BioVector® Nav1.9 稳定表达细胞株（人源/鼠源电压门控钠离子通道 Nav1.9） / BioVector® Nav1.9 Stable Expression Cell Line (Human/Mouse Voltage-Gated Sodium Channel Nav1.9)

一、 背景与来源说明 / Background & Origin

BioVector® Nav1.9 稳定表达细胞株（人源/鼠源电压门控钠离子通道 Nav1.9 稳定过表达细胞系）是以 BioVector® HEK293 或 BioVector® CHO-K1 为宿主细胞，通过稳转构建高水平稳定表达 $Na_V1.9$$\alpha$ 亚基（SCN11A 基因编码）的重组离子通道细胞模型。

Nav1.9 属于耐河豚毒素（TTX-resistant, TTX-R）的电压门控钠离子通道，主要表达于小直径初级感觉神经元（如背根神经节 DRG 神经元）。其电生理特性表现为极慢的激活/失活动力学以及显著的超极化激活阈值，产生持久的窗口电流（Window current），在设置神经元静息膜电位及动作电位阈值中起关键作用。由于 Nav1.9 与外周痛觉传导（如病理性疼痛、炎症痛、内脏痛及小纤维神经病）密切相关，该稳定表达细胞株是突破原代 DRG 神经元分离限制、用于 Nav1.9 通道靶向镇痛药物筛选与电生理评估的理想工具。

二、 细胞生物学特征 / Biological Characteristics

• 宿主背景 / Host Cell: 人胚肾细胞（HEK293）或 中国仓鼠卵巢细胞（CHO-K1）

• 靶基因 / Target Gene: 人源 SCN11A (Human $Na_V1.9$) / 鼠源 Scn11a (Mouse $Na_V1.9$)

• 生物安全等级 / Biosafety Level: BSL-1

• 生长特性 / Growth Properties: 贴壁生长（Adherent），呈铺路石状或多角形上皮/成纤维细胞样形态

• 筛选抗性 / Selection Antibiotic: BioVector® Geneticin (G418) 或 BioVector® Puromycin

• 电生理特性 / Electrophysiological Properties:

  • TTX 抗性: 对河豚毒素（TTX）耐受（$IC_{50} > 10\ \mu\text{M}$）

  • 动力学特征: 慢激活与慢失活超极化钠电流，显著的持久电流（Persistent Current）

• 主要应用 / Key Applications: Nav1.9 靶向小分子阻滞剂与激活剂筛选、自动全细胞膜片钳（Automated Patch Clamp, APC）药物安评、镇痛药物作用机制研究、离子通道结构与功能分析

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

1. 推荐培养基 / Complete Growth Medium

• 基础培养基: BioVector® High-Glucose DMEM (用于 HEK293 宿主) 或 BioVector® DMEM/F-12 Medium (用于 CHO-K1 宿主)

• 添加成分: 10% 灭活 BioVector® Fetal Bovine Serum (FBS) + 1% 双抗（Penicillin-Streptomycin）

• 维持抗性: 100–400 $\mu\text{g/mL}$BioVector® Geneticin (G418)（用于保持基因高水平表达）

• 培养环境: 37 摄氏度，5% $\text{CO}_2$，高湿度培养箱

2. 复苏与传代操作 / Thawing & Subculture

1. 复苏: 将冻存管快速置于 37 摄氏度水浴中融化（1–2 分钟）。加入 8–10 mL 预热无抗性的 BioVector® Complete Growth Medium，120 x g 离心 5 分钟，弃上清后重悬接种于培养瓶中。初次复苏不加筛选抗性，待细胞贴壁恢复 24 小时后再加入筛选抗性。

2. 消化传代:

   • 细胞汇合度达到 70%–80% 时进行传代（切勿让细胞过度生长，否则易影响离子通道电生理表达）。

   • 用无钙镁 DPBS 洗涤后，加入 0.05% 或 0.25% Trypsin-EDTA 37 摄氏度消化 1–2 分钟，加入含血清培养基终止消化，轻柔吹打分散。

