灵芝孢子对哮喘豚鼠肥大细胞作用机制的研究
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摘要
目的:建立哮喘动物模型,探讨灵芝孢子对哮喘豚鼠肥大细胞激活机制的调控作用。
方法:10%卵蛋白腹腔注射致敏后雾化吸入1%卵蛋白诱导建立哮喘动物模型。将30只健康豚鼠按随机数字表分为生理盐水对照组(A),哮喘模型组(B),灵芝孢子组(C)三组,每组10只,分别用生理盐水,灵芝孢子对各组豚鼠进行灌胃干预治疗15天。测定哮喘豚鼠的引喘潜伏期,肺泡灌洗液计数白细胞总数及分类,肺组织H-E染色和免疫组化观察肺组织病理变化及肥大细胞类胰蛋白酶分布情况。
结果: 1、灵芝孢子组哮喘豚鼠的引喘潜伏期明显较哮喘模型组延长。2、哮喘组肺泡灌洗液白细胞总数及嗜酸性粒细胞比例较生理盐水组增高,肺组织炎性病变明显;灵芝组肺泡灌洗液白细胞总数、嗜酸性粒细胞比例及肺组织炎性病变明显较哮喘组降低或减轻。3、哮喘组肥大细胞类胰蛋白酶染色阳性的细胞计数明显较生理盐水组增多,主要分布在气道粘膜下,肺泡间隔及血管周围;灵芝孢子组类胰蛋白酶染色阳性的肥大细胞计数较哮喘组显著减少。
结论:1、单纯卵蛋白致敏和雾化能够成功复制出哮喘动物模型。2、灵芝孢子有延长哮喘豚鼠引喘潜伏期,减轻肺组织炎性病变,抑制肥大细胞激活释放类胰蛋白酶的作用。
Objective: To establish asthma animal model and investigate the effect of Ganoderma spores on mast cell in asthmatic guinen pigs .
Methods: The asthmatic guinea pigs model was set up by intraperitoneal injection of 10% OVA(ovalbumin) for sensitizing and exposure to aerosolize 1% OVA for inducing asthma. 30 guinea pigs were divided into 3 groups by random number meter : a NS group(A), an asthma group(B) and a ganoderma spores group(C),with 10 guinea pigs in each. NS or ganoderma spores were infused into stomach of guinen pigs for fifteen days. The incubation period of asthma was detected , and the total cell numbers and classification in bronchoalveola lavage fluid(BALF) were studied .The lung tissues were stained with Hematoxylin-Eosin(H-E) to observe the pathological changes and with anti-human tryptase antibody(AAI) by using immunohistochemical technique to count the mast cell tryptase (MCT) .
Results:(1) Compared with asthma group, ganoderma spores could markedly prolong the incubation period of asthma,and the mast cell tryptases counts in ganoderma spores group decreased significantly.Meanwhile, the total cell numbers and the percentage of eosinophil of BALF in ganoderma spores group were lower than those in asthma group and the inflammation response in lung tissue were significantly slighter. (2) Compared with NS group, asthma group had more leukocytes,higher percentage of eosinophil in BALF and more mast cell tryptase expression in airway submucosa, alveolar walls and vascular surrounding.The inflammation response in lung tissue of asthma group was evident.
conclusion:(1)Intraperitoneal injection and atomization of OVA could duplicate asthma animal model successfully.(2) Ganoderma spores could prolong the incubation period of asthma , obviously relieve inflammation response in airway and lung tissue,and also depress mast cell activation.
方法:10%卵蛋白腹腔注射致敏后雾化吸入1%卵蛋白诱导建立哮喘动物模型。将30只健康豚鼠按随机数字表分为生理盐水对照组(A),哮喘模型组(B),灵芝孢子组(C)三组,每组10只,分别用生理盐水,灵芝孢子对各组豚鼠进行灌胃干预治疗15天。测定哮喘豚鼠的引喘潜伏期,肺泡灌洗液计数白细胞总数及分类,肺组织H-E染色和免疫组化观察肺组织病理变化及肥大细胞类胰蛋白酶分布情况。
结果: 1、灵芝孢子组哮喘豚鼠的引喘潜伏期明显较哮喘模型组延长。2、哮喘组肺泡灌洗液白细胞总数及嗜酸性粒细胞比例较生理盐水组增高,肺组织炎性病变明显;灵芝组肺泡灌洗液白细胞总数、嗜酸性粒细胞比例及肺组织炎性病变明显较哮喘组降低或减轻。3、哮喘组肥大细胞类胰蛋白酶染色阳性的细胞计数明显较生理盐水组增多,主要分布在气道粘膜下,肺泡间隔及血管周围;灵芝孢子组类胰蛋白酶染色阳性的肥大细胞计数较哮喘组显著减少。
结论:1、单纯卵蛋白致敏和雾化能够成功复制出哮喘动物模型。2、灵芝孢子有延长哮喘豚鼠引喘潜伏期,减轻肺组织炎性病变,抑制肥大细胞激活释放类胰蛋白酶的作用。
Objective: To establish asthma animal model and investigate the effect of Ganoderma spores on mast cell in asthmatic guinen pigs .
