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Mast Cell Dependent Airway Hyperresponsiveness (AHR)
肥大细胞导致的气道高反应性小鼠模型(AHR)   

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Abstract

Asthma is a complex phenotype that involves multiple mechanisms, including adaptive and innate immunity as well as physiological and mechanical changes in the airways. In the models of asthma induced by sensitization and aerosolized allergen exposure in the absence of adjuvant, mast cells facilitate the development of in ammation and airway hyper-responsiveness. This model is useful to analysis of function of mast cells in AHR.

Materials and Reagents

  1. Mice (C57BL/6)
    Note: BALB/c is also appropriate to this experiment.
  2. Ovalbumin (OVA) (grade V) (Sigma Aldrich, catalog number: A5503 )
  3. Pentobarbital (Sigma-Aldrich, catalog number: P3761 )
  4. Acetylcholine chloride (Sigma-Aldrich, catalog number: A6625 )
  5. Phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number: 20012 )
  6. Wright–Giemsa stain (MUTO PURE CHEMICALS CO.,LTD, catalog number: 15022 )
  7. Saline

Equipment

  1. Syringe (1 ml) (Terumo Medical Corporation, catalog number: SS-01T2525 )
  2. Needle (26G1/2)
  3. Ultrasonic nebulizer (NIHON KOHDEN CORPORATION, catalog number: TUR-3200 )
  4. Differential pressure transducer (NIHON KOHDEN CORPORATION, catalog number: TP-602T )
  5. Light microscope (OLYMPUS, model: CX-41 )
  6. Tracheal cannula (Natume, catalog number: SP31 )
  7. Cytospin (Shandon, model: Cytospin 3 )
  8. Centrifuges

Procedure

  1. Priming and challenge
    1. Mice were immunized by intraperitoneally injection of OVA (10 μg/mouse) eight times at 2-day intervals (priming) for 2 week.
    2. On day 26 after the last injection, mice were treated with aerosolized OVA (1%) for 20 min per day (challenge).
    3. Repeat an aerosol challenge on day 27 and 28.

  2. Measurement of airway responsiveness
    1. On day 30, 36 h after the last aerosol challenge, mice were anesthetized with a pentobarbital (50 mg/kg) intraperitoneally.
    2. Cannula was inserted by opening a direct airway through an incision in the trachea (see Figure1).
    3. Stepwise increases in acetylcholine dose (0.6 to 20 mg/ml) were given with an ultrasonic nebulizer.
    4. Airway opening pressure was recorded continuously.
      *The data were expressed as the provocative concentration 150 (PC150), the concentration at which airway pressure was 150% of its baseline value. PC150 was calculated by log-linear interpolation for individual animals.


      Figure 1. Cannula was inserted into a direct airway through an incision in the trachea after opening

  3. Bronchoalveolar lavage and cell counting
    1. Mice were given a lethal dose of pentobarbital (120 mg/kg, i.p.), and the lungs were gently lavaged three times with PBS at a pressure of 25 cm H2O via the tracheal cannula.
    2. Total cell counts were determined by light microscopy.
    3. The lavage fluid was centrifuged at 800 rpm for 5 min at 4 °C.
    4. The cell pellet was resuspended in saline, and cytospin preparations were made.
    5. Differential counts on 200 cells were performed by light microscopy using Wright-Giemsa stain.

Acknowledgments

This protocol was developed and implemented by Dr. Masato Kubo at Division of Molecular Pathology, Tokyo University of Science, Chiba, Japan and Dr. Hiromasa Inoue at Department of Pulmonary Medicine, Kagoshima University, Kagoshima, Japan. This work was supported by a Grant-in-Aid-of-Scientific Research in Priority Areas of the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation.

References

  1. Sawaguchi, M., Tanaka, S., Nakatani, Y., Harada, Y., Mukai, K., Matsunaga, Y., Ishiwata, K., Oboki, K., Kambayashi, T., Watanabe, N., Karasuyama, H., Nakae, S., Inoue, H. and Kubo, M. (2012). Role of mast cells and basophils in IgE responses and in allergic airway hyperresponsiveness. J Immunol 188(4): 1809-1818.
  2. Taube, C., Wei, X., Swasey, C. H., Joetham, A., Zarini, S., Lively, T., Takeda, K., Loader, J., Miyahara, N., Kodama, T., Shultz, L. D., Donaldson, D. D., Hamelmann, E. H., Dakhama, A. and Gelfand, E. W. (2004). Mast cells, Fc epsilon RI, and IL-13 are required for development of airway hyperresponsiveness after aerosolized allergen exposure in the absence of adjuvant. J Immunol 172(10): 6398-6406.