   • 按 1:3 至 1:6 比例传代。

3. 冻存保存 / Cryopreservation

• 冻存液配方: 90% BioVector® FBS + 10% DMSO 或 70% BioVector® Basal Medium + 20% BioVector® FBS + 10% DMSO

• 保存条件: 程序降温后长期保存于液氮（-196 摄氏度）。

四、 技术指标简表 / Technical Data Summary

BioVector® Nav1.9 稳定表达细胞株（人源/鼠源电压门控钠离子通道 Nav1.9） / BioVector® Nav1.9 Stable Expression Cell Line (Human/Mouse Voltage-Gated Sodium Channel Nav1.9)

1. Background and Origin

BioVector® Nav1.9 Stable Expression Cell Line is a recombinant ion channel model generated by stably overexpressing the $Na_V1.9$$\alpha$ subunit (encoded by the SCN11A gene) in host cells such as BioVector® HEK293 or BioVector® CHO-K1.

$Na_V1.9$ is a tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel predominantly expressed in small-diameter primary nociceptive dorsal root ganglion (DRG) neurons. It exhibits ultra-slow activation and inactivation kinetics with a hyperpolarized activation threshold, generating a prominent persistent "window current" that regulates resting membrane potential and firing thresholds. Because $Na_V1.9$ plays an essential role in pain pathways (inflammatory, neuropathic, and visceral pain), this stable cell line serves as a robust platform for $Na_V1.9$-targeted drug discovery and electrophysiological profiling.

2. Biological Characteristics

• Host Background: Human Embryonic Kidney (HEK293) or Chinese Hamster Ovary (CHO-K1)

• Target Gene: Human SCN11A ($Na_V1.9$) / Mouse Scn11a ($Na_V1.9$)

• Biosafety Level: BSL-1

• Growth Properties: Adherent; cobblestone epithelial or fibroblastic morphology

• Selection Marker: BioVector® Geneticin (G418) or BioVector® Puromycin

• Electrophysiological Features:

  • TTX Resistance: Resistant to tetrodotoxin ($IC_{50} > 10\ \mu\text{M}$)

  • Kinetics: Slow activation/inactivation with hyperpolarized voltage dependence and persistent inward current

• Primary Applications: High-throughput screening (HTS) of $Na_V1.9$ blockers/activators, automated patch-clamp (APC) assays, analgesic drug evaluation, and channel structure-function studies

3. Culture and Handling Protocols

Recommended Growth Medium

• Basal Medium: BioVector® High-Glucose DMEM (for HEK293 host) or BioVector® DMEM/F-12 Medium (for CHO-K1 host)

• Supplements: 10% heat-inactivated BioVector® Fetal Bovine Serum (FBS) + 1% Penicillin-Streptomycin

• Selection Antibiotics: 100–400 $\mu\text{g/mL}$BioVector® Geneticin (G418) to maintain expression

• Culture Conditions: 37 degrees Celsius, 5% $\text{CO}_2$, humidified incubator

Thawing and Subculture Protocol

1. Thawing: Thaw rapidly in a 37 degrees Celsius water bath. Resuspend in 8–10 mL pre-warmed antibiotic-free BioVector® Complete Growth Medium, centrifuge at 120 x g for 5 minutes, and seed into culture flasks. Add selection antibiotics 24 hours post-plating.

2. Passaging:

   • Subculture at 70–80% confluence (avoid overgrowth to maintain optimal electrophysiological current density).

   • Wash with DPBS, treat with 0.05% or 0.25% Trypsin-EDTA for 1–2 minutes at 37 degrees Celsius, neutralize with serum-containing medium, and gently disperse.

   • Split ratio: 1:3 to 1:6.

Cryopreservation

• Freezing Medium: 90% BioVector® FBS + 10% DMSO or 70% BioVector® Basal Medium + 20% BioVector® FBS + 10% DMSO

• Storage: Liquid nitrogen (-196 degrees Celsius).

4. Technical Data Summary

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