Methods: The asthmatic guinea pigs model was set up by intraperitoneal injection of 10% OVA(ovalbumin) for sensitizing and exposure to aerosolize 1% OVA for inducing asthma. 30 guinea pigs were divided into 3 groups by random number meter : a NS group(A), an asthma group(B) and a ganoderma spores group(C),with 10 guinea pigs in each. NS or ganoderma spores were infused into stomach of guinen pigs for fifteen days. The incubation period of asthma was detected , and the total cell numbers and classification in bronchoalveola lavage fluid(BALF) were studied .The lung tissues were stained with Hematoxylin-Eosin(H-E) to observe the pathological changes and with anti-human tryptase antibody(AAI) by using immunohistochemical technique to count the mast cell tryptase (MCT) .
Results:(1) Compared with asthma group, ganoderma spores could markedly prolong the incubation period of asthma,and the mast cell tryptases counts in ganoderma spores group decreased significantly.Meanwhile, the total cell numbers and the percentage of eosinophil of BALF in ganoderma spores group were lower than those in asthma group and the inflammation response in lung tissue were significantly slighter. (2) Compared with NS group, asthma group had more leukocytes,higher percentage of eosinophil in BALF and more mast cell tryptase expression in airway submucosa, alveolar walls and vascular surrounding.The inflammation response in lung tissue of asthma group was evident.
conclusion:(1)Intraperitoneal injection and atomization of OVA could duplicate asthma animal model successfully.(2) Ganoderma spores could prolong the incubation period of asthma , obviously relieve inflammation response in airway and lung tissue,and also depress mast cell activation.
引文
[1] 叶任高,陆再英.内科学[M].第六版.北京:人民卫生出版社,2003 .64-73
[2] Hallgren J,Pejler G. Biology of mast cell tryptase.An inflammatory mediator[J]. FEBS Journal.2006,273 (9):1871–1895
[3] Barned PJ. Pathophysiology of asthma[J]. Br J Clin Pharmacol. 1996,42: 3–10
[4] Centers for disease control and prevention. Asthma mortality and hospitalization among children and young adults- United States 1998 -1993[J].MMWR Morb Mortal Wkly Rep.1996,45:350—353
[5] 顾立刚,赵仲生,陶君娣,等. 薄盖灵芝对小鼠内外免疫反应的实验研究[J] .上海免疫学杂志.1989,9(3):145-147
[6] 张罗修,盛惊州,薛迎旦,等.日本灵芝对抗体形成细胞及变态反应的影响[J].中药药理与临床.1992,8(2):10-14
[7] Kohda H,Toknmoto W,Sakamoto K,et al. The biologically active constituents of Ganoderma lucidum (Fr.) Karst. Histamine release-inhibitory triterpenes[J]. Chem Pharm Bull (Tokyo).1985, 33(4):1367-1374
[8] 龚志锦.常用的特殊染色方法-甲苯氨蓝染色[A] .见:王伯远编. 病理学技术[C].北京:人民卫生出版社,2002.172
[9] 刘传合,薛全福,陈育智,等.支气管哮喘动物模型的研究现状[J] .中华结核与呼吸杂志.2000,23(11):647-649
[10]Akihisa T, Nakamura Y, Tagata M, et al.Anti-inflammatory and anti-tumor promoting effects of triterpene acids and sterols from the fungus Ganoderma lucidum[J]. 2007, 4(2): 224-231
[11]Hajjaj H, Mace C, Roberts M, et al. Effect of 26-oxygenosterols from Ganoderma lucidum and their activity as cholesterol synthesis inhibitors[J]. 2005,71(7): 3653-3658
[12]宁安红,曾婧,黄敏,等.灵芝多糖对小鼠肿瘤的抑制作用及免疫指标的观察[J] .肿瘤研究与临床.2004,16(3):147-149
[13]王林,王玉红,章克昌,等.灵芝中药发酵液对慢性支气管炎疗效的研究[J] .中国食用菌.2004,23(5):39-41
[14]Bradding P,Walls AF,Holgate ST,et al. The role of the mast cell in the pathophysiology of asthma[J]. J Allergy Clin Immunol. 2006,117(6):1277-1284
[15] He S,Walls AF. Human mast cell tryptase:a stimulus of microvascular leakage and mast cell activation[J]. Eur J Pharmacol.1997,328(1):89—97
[16] Payne V,Kam PC. Mast cell tryptase:a review of its physiology and clinical significance[J]. Anaesthesia.2004,59(7):695-703
