BioVector® ROSA KIT D816V 人肥大细胞株 / ROSA KIT D816V Human Mast Cell Line

一、 背景与来源说明 / Background & Origin

ROSA KIT D816V 是一种来源于人脐带血 CD34+ 祖细胞经基因工程表达或筛选获得的细胞模型，转入并表达了驱动型 KIT D816V 突变基因。在系统性肥大细胞增多症（Systemic Mastocytosis, SM）以及肥大细胞白血病（Mast Cell Leukemia, MCL）中，KIT D816V 突变会导致受体酪氨酸激酶发生配体非依赖性的持续自我激活。ROSA KIT D816V 细胞株是研究系统性肥大细胞增多症发病机制、KIT D816V 介导的下游信号转导、酪氨酸激酶抑制剂（TKI，如米哚妥林 Midostaurin、阿瓦替尼 Avapritinib）耐药与药效评估的国际标准人源肥大细胞模型。

二、 细胞生物学特征 / Biological Characteristics

• 组织来源 / Tissue Origin：人造血祖细胞 derived 人肥大细胞 (Human Mast Cell Line)

• 生长特性 / Growth Properties：主要呈悬浮生长（Suspension）或部分弱贴壁生长

• 关键突变 / Key Mutation：KIT 外显子 17 D816V 激活型突变 (KIT D816V)

• 细胞标记物 / Cell Markers：

  • 肥大细胞受体：CD117 (KIT)、Fc epsilon RI (高亲和力 IgE 受体)

  • 颗粒特异性蛋白：类 tryptase (肥大细胞类 tryptase)

• 应用领域 / Applications：系统性肥大细胞增多症 (SM) 病理机制、KIT D816V 靶向抑制剂筛选、肥大细胞脱颗粒与变态反应信号转导研究

三、 培养条件与传代指南 / Culture & Handling Protocols

1. 完全培养基配方 / Medium Formulation

• 基础培养基 / Basal Medium：IMDM 或 RPMI-1640 基础培养基

• 血清与添加剂 / Serum & Supplements：

  • 10% 胎牛血清 (FBS)

  • 人干细胞因子 (rhSCF, 建议 10 至 50 ng/mL，依特定株系对 SCF 依赖性而定)

• 抗生素 / Antibiotics：100 U/mL 青霉素 + 100 ug/mL 链霉素

2. 培养与传代操作 / Incubation & Subculture

• 培养环境 / Conditions：37 摄氏度，5% CO2，饱和湿度

• 密度管理 / Confluence：保持细胞密度在 2 x 10^5 至 1 x 10^6 cells/mL 之间，避免细胞密度过低导致生长停滞

• 传代步骤 / Procedure：

  1. 收集含有细胞的培养悬液于离心管中。

  2. 以 200 x g 离心 5 分钟，弃去旧培养基。

  3. 加入新鲜完全培养基重悬细胞沉淀。

  4. 按照 1:2 至 1:4 的比例稀释接种至新培养瓶中。

四、 技术指标简表 / Technical Data Summary

BioVector® ROSA KIT D816V Human Mast Cell Line

1. Background and Origin

ROSA KIT D816V is a human mast cell line engineered or selected to express the constitutive KIT D816V activation mutation. In systemic mastocytosis (SM) and mast cell leukemia (MCL), the KIT D816V mutation causes ligand-independent, persistent activation of the receptor tyrosine kinase. ROSA KIT D816V serves as a key cellular model for investigating SM pathophysiology, downstream oncogenic signaling, and evaluating tyrosine kinase inhibitors (TKIs, such as Midostaurin and Avapritinib).

2. Biological Characteristics

• Tissue Origin: Human hematopoietic progenitor-derived mast cell line

• Growth Properties: Suspension or loosely adherent round cells

• Key Mutation: KIT Exon 17 D816V gain-of-function mutation

• Cell Markers:

  • Surface receptors: CD117 (KIT), Fc epsilon RI (high-affinity IgE receptor)

  • Granule proteins: Mast cell Tryptase

• Applications: Systemic mastocytosis research, targeted drug discovery against KIT D816V, mast cell degranulation, and allergy signaling pathway assays

3. Culture and Handling Protocols

Culture Medium Formulation

• Basal Medium: IMDM or RPMI-1640

• Serum and Additives: 10 percent Fetal Bovine Serum (FBS), supplemented with recombinant human Stem Cell Factor (rhSCF, 10 to 50 ng/mL as required)

• Antibiotics: 100 U/mL Penicillin and 100 ug/mL Streptomycin

Incubation and Subculture Guide

• Incubation Conditions: 37 degrees Celsius, 5 percent CO2, humidified atmosphere

• Density Management: Maintain cell density between 2 x 10^5 and 1 x 10^6 cells/mL to ensure exponential proliferation

• Subculture Procedure:

  1. Collect cell suspension into a sterile centrifuge tube.

  2. Centrifuge at 200 x g for 5 minutes and aspirate spent supernatant.

  3. Resuspend cell pellet in fresh complete medium.

  4. Dilute and seed cells at a ratio of 1:2 to 1:4 into fresh culture vessels.

4. Technical Data Summary

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