简介

哮喘是一种复杂的表型,涉及多种机制,包括适应性和先天性免疫以及气道的生理和机械变化。 在不存在佐剂的情况下,在致敏和气雾化的过敏原暴露诱导的哮喘模型中,肥大细胞促进了炎症和气道高反应性的发展。 该模型可用于分析AHR中肥大细胞的功能。

材料和试剂

  1. 小鼠(C57BL/6)
    注意:BALB/c也适合此实验。
  2. 卵白蛋白(OVA)(等级V)(Sigma Aldrich,目录号:A5503)
  3. 戊巴比妥(Sigma-Aldrich,目录号:P3761)
  4. 乙酰胆碱氯化物(Sigma-Aldrich,目录号:A6625)
  5. 磷酸盐缓冲盐水(PBS)(Life Technologies,Gibco ,目录号:20012)
  6. Wright-Giemsa染料(MUTO PURE CHEMICALS CO。,LTD,目录号:15022)
  7. 盐水

设备

  1. 注射器(1ml)(Terumo Medical Corporation,目录号:SS-01T2525)
  2. 针(26G1/2)
  3. 超声雾化器(NIHON KOHDEN CORPORATION,目录号:TUR-3200)
  4. 差压压力传感器(NIHON KOHDEN CORPORATION,目录号:TP-602T)
  5. 光学显微镜(OLYMPUS,型号:CX-41)
  6. 气管套管(Natume,目录号:SP31)
  7. Cytospin(Shandon,型号:Cytospin 3)
  8. 离心机

程序

  1. 启动和挑战
    1. 通过以2天的间隔(引发)腹膜内注射OVA(10μg/小鼠)8次来免疫小鼠2周。
    2. 在最后一次注射后第26天,用雾化的OVA(1%)每天处理小鼠20分钟(攻击)。
    3. 在第27天和28天重复气溶胶挑战。

  2. 气道反应性的测量
    1. 在第30天,在最后一次气溶胶攻击后36小时,用戊巴比妥(50mg/kg)腹膜内麻醉小鼠。
    2. 通过在气管中的切口打开直接气道插入插管(参见图1)
    3. 用超声雾化器逐步增加乙酰胆碱剂量(0.6至20mg/ml)
    4. 持续记录气道开口压力。
      *数据表示为激发浓度150(PC150),气道压力为其基线值的150%的浓度。 通过对于单个动物的对数线性插值计算PC150

      图1.打开后,插管通过气管切口插入直接气道

  3. 支气管肺泡灌洗和细胞计数
    1. 给小鼠戊巴比妥(120mg/kg,ip)的致死剂量,并且通过气管套管在25cm H 2 O O的压力下用PBS轻轻地灌洗肺三次。 />
    2. 通过光学显微镜测定总细胞计数
    3. 将灌洗液在4℃下以800rpm离心5分钟
    4. 将细胞沉淀重悬于盐水中,制备细胞离心涂片制备物
    5. 通过使用Wright-Giemsa染色的光学显微镜进行200个细胞的差异计数

致谢

该协议由日本千叶县东京科技大学分子病理学部的Masato Kubo博士和日本鹿儿岛鹿儿岛大学肺科医院Hiromasa Inoue博士开发和实施。这项工作得到了日本教育,文化,体育,科学和技术部优先领域的科学研究资助和国家研究所健康科学基础研究促进计划的支持的生物医学创新。

参考文献

  1. Sawaguchi,M.,Tanaka,S.,Nakatani,Y.,Harada,Y.,Mukai,K.,Matsunaga,Y.,Ishiwata,K.,Oboki,K.,Kambayashi,T.,Watanabe, Karasuyama,H.,Nakae,S.,Inoue,H。和Kubo,M。(2012)。 肥大细胞和嗜碱性粒细胞在IgE反应和过敏性气道高反应性中的作用 J Immunol 188(4):1809-1818。
  2. Taube,C.,Wei,X.,Swasey,CH,Joetham,A.,Zarini,S.,Lively,T.,Takeda,K.,Loader,J.,Miyahara,N.,Kodama,T.,Shultz ,LD,Donaldson,DD,Hamelmann,EH,Dakhama,A。和Gelfand,EW(2004)。 肥大细胞,FcεRI和IL-13是气溶胶过敏原后气道高反应性发展所必需的 在缺乏佐剂的情况下暴露。 172(10):6398-6406。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Motomura, Y. and Kubo, M. (2012). Mast Cell Dependent Airway Hyperresponsiveness (AHR). Bio-protocol 2(19): e267. DOI: 10.21769/BioProtoc.267.
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