulating nerve growth factor levels are increased in humans with allergic diseases and asthma. Proc Natl Acad Sci U S A.1996.93(20):10955-60.
[67]Olgart HC, Frossard N. Nerve growth factor and asthma. Pulm Pharmacol Ther.2002.15(1):51-60.
[68]Olgart C, Frossard N. Human lung fibroblasts secrete nerve growth factor: effect of inflammatory cytokines and glucocorticoids. Eur Respir J.2001. 18(1):115-21.
[69]Carr MJ, Hunter DD, Undem BJ. Neurotrophins and asthma. Curr Opin Pulm Med.2001.7(1):1-7.
[70]Fox AJ, Patel HJ, Barnes PJ, Belvisi MG. Release of nerve growth factor by human pulmonary epithelial cells:role in airway inflammatory diseases. Eur J Pharmacol.2001.424(2):159-62.
[71]Bonini S, Lambiase A, Bonini S, Levi-Schaffer F, Aloe L. Nerve growth factor: an important molecule in allergic inflammation and tissue remodelling. Int Arch Allergy Immunol.1999.118(2-4):159-62.
[72]Braun A, Quarcoo D, Schulte-Herbruggen O, Lommatzsch M, Hoyle G, Renz H. Nerve growth factor induces airway hyperresponsiveness in mice. Int Arch Allergy Immunol.2001.124(1-3):205-7.
[73]Quarcoo D, Schulte-Herbruggen O, Lommatzsch M, et al. Nerve growth factor induces increased airway inflammation via a neuropeptide-dependent mechanism in a transgenic animal model of allergic airway inflammation. Clin Exp Allergy.2004.34(7):1146-51.
[74]朱锦琪,冯俊涛,胡成平等.神经生长因子、白血病抑制因子调控支气管哮喘大鼠神经源性炎症机制的研究.中华结核和呼吸杂志.2006.29(6)：376-380.
[75]Kubota T, Koga K, Araki H, et al. [The relationships of mononuclear leukocyte beta-adrenergic receptors to aerobic capacity and exercise-induced asthma in asthmatic children]. Arerugi.2000.49(1):40-51.
[76]Counil FP, Varray A, Karila C, Hayot M, Voisin M, Prefaut C. Wingate test performance in children with asthma:aerobic or anaerobic limitation. Med Sci Sports Exerc.1997.29(4):430-5.
[77]Greene LA, Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A.1976.73(7):2424-8.
[78]吴克复,马小彤,宋玉华.细胞的功能冗余性及其意义.生物化学与生物物理进展.2004.31(12)：1054-1057.
[79]Unsicker K, Krisch B, Otten U, Thoenen H. Nerve growth factor-induced fiber outgrowth from isolated rat adrenal chromaffin cells:impairment by glucocorticoids. Proc Natl Acad Sci U S A.1978.75(7):3498-502.
[80]Unsicker K, Krisch B, Otten U, Thoenen H. [Nerve growth factor-induced axon growth and its antagonism with dexamethasone:in vitro studies on adrenal medulla cells]. Verh Anat Ges.1978. (72):371-2.
[81]Unsicker K, Krisch B, Otten U, Thoenen H. Nerve growth factor-induced fiber outgrowth from isolated rat adrenal chromaffin cells:impairment by glucocorticoids. Proc Natl Acad Sci U S A.1978.75(7):3498-502.
[1]刘玉梅,张自强.微球制剂临床应用进展.齐鲁药事.2006,25(4)：234-236
[2]韩敏,苏秀霞,李仲谨.载药微球制剂的研究进展.应用化工.2007,36(5)：493-495
[3]Longo W E, Iw ata H.Preparation of hydrophilic album in microspheres using polymeric dispersing agents. J Pharm Sci.1982,71(9):1323-1328.
[4]李凤前,陆彬,曾仁杰.地塞米松磷酸盐牛血清清蛋白微球的研制.中国医药工业杂志.1999,30(3)：113-115.
[5]丁红,刑桂琴,谢茵.阿霉素明胶微球的制备与特性研究.中国医院药学杂志.2000,20(7)：387-389.
[6]方华丰,周宜开.壳聚糖微球的研究进展.国外医药合成药生化药制剂分册.1999,20(5)：315-318.
[7]张珠.利福平壳聚糖蛋白微球的制备及性能研究.武汉理工大学学报.2001,23(1)：21-23.
[8]许天开,赵树杰.PHB在生物医学中的应用研究进展.应用与环境生物学报.1995,7(1)：85-88.
