组成图示
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传感器类型
全细胞生物传感器
检测对象
胞外 ATP（ATP），样品基质：培养细胞浴液（GENC 单层上方）
检测原理
机械刺激 GENC 后，Cx40 半通道开放并向浴液释放 ATP。ATP 扩散至 PC12 生物传感器细胞表面，结合嘌呤能 P2 受体（P2X/P2Y），激活受体并引起 PC12 胞内钙升高。PC12 细胞预先负载 Fluo-4 AM 和 Fura red AM，钙结合后改变荧光发射；共聚焦显微镜以 488 nm 激发，分别检测 Fluo-4（520±20 nm）和 Fura red（约 600 nm）信号，通过比率成像和剂量-响应校准将 PC12 钙响应转换为局部胞外 ATP 浓度。ATP 浓度越高，P2 受体激活越强，PC12 钙升高和荧光比率变化越大；Cx40 敲低减少 ATP 释放，使传感器响应消失。
检测灵敏度
原文未报告 LOD、线性范围、灵敏度斜率或相关系数。
效应效果
PC12 生物传感器以钙响应读出，suramin 可阻断响应，表明对嘌呤能 ATP 信号具有选择性；ATP 清除酶处理后钙波受限，洗脱后速度恢复至 9.6±0.5 μm/s、Δ[Ca2+]i 恢复至 394±39 nM，提示可逆。Cx40 siRNA 使胞外 ATP 升高从 76±2 μM 降至 6±1 μM，钙波速度降至 2.0±0.3 μm/s。GENC 钙波在 400 μm 内无衰减，速度 7.9±0.3 μm/s，Δ[Ca2+]i 599±58 nM；MMDD1 细胞则快速衰减。作者认为 Cx40 半通道介导的 ATP 释放和嘌呤能钙信号是肾小球内皮钙波关键，可能参与肾小球滤过和肾素释放调控。
传感器的构成
- 基底/培养表面：玻璃盖玻片（glass coverslip），承载 GENC 单层并提供成像表面。
- 样品细胞层：肾小球内皮细胞（GENC）单层，受机械刺激后通过 Cx40 半通道释放 ATP。
- 生物传感器细胞：PC12 细胞，表达嘌呤能 P2 受体，作为 ATP 全细胞生物传感器。
- 识别元件：PC12 细胞表面嘌呤能 P2 受体（P2X/P2Y），结合胞外 ATP 并启动钙信号。
- 信号标记物：钙离子荧光指示剂 Fluo-4 AM 与 Fura red AM（各 10 μM），响应 PC12 胞内钙升高。
- 换能/读出：Leica TCS SP2 AOBS MP 共聚焦显微镜，488 nm 激发，Fluo-4 520±20 nm 与 Fura red 约 600 nm 发射，比率成像。
- 对照/验证：suramin（100 μM）阻断 P2 受体以确认 ATP 特异性；apyrase/hexokinase（各 50 U/ml）清除 ATP 用于验证。
中文摘要
内皮细胞内钙（[Ca2+]i）在球旁血管功能中重要。本研究鉴定培养肾小球内皮细胞（GENC）中内皮钙波的存在及分子要素。GENC 培养于玻璃盖玻片，负载 Fluo-4/Fura red，用荧光共聚焦显微镜进行比率 [Ca2+]i 成像。机械刺激单个 GENC 使 [Ca2+]i 升高约 9 倍，以 7.9±0.3 μm/s 再生性传播至少 400 μm，幅度、动力学和速度无衰减。尼莫地平无影响，去除胞外钙使 Δ[Ca2+]i 降低约 50%。β-甘草次酸或 Cx40 siRNA 几乎完全消除 Δ[Ca2+]i 和钙波；apyrase/hexokinase 清除 ATP 或 suramin 阻断 P2 受体也产生类似阻断。刮除细胞形成无接触线不阻止传播。ATP 生物传感器检测到钙波期间胞外 ATP 升高（Δ=76±2 μM），Cx40 siRNA 后消失（Δ=6±1 μM）。提示 Cx40 半通道和胞外 ATP 是肾小球内皮钙波关键要素。
英文摘要
Endothelial intracellular calcium ([Ca(2+)](i)) plays an important role in the function of the juxtaglomerular vasculature. The present studies aimed to identify the existence and molecular elements of an endothelial calcium wave in cultured glomerular endothelial cells (GENC). GENCs on glass coverslips were loaded with Fluo-4/Fura red, and ratiometric [Ca(2+)](i) imaging was performed using fluorescence confocal microscopy. Mechanical stimulation of a single GENC caused a nine-fold increase in [Ca(2+)](i), which propagated from cell to cell throughout the monolayer (7.9 +/- 0.3 microm/s) in a regenerative manner (without decrement of amplitude, kinetics, and speed) over distances >400 microm. Inhibition of voltage-dependent calcium channels with nifedipine had no effect on the above parameters, but the removal of extracellular calcium reduced Delta[Ca(2+)](i) by 50%. Importantly, the gap junction uncoupler alpha-glycyrrhetinic acid or knockdown of connexin 40 (Cx40) by transfecting GENCs with Cx40 short interfering RNA (siRNA) almost completely eliminated Delta[Ca(2+)](i) and the calcium wave. Breakdown of extracellular ATP using a scavenger cocktail (apyrase and hexokinase) or nonselective inhibition of purinergic P2 receptors with suramin, had similar blocking effects. Scraping cells off along a line eliminated physical contact between cells but did not effect calcium wave propagation. Using an ATP biosensor technique, we detected a significant elevation in extracellular ATP (Delta = 76 +/- 2 microM) during calcium wave propagation, which was abolished by Cx40 siRNA treatment (Delta = 6 +/- 1 microM). These studies suggest that connexin 40 hemichannels and extracellular ATP are key molecular elements of the glomerular endothelial calcium wave, which may serve important juxtaglomerular functions.