[17] He S,Gaca MDA,Walls AF. A role for tryptase in the activation of human mast cells: modulation of histamine release by tryptase and inhibitors of tryptase[J]. Journal of Pharmacology and Experimental Therapeutics.1998,286(1): 289–297
[18] Vliagoftis H,Lacy P,Luy B,et al. Mast cell tryptase activates peripheral blood eosinophils to release granule-associated enzymes[J]. Int Arch Allergy Immunol. 2004,135(3):196–204
[19] Huang C, De Sanctis GT, et al. Evaluation of the substrate specificity of human mast cell tryptase beta I and demonstration of its importance in bacterial infections of the lung[J].J Biol Chem .2001,276(28): 26276–26284
[20] He S,Peng Q,Walls AF,et al.Potent induction of a neutrophil and eosinoplhil -rich infiltrate in vivo by human mast cell tryptase:selective enhan cement of eosinoplfil recruitment by lfistamine[J].J Immunol.1997,159(12):6216—6225
[21] Clark JM,Abraham WM,Fishman CE,et al.Tryptase inhibitors block allergen- induced airway and inflammatory responses in allergic sheep[J].Am J Respir Crit Care Med . 1995,152(6 pt 1):2076–2083
[22]Kirby JG,Hargreave FE,Gleich GJ,et al.Bronchoatveolar cell profiles of asthmatic and no-asthmatic subjectsl[J]. Am Rev Respir Dis.1987,136(2):379 -383
[23]Wenzel SE,Fowler AA,Schwartz LB.Activation of pulmonary mast cells by bronchoalveolar allergen challenge[J].Am Rev Respir Dis 1988,137(4):1002-.1008
[24]林小平,何韶衡,赵海涛.哮喘支气管黏膜中类胰蛋白酶的表达增强[J].免疫学杂志.2003, 19(5):375-377
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[2] Schwartz LB,Lewis RA,Austen KF.Tryptase from human pulmonary mast cells. Purification and characterization[J]. J Biol Chem .1981,256(22):11939–11943
[3]Hallgren J,Pejler G.Biology of mast cell tryptase.An inflammatory mediator[J] FEBS Journal.2006,273 (9):1871–1895
[4]Pereira PJ,Bergner A,Macedo-Ribeiro S,et al.Human beta-tryptase is a ring-like tetramer with active sites facing a central pore[J].Nature.1998,392(6673): 306–311
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[7]Hallgren J, Karlson U, Poorafshar M, et al. Mechanism for activation of mouse mast cell tryptase: dependence on heparin and acidic pH for formation of active tetramers of mouse mast cell protease 6[J].Biochemistry.2000,39(42):13068–13077
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[2] Hallgren J,Pejler G. Biology of mast cell tryptase.An inflammatory mediator[J]. FEBS Journal.2006,273 (9):1871–1895
[3] Barned PJ. Pathophysiology of asthma[J]. Br J Clin Pharmacol. 1996,42: 3–10
[4] Centers for disease control and prevention. Asthma mortality and hospitalization among children and young adults- United States 1998 -1993[J].MMWR Morb Mortal Wkly Rep.1996,45:350—353
[5] 顾立刚,赵仲生,陶君娣,等. 薄盖灵芝对小鼠内外免疫反应的实验研究[J] .上海免疫学杂志.1989,9(3):145-147
[6] 张罗修,盛惊州,薛迎旦,等.日本灵芝对抗体形成细胞及变态反应的影响[J].中药药理与临床.1992,8(2):10-14
[7] Kohda H,Toknmoto W,Sakamoto K,et al. The biologically active constituents of Ganoderma lucidum (Fr.) Karst. Histamine release-inhibitory triterpenes[J]. Chem Pharm Bull (Tokyo).1985, 33(4):1367-1374
[8] 龚志锦.常用的特殊染色方法-甲苯氨蓝染色[A] .见:王伯远编. 病理学技术[C].北京:人民卫生出版社,2002.172
[9] 刘传合,薛全福,陈育智,等.支气管哮喘动物模型的研究现状[J] .中华结核与呼吸杂志.2000,23(11):647-649
[10]Akihisa T, Nakamura Y, Tagata M, et al.Anti-inflammatory and anti-tumor promoting effects of triterpene acids and sterols from the fungus Ganoderma lucidum[J]. 2007, 4(2): 224-231
[11]Hajjaj H, Mace C, Roberts M, et al. Effect of 26-oxygenosterols from Ganoderma lucidum and their activity as cholesterol synthesis inhibitors[J]. 2005,71(7): 3653-3658
[12]宁安红,曾婧,黄敏,等.灵芝多糖对小鼠肿瘤的抑制作用及免疫指标的观察[J] .肿瘤研究与临床.2004,16(3):147-149
[13]王林,王玉红,章克昌,等.灵芝中药发酵液对慢性支气管炎疗效的研究[J] .中国食用菌.2004,23(5):39-41
[14]Bradding P,Walls AF,Holgate ST,et al. The role of the mast cell in the pathophysiology of asthma[J]. J Allergy Clin Immunol. 2006,117(6):1277-1284