[9]王正容,陆彬,杨红.左炔诺孕酮-聚-3-羟基丁酸酯缓释微球的研究.药学学报,1 999,34(1)：57-59.
[10]刘哲鹏,潘俊,陆伟跃.生物黏附微球研究及前景.国外医药合成药生化药制剂 分册.2001,22(2)：111-114.
[11]张瑶. 缓释微球制剂的研究进展.2004,23(11)：843-844
[12]周新腾,潘卫三,张汝华.局部麻醉药生物可降解缓释微球的研究进展.中国新药杂志.2001,10(9)：658-660.
[13]董爱梅,张建强.眼用微球制剂研究进展.齐鲁药事.2005,24(1)：36-38.
[14]王红梅,江燕,王静.鼻腔给药新剂型初探.方药研究,2002,(2)：53.
[15]王晶,王勤,李静.聚乳酸微球制剂的研究概况.山东生物医学工程,1999.18(3)：50-52
[16]甘莉,鲜平.前列腺素E1脂微球制剂治疗糖尿病周围神经病变的疗效观察.临床医药实践杂志.2004,13(2)：126-27.
[17]易玉新,杨宇,屈晓冰,等.前列腺素E1脂微球制剂治疗老年脑梗塞的疗效.中日友好医院学报.2004,18(1)：20-22.
[18]顾慧华,田力,薛来英.前列腺素El脂微球载体制剂治疗慢性重型肝炎的临床研究.中国现代应用药学杂志.2004,21(1)：69-70.
[19]邵景范,罗永湘.神经生长因子的载体及其控释系统.中华显微外科杂志.1995,18(4)：303-305.
[20]蒋涛,任先军.神经营养因子及其微球制剂.中国药师.2006,9(5)：457-458
[21]黄芳,费俭,刘晓等.神经营养因子.细胞生物学杂志.1996,18：17-20
[22]Shahrooz R, Justin OH, Zhong CT, et al. Induction of apoptosis by the low-affinity NGF receptor.Science,1993,261:345-348
[23]Barrett GL, Bartlett PF. The p75 nerve growth factor receptor mediated survival or death depending on the stage of sensory neuron development.Proc Nail Acad Sci USA,1994,91:6501-6505
[24]邓小龙,陈浩凡.几种可生物降解材料在胶体微粒给药系统中的应用.中国药师,2002,5(8)：492-494
[25]Camarata PJ, Suryanarayanan R,Turner DA, et al. Sustained release of nerve growth factor from biodegradable polymer microspheres. Neurosurgery, 1992,30(3):313-319
[26]Pean JM, Venier-Julienne MC, Boury F, et al. NGF release from poly (D,L-lactide-co-glycolide) microspheres. Efect of some formulation parameters on encapsulated NGF stability. J Control Release,1998,56 (1-3):175-187
[27]Menei P,Pean JM,Neriere-Dafuin V,et al.Intracerebralimplantation of NGF-releasing biodegradable Microspheres Protects striatum against excitotoxic damage. Exp Neural,2000,161(1):259-272
[28]Aubert-Pouessel A,Venier-Julienne AC,Clavreul A,et al.In vitro study of GDNF release From Biodegradable PLGA microspheres. J Control Release,2004, 95(3):463-475
[29]Jollivet C,Aubert-Pouessel A,ClavreulA,et al. Long-term effect of intra-striatal glial cell line-derived neurotrophic factor-releasing microspheres in a partial rat model of Parkinson's disease. Neurosci Lett,2004,356(3):207-210
[30]Jolivet C, Aubert-Pouessel A, Clavreul A, et al. Striatal implantation of GDNF releasing biodegradable microspheres promotes recovery of motor function in a partial model of Parkinson's disease.Biornaterials,2004,2 5(5):933-942
[31]Maysinger D, Krieglstein K, Filipovic-Greic J, et al. Microencapsulated ciliary neurotrophic factor:physical properties and biological activities. Exp Neurol, 1996,138(2):177-188
[32]Mittal S, Cohen A, Maysinger D. In vitro effects of brain derived neurotrophic fact or released from microspheres.Neuronreport,1994,5(18):2577-2582
[33]Rellinger RJ, Mailliard RB, Barksdale EMJ.Neurobla stoma and dendritic cell function. Semin Pediatr Surg,2004,13(1):61.
[34]李爱敏,孙洪亮,张锦华.Caspase-8沉默-神经母细胞瘤耐药新机制.国外医学-儿科学分册,2004,31(3)：165.
