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OVA 致敏激发过敏性气道炎症模型（文献参考）

卵白蛋白（OVA）蛋白抗原与氢氧化铝 Th2 佐剂致敏、随后反复气道激发诱导的急性过敏性气道炎症范式，是该领域最早且应用最广的模型之一；重度与轻度 OVA 方案产生不同的急性与长期效应：重度方案在恢复 4 个月后仍存在肺功能损害、衰减率降低、胶原沉积与弹性蛋白减少，长期重塑结局与屋尘螨（HDM）模型不同（HDM 无组织学异常但肺面积更小、膈肌运动更大）。

Study design

以预先生成的 cDNA 为输入的 SYBR Green 实时定量 PCR 检测。每孔加入 10 ng cDNA，技术三重复；使用 PowerUp SYBR Green Master Mix 与 5 µM 正反向引物；按源方案循环条件扩增并读取 CT，以看家基因和参考实验组进行 ΔΔCT 分析。具体基因、引物和生物学分组按各研究设计确定。

Outcome measures

获得可供光学显微镜观察的 H&E 染色组织切片：细胞核蓝紫色、细胞质与结缔组织纤维粉红色，用于观察组织中细胞的整体结构与形态细节。本方案本身不产出病理评分数值，也不提供特定分子标志物的定量结果。

各样品目标序列的 CT 值；以看家基因归一化并以参考实验组计算 ΔΔCT/相对表达量。本方案不包含具体基因或疾病特异结论，输出判读依赖所选的引物、看家基因和实验分组。

Experimental animals

Hematoxylin and Eosin (H&E) staining for histopathology
Age
来源未报告周龄（体重 14.9–17.3 g）
Sex
Female
Tissue
lung（肺，含主支气管）
Hematoxylin and Eosin (H&E) staining for histopathology
Age
来源未报告周龄（体重 14.9–17.3 g）
Sex
Female
Tissue
lung（肺，含主支气管）
Hematoxylin and Eosin (H&E) staining for histopathology
Age
来源未报告周龄（体重 14.9–17.3 g）
Sex
Female
Tissue
lung（肺，含主支气管）
Hematoxylin and Eosin (H&E) staining for histopathology
Age
未记载周龄，终点取材组织切片 HE 判读（离体）
Sex
Female
Tissue
lung（肺，含主支气管）

Experimental procedures

脱蜡（Dewaxing） 石蜡切片置于二甲苯中脱蜡 3 次，每次 00:03:00。 复水（Rehydration） 石蜡切片依次置于 100% 乙醇中 3 次、每次 00:03:00，再置于 95% 乙醇中 1 次、00:03:00，随后用水漂洗。 苏木素核染色（Hematoxylin staining） 石蜡切片以苏木素染液染色 00:00:20，随后在流水下冲洗 00:01:00。 分化与返蓝（Differentiation and bluing） 石蜡切片置于返蓝液中 00:01:00，随后在流水下冲洗 00:01:00；此步使细胞核呈现蓝色。 伊红染色（Eosin staining） 石蜡切片以伊红复染 00:00:10；此步使细胞质成分与结缔组织纤维呈现粉红色。 脱水与透明（Dehydration and Clearing） 石蜡切片依次置于 95% 乙醇中 00:02:00、100% 乙醇中 2 次、每次 00:02:00，随后置于二甲苯中 2 次、每次 00:02:00。 封片（Mounting） 石蜡切片以 Permount 封片介质加盖玻片封固。

qPCR 反应体系 **Option 2****: SYBR qPCR** qPCR 反应体系 Prepare SYBR Master mix (ul per well): 1. 5ul PowerUp SYBR Green Master Mix (#A25776, Applied Biosystem-ThermoFisher) 1. 1ul Primers (Forward and Reverse, diluted in Nuclease-free water at 5µM) qPCR 反应体系 Add 6ul of SYBR Master mix to each well with an automatic pipette (Multipette E3, Eppendorf) qPCR 反应体系 Add 4ul of cDNA per well (10ng cDNA) in technical triplicates to each well of LightCycler 480 Multiwell 384-plate (#4729749001, Roche Diagnostics) 热循环 Perform PCR using the following cycling conditions in a LightCycler® 480 System (Roche): 1. 50˚C for 2min 1. 95˚C for 2min 1. 95˚C for 15s + 60˚C for 1min (x40cycles) 数据分析 Using the LightCycler Software obtain the threshold cycles (CT) signals from each samples. 数据分析 Perform ΔΔCT-method to analyze the expression data using the endogenous control genes and the reference experimental group.

Model identity

Model name
OVA 致敏激发过敏性气道炎症模型（文献参考）
Species
mouse
Taxonomy ID
10090
Strain
BALB/c
Genetic background
BALB/c 在 MGI 中记录为品系家系（strain family，含 200 余个亚系与衍生记录），亚系间存在已知遗传差异；未标注亚系的 BALB/c，其具体遗传构成取决于实际使用的繁育群体。

Biological characteristics

Research questions
过敏性气道炎症机制与干预评价；OVA 与 HDM 范式间长期肺功能与重塑结局的比较。
Limitations
文献参考模型，不代表公司实际保有、供应或质检；模型以急性炎症范式为主，其复现人类疾病的能力长期存在争议，小鼠模型复现慢性过敏性炎症存在已知局限；长期肺功能与重塑结局随方案强度与致敏原范式显著不同（重度 OVA 与 HDM 结局相异），模型选择与评定时间点是影响结论外推的核心条件。

Experimental methods

Experimental protocols

  • Hematoxylin and Eosin (H&E) staining for histopathology

    Hematoxylin and eosin (H&E) staining of fixed, paraffin-embedded tissue sections (4 μm). The workflow covers dewaxing, rehydration, hematoxylin nuclear staining, bluing, eosin counterstaining, dehydration and clearing, and coverslipping, yielding blue-purple nuclei and pink cytoplasm and connective tissue fibers for light-microscopic assessment of cellular and tissue morphology.