[15] He S,Walls AF. Human mast cell tryptase:a stimulus of microvascular leakage and mast cell activation[J]. Eur J Pharmacol.1997,328(1):89—97
[16] Payne V,Kam PC. Mast cell tryptase:a review of its physiology and clinical significance[J]. Anaesthesia.2004,59(7):695-703
[17] He S,Gaca MDA,Walls AF. A role for tryptase in the activation of human mast cells: modulation of histamine release by tryptase and inhibitors of tryptase[J]. Journal of Pharmacology and Experimental Therapeutics.1998,286(1): 289–297
[18] Vliagoftis H,Lacy P,Luy B,et al. Mast cell tryptase activates peripheral blood eosinophils to release granule-associated enzymes[J]. Int Arch Allergy Immunol. 2004,135(3):196–204
[19] Huang C, De Sanctis GT, et al. Evaluation of the substrate specificity of human mast cell tryptase beta I and demonstration of its importance in bacterial infections of the lung[J].J Biol Chem .2001,276(28): 26276–26284
[20] He S,Peng Q,Walls AF,et al.Potent induction of a neutrophil and eosinoplhil -rich infiltrate in vivo by human mast cell tryptase:selective enhan cement of eosinoplfil recruitment by lfistamine[J].J Immunol.1997,159(12):6216—6225
[21] Clark JM,Abraham WM,Fishman CE,et al.Tryptase inhibitors block allergen- induced airway and inflammatory responses in allergic sheep[J].Am J Respir Crit Care Med . 1995,152(6 pt 1):2076–2083
[22]Kirby JG,Hargreave FE,Gleich GJ,et al.Bronchoatveolar cell profiles of asthmatic and no-asthmatic subjectsl[J]. Am Rev Respir Dis.1987,136(2):379 -383
[23]Wenzel SE,Fowler AA,Schwartz LB.Activation of pulmonary mast cells by bronchoalveolar allergen challenge[J].Am Rev Respir Dis 1988,137(4):1002-.1008
[24]林小平,何韶衡,赵海涛.哮喘支气管黏膜中类胰蛋白酶的表达增强[J].免疫学杂志.2003, 19(5):375-377
[1] Bradding P,Walls AF.Holgate ST,et al.The role of the mast cell in the pathophysiology of asthma[J].J Allergy Clin Immunol. 2006 ,117(6): 1277 -1284
[2] Schwartz LB,Lewis RA,Austen KF.Tryptase from human pulmonary mast cells. Purification and characterization[J]. J Biol Chem .1981,256(22):11939–11943
[3]Hallgren J,Pejler G.Biology of mast cell tryptase.An inflammatory mediator[J] FEBS Journal.2006,273 (9):1871–1895
[4]Pereira PJ,Bergner A,Macedo-Ribeiro S,et al.Human beta-tryptase is a ring-like tetramer with active sites facing a central pore[J].Nature.1998,392(6673): 306–311
[5]Schwartz LB.Clinical utility of tryptase levels in systemic mastocytosis and associated hematologic disorders[J].Leukaemia Research .2001,25(7): 553–562.
[6]Huang C,Sanctis GT.Evaluation of the substrate specificity of human mast cell tryptase beta I and demonstration of its importance in bacterial infections of the lung. J Biol Chem. 2001,276(28):26276–26284
[7]Hallgren J, Karlson U, Poorafshar M, et al. Mechanism for activation of mouse mast cell tryptase: dependence on heparin and acidic pH for formation of active tetramers of mouse mast cell protease 6[J].Biochemistry.2000,39(42):13068–13077
[8]Vliagoftis H,Lacy P,Luy B,et al.Mast cell tryptase activates peripheral blood eosinophils to release granule-associated enzymes[J].Int Arch Allergy Immunol .2004,135(3):196–204
[9]He S,Peng Q,Walls AF.Potent induction of a neutrophil and eosinoplhil– rich infiltrate in vivo by human mast cell tryptase:selective enhan cement of eosinoplfil recruitment by lfistamine[J].J Immunol.1997,159(12):6216—6225
[10]Huang C,Friend DS,Qiu WT,et al.Induction of a selective and persistent extravas- ation of neutrophils into the peritoneal cavity by tryptase mouse mast cell protease 6[J].J Immunol . 1998,160(4): 1910–1919
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