[35]高强,董倩,吕振华,等.转神经生长因子基因诱导神经母细胞瘤分化的研究.中华小儿外科杂志,2005,26(1)：34.
[36]陆莹,赵宝东,赵春玉.NGF对缺血损伤引起大鼠海马CA1区神经细胞凋亡的作用.锦州医学院学报,2004,3(1)：21.
[37]Hoyle, G. W., Graham, R. M., Finkelstein, J. B., Nguyen, K. P., Gozal, D.,& Friedman, M. Hyperinnervation of the airways in transgenic mice overexpressing nerve growth factor. Am J Respir Cell Mol Biol. (1998).18(2),149-157.
[38]Hunter, D. D., Myers, A. C.,& Undem, B. J. Nerve growth factorinduced phenotypic switch in guinea pig airway sensory neurons.Am J Respir Crit Care Med,(2000).161(6),1985-1990.
[39]Dinh, Q. T., Groneberg, D. A., Peiser, C., Springer, J., Joachim, R. A., Arck, P. C.,et al. Nerve growth factor-induced substance P in capsaicininsensitive vagal neurons innervating the lower mouse airway. Clin Exp Allergy.(2004).34(9), 1474-1479.
[40]Nassenstein, C., Dawbarn, D., Pollock, K., Allen, S. J., Erpenbeck, V. J., Spies, E., et al. Pulmonary distribution, regulation, and functional role of Trk receptors in a murine model of asthma. J Allergy Clin Immunol. (2006a).118(3),597-605.
[41]Micera, A., Vigneti, E., Pickholtz, D., Reich, R., Pappo, O., Bonini, S., et al.Nerve growth factor displays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair.Proc Natl Acad Sci USA.(2001).98(11),6162-6167.
[42]Kohyama, T., Liu, X., Wen, F. Q., Kobayashi, T., Abe, S., Ertl, R., et al. Nerve growth factor stimulates fibronectin-induced fibroblast migration.J Lab Clin Med. (2002).140(5),329-335.
[43]Chevet, E., Lemaitre, G., Janjic, N., Barritault, D., Bikfalvi, A.,&Katinka, M. D.Fibroblast growth factor receptors participate in the control of mitogen-activated protein kinase activity during nerve growth factor-induced neuronal differentiation of PC12 cells. J Biol Chem.(1999).274(30), 20901-20908.
[44]Cosgaya, J. M.,& Aranda, A.. Nerve growth factor regulates transforming growth factor-beta 1 gene expression by both transcriptional and posttranscriptional mechanisms in PC12 cells. J Neurochem. (1995)65(6),2484-2490.
[45]Sin, A. Z., Roche, E. M., Togias, A., Lichtenstein, L. M.,& Schroeder, J. T. Nerve growth factor or IL-3 induces more IL-13 production from basophils of allergic subjects than from basophils of nonallergic subjects.J Allergy Clin Immunol.(2001).108(3),387-393.
[46]Noga,O., Englmann, C., Hanf, G., Grutzkau, A., Guhl, S., & Kunkel, G. Activation of the specific neurotrophin receptors TrkA, TrkB and TrkC influences the function of eosinophils. Clin Exp Allergy. (2002).32(9), 1348-1354.
[47]Nithya, M., Suguna, L.,& Rose, C. The effect of nerve growth factor on the early responses during the process of wound healing. Biochim Biophys Acta.(2003).1620(1-3),25-31.
[48]Takahashi, H., Uno, S., Watanabe, Y., Arakawa, K.,& Nakagawa, S. Expression of nerve growth factor-induced type 1 plasminogen activator inhibitor (PAI-1) mRNA is inhibited by genistein and wortmannin. Neuroreport.(2000).11(5), 1111-1115.
[49]Rahbek, U. L., Dissing, S., Thomassen, C., Hansen, A. J.,& Tritsaris, K. Nerve growth factor activates aorta endothelial cells causing PI3K/Akt- and ERK-dependent migration. Pflugers Arch.(2005).450(5),355-361.
[50]Raychaudhuri, S. K., Raychaudhuri, S. P.,Weltman, H., & Farber, E.M. Effect of nerve growth factor on endothelial cell biology:proliferation and adherence molecule expression on human dermal microvascular endothelial cells. Arch Dermatol Res. (2001).293(6),291-295.