  • cDNA 模板的实时定量 PCR（qPCR，SYBR Green 分支）

    This protocol adopts the SYBR Green branch of the protocols.io qPCR v1 record. The input is pre-generated cDNA (10 ng per well); RNA extraction and reverse transcription are not included. PowerUp SYBR Green Master Mix and forward/reverse primers are used on a LightCycler 480 with 50˚C for 2 min, 95˚C for 2 min, and 40 cycles of 95˚C for 15 s plus 60˚C for 1 min. CT values are read and analysed by the ΔΔCT method. No specific genes, primer sequences or disease-model readouts are asserted.

  • Hematoxylin and Eosin (H&E) staining for histopathology

    Hematoxylin and eosin (H&E) staining of fixed, paraffin-embedded tissue sections (4 μm). The workflow covers dewaxing, rehydration, hematoxylin nuclear staining, bluing, eosin counterstaining, dehydration and clearing, and coverslipping, yielding blue-purple nuclei and pink cytoplasm and connective tissue fibers for light-microscopic assessment of cellular and tissue morphology.

  • Hematoxylin and Eosin (H&E) staining for histopathology

    Hematoxylin and eosin (H&E) staining of fixed, paraffin-embedded tissue sections (4 μm). The workflow covers dewaxing, rehydration, hematoxylin nuclear staining, bluing, eosin counterstaining, dehydration and clearing, and coverslipping, yielding blue-purple nuclei and pink cytoplasm and connective tissue fibers for light-microscopic assessment of cellular and tissue morphology.

  • Hematoxylin and Eosin (H&E) staining for histopathology

    Hematoxylin and eosin (H&E) staining of fixed, paraffin-embedded tissue sections (4 μm). The workflow covers dewaxing, rehydration, hematoxylin nuclear staining, bluing, eosin counterstaining, dehydration and clearing, and coverslipping, yielding blue-purple nuclei and pink cytoplasm and connective tissue fibers for light-microscopic assessment of cellular and tissue morphology.

Reagents and materials

References

  1. Markus MA, Albers J, Alves F, Dullin C. Distinct long-term effects on lung function and airway remodeling in ovalbumin and house dust mite mouse models of experimental asthma. Sci Rep. 2026;16(1):12737. DOI:10.1038/s41598-026-47822-x; PMID:42000816; PMCID:PMC13091909. 原文逐句支持：'One of the oldest and most widely used models is the ovalbumin (OVA)-induced acute allergic inflammation model, in which mice are sensitized with the protein antigen OVA combined with the pro-T-Helper cell (Th2) adjuvant aluminum hydroxide'；方法逐字记载 'Female BALB/c mice (8–9 weeks old) were purchased from Charles River Laboratories'，支持 strain=BALB/c（该采购记录为原文实验条件，非公司来源）；'their ability to reproduce human disease remains controversial' 与 mouse models 复现慢性过敏性炎症的 known limitations；重度 OVA（SAA）较轻度（MAA）急性与长期效应更强，恢复 4 个月后 SAA 衰减率降低、胶原沉积、弹性蛋白减少，HDM 无组织学异常但肺面积更小、膈肌运动更大。
  2. MGI strain record BALB/c (MGI:2161072): Attributes: strain family; Strain Family Members: 204。支持品系家系界定与不指定亚系。
  3. NCBI Taxonomy record 10090: Mus musculus (house mouse), rank species.
  4. MGI Inbred Strains documentation — BALB: 'Three major substrains trace back to before 1940'; 'Data on genetic markers suggest that these substrains have diverged largely through mutation or residual heterozygosity rather than genetic contamination.'。支持 BALB/c 亚系间遗传差异。
  5. Nathalia L. Oliveira; Samantha Gruenheid. Hematoxylin and Eosin (H&E) staining for histopathology. protocols.io, v1, 2026-07-31. DOI: 10.17504/protocols.io.kxygxr9jdg8j
  6. Epredia Signature Series Hematoxylin (7221), 产品信息页, Fisher Scientific
  7. Epredia Signature Series Bluing Reagent (7301), 产品信息页, Fisher Scientific
  8. Epredia Signature Series Eosin Y (7111), 产品信息页, Fisher Scientific
  9. Xylene, Fisherbrand HistoPrep (HC7001GAL), 产品信息页, Fisher Scientific
  10. Fisher Chemical Permount Mounting Medium (SP15 产品族), 产品信息页, Fisher Scientific
  11. Ethanol (CID 702, CAS 64-17-5), PubChem 化合物标准记录
  12. Miquel Vila. Gene expression analysis by quantitative Real-Time PCR (qPCR). protocols.io. https://doi.org/10.17504/protocols.io.ewov1oyz2lr2/v1
  13. Bustin SA, et al. MIQE précis: Practical implementation of minimum standard guidelines for quantitative real-time PCR experiments. BMC Molecular Biology 2010;11:74. PMID 20858237. doi:10.1186/1471-2199-11-74
  14. ARRIVE 2.0 guidelines
Model consultation