[51]Cantarella, G., Lempereur, L., Presta, M., Ribatti, D., Lombardo,G., Lazarovici, P., et al. Nerve growth factor-endothelial cell interaction leads to angiogenesis in vitro and in vivo. FASEB J. (2002).16(10):1307-1309.
[52]Moser, K. V., Reindl, M., Blasig, I.,& Humpel, C. Brain capillary endothelial cells proliferate in response to NGF, express NGF receptors and secrete NGF after inflammation. Brain Res. (2004).1017(1-2):53-60.
[53]Dolle, J. P., Rezvan, A., Allen, F. D., Lazarovici, P.,& Lelkes, P. I. Nerve growth factor-induced migration of endothelial cells. J Pharmacol Exp Ther.(2005).315(3),1220-1227.
[54]Donovan, M. J., Miranda, R. C., Kraemer, R., McCaffrey, T. A., Tessarollo, L.,Mahadeo, D., et al. Neurotrophin and neurotrophin receptors in vascular smooth muscle cells. Regulation of expression in response to injury. Am J Pathol. (1995).147(2),309-324.
[55]Kraemer, R., Nguyen, H., March, K. L.,& Hempstead, B. NGF activates similar intracellular signaling pathways in vascular smooth muscle cells as PDGF-BB but elicits different biological responses. Arterioscler Thromb Vasc Biol. (1999).19(4),1041-1050.
[56]Calza, L., Giardino, L., Giuliani, A., Aloe, L., & Levi-Montalcini, R. Nerve growth factor control of neuronal expression of angiogenetic and vasoactive factors. Proc Natl Acad Sci U S A.(2001).98(7),4160-4165.
[57]Freund-Michel, V., Bertrand, C.,& Frossard, N. TrkA signalling pathways in human airway smooth muscle cell proliferation. Cell Signal.(2006).18(5), 621-627.
[58]Watson, J. J., Fahey, M. S., van den Worm, E., Engels, F., Nijkamp, F. P.,Stroemer, F., et al. TrkAd5:A novel therapeutic agent for treatmentof inflammatory pain and asthma. J Pharmacol Exp Ther.(2006).316(3),1122-1129.
[59]Freund-Michel V, Frossard N. The nerve growth factor and its receptors in airway inflammatory diseas.Pharmacol Ther,2008,117(1):52.
[60]唐晓梅,汪哗.神经生长因子治疗Bell's面瘫临床疗效观察.临床医学,2008,28(6)：23.
[61]李志飞.神经生长因子佐治难治性Tourette综合症疗观察.现代实用医学,2006,18(8)：548.
[62]王国华,严超英,武辉.外源性神经生长因子治疗中度新生儿缺氧缺血性脑病临床疗效观察及探讨治疗时间窗.中国妇幼保健,2008,23(10)：642.
[63]刘洪波,方树友,张丙谦等.神经生长因子治疗格林巴利综合征116例疗效观察.山东医药,2007,(10)：38.
[64]Paryaneh TA. Nerve growth factor prevents demylinon, cell death and progression of the disease in experimental allergic encephalomyelitis.Iran J Allergy Ashma Immunol,2006,5(4):177.
[65]Bella AJ, Lin G, Lin CS, et al. Nerve growth fact modulation of the cavernous nerve response to injury. J Sex Med,2009,6(Suppl3):34
[66]Mufson EJ, Counts SE, Perez SE,et al. Cholinergic System cluring the progression of Alzheimer's disease:therapeutic implications. Expert Rev Neurother,2008,8(11):1703.
[67]刘雪纯,余国正.神经生长因子治疗糖尿病周围神经病变临床观察.中国冶金工业医学杂志,2006,23(6)：661.
[68]Ieda M, Kimura K, Kanazawa H,et al. Regulation cardiac nerves:a new paradigm in the management sudelen cardiac death. Curr Med Chem, 2008,1(17):1 731.
[69]陈萍.神经生长因子对外伤性视神经视网膜挫伤的疗效观察.首都医药,2007,6(1):20.
[70]Sun W,Lin H,Chen B,et al. Collagen scaffolds loaded with collagen-binding NGF-beta accelerate ulcer healing.J Biomed Mater Res A,2009,3(12):50.
[71]黄荣道,杨杰,陈柳林.支气管镜局部用药治疗复发性肺结核27例疗效观察.微创医学,2008,3 (4):376-377
[72]崔福德等.药剂学.中国医药科技出版社.2002.153
[73]王亚秋.口腔局部用药浅析.上海医药.2001,22(12)：548-549