离体 尿路上皮细胞中机械活化的CA2+ 瞬变分析
Summary
该协议描述了一种使用荧光Ca2 + 传感器GCaMP5G评估天然尿路上皮细胞中机械激活离子通道功能的方法。
Abstract
机械激活离子通道是将机械刺激（如拉伸力或剪切力）转换为电和生化信号的生物换能器。在哺乳动物中，机械激活通道对于检测触觉、听觉、红细胞体积调节、基础血压调节和膀胱充盈感等各种过程中的外部和内部刺激至关重要。虽然机械激活离子通道的功能已在体外环境中使用膜片钳技术进行了广泛研究，但评估它们在天然环境中的功能仍然是一项艰巨的任务，通常是因为这些通道表达位点（例如，传入末端、Merkel 细胞、压力感受器和肾小管）的访问受限或难以应用膜片钳技术（例如 尿路上皮伞细胞的顶端表面）。该协议描述了一种在离体尿路上皮制剂中使用荧光传感器GCaMP5G评估机械诱发的Ca 2 +瞬变的程序，该技术可以很容易地适用于研究其他天然组织制剂中的机械诱发Ca2 +事件。
Introduction
当尿滤液通过肾单位时，尿道中的上皮细胞受到机械力，尿液被泵出肾盂并通过输尿管储存在膀胱中。人们早就认识到，液体对泌尿道上皮细胞施加的机械力（例如剪切应力和拉伸）调节近端肾小管中蛋白质和远端肾单位中溶质的重吸收1，2，3，4，5，6，7，8，9，10，11，12，13，以及尿液在膀胱中的储存和排尿14，15，16，17。
机械刺激转化为电和生化信号的过程称为机械转导，由响应细胞结构变形或相关细胞外基质变形的蛋白质介导18，19，20，21。机械激活离子通道的独特之处在于，它们响应膜张力、压力或剪切应力的变化从封闭状态转变为开放渗透状态18，19，20，21，22。此外，Ca 2+瞬变可以通过整合素介导的机械转导或通过激活细胞-细胞连接处的机械响应粘附系统23，24，25，26来启动。离子通道功能通常使用膜片钳技术进行评估，该技术涉及在细胞膜和贴片移液器27之间形成千兆欧密封。然而，位于具有致密细胞外基质（例如肾小管）或被物理屏障（例如糖萼）包围的深层组织层中的细胞很难用玻璃微量移液器进入。同样，嵌入的细胞或作为机械稳定性差的组织组成部分的细胞（例如尿路上皮）不能轻易地用膜片钳技术进行研究。由于许多机械激活的离子通道可渗透到Ca 2+，因此另一种方法是使用Ca2+敏感染料或遗传编码钙指示剂（GECIs）如GCaMP）通过荧光显微镜评估其活性。最近在蛋白质工程方面的努力显着增加了GECIs28，29，30的动态范围，灵敏度和响应，遗传学的进步使其在特定细胞群中表达，使其非常适合研究机械转导。
尿路上皮是覆盖膀胱内部的分层上皮，起到屏障的作用，防止尿溶质扩散到膀胱间质中，但也起到换能器的作用，感知膀胱充盈并将这些事件传达给下面的神经和肌肉组织16。先前的研究表明，尿路上皮和下层组织之间的通信需要机械激活的离子通道Piezo1和Piezo231。为了评估尿路上皮细胞中机械诱导的Ca 2+瞬变，开发了一种新技术，该技术使用腺病毒基因转移在尿路上皮细胞中表达Ca2+传感器GCaMP5G。该技术采用粘膜片制备，可轻松进入最外层的伞状细胞层和计算机辅助系统，用于用封闭的玻璃微量移液管同时机械刺激单个细胞并记录荧光随时间的变化。
Protocol
动物的护理和处理是根据匹兹堡大学机构动物护理和使用委员会进行的。雌性，2-4个月大的C57Bl / 6J小鼠用于本研究。小鼠是商业获得的（见 材料表）。 1. 设备组装与设置 使用配备高分辨率相机和稳定光源的正置显微镜进行Ca2+ 成像（参见 材料表）。使用显微镜兼容软件采集图像，该软件允许通过USB数字I / O设备 <em…
Representative Results
本协议描述了一种使用荧光Ca 2 +传感器GCaMP5G评估伞形细胞中机械诱发的Ca2 +瞬变的技术。腺病毒转导用于在尿路上皮细胞中表达GCaMP5G，因为它的效率高，并且产生更高的表达水平。来自转导膀胱的染色冷冻切片的荧光图像如图2D所示。对于这些实验，GCaMP5G在伞细胞层中的表达最高。在戳伞细胞的实验中捕获的一系列代表性图像如图3A所…
Discussion
所有生物体，似乎大多数细胞类型，都表达响应机械刺激的离子通道20，33，34，35，36，37。这些机械激活通道的功能主要通过膜片钳技术进行评估。然而，由于可及性问题，机械激活离子通道的膜片钳研究在很大程度上仅限于解离的细胞和细胞系。…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH拨款R01DK119183（G.A.和MDC）和S10OD028596（G.A.）以及匹兹堡肾脏研究中心的细胞生理学和模式生物肾脏成像核心（P30DK079307）的支持。
Materials
| 20x Objective | Olympus | UMPlanFL N | |
| 24 G ¾” catheter | Medline | Suresite IV slide | |
| 4x Objective | Olympus | UPlanFL N | |
| Analog/digital converter | Molecular Devices | Digidata 1440A | |
| Anti-GFP antibody | Abcam | Ab6556 | |
| Beam splitter | Chroma | T495lpxr | |
| Bipolar temperature controller | Warner Instruments | TC-344B | |
| CaCl2 | Fluka | 21114-1L | 1 M solution |
| cellSens software | Olympus | Imaging software | |
| CMOS camera | Hamamatsu | ORCA fusion | |
| Donkey anti-rabbit conjugated to Alexa Fluor 488 | Jackson ImmunoResearch | 711-545-152 | |
| Excel | Microsoft Corporation | ||
| Filter | Chroma | ET470/40X | |
| Glass capillaries Corning 8250 glass | Warner Instruments | G85150T-4 | |
| Glucose | Sigma | G8270 | |
| HEPES | Sigma | H4034 | |
| Inline heater | Warner Instruments | SH-27B | |
| KCl | Sigma | 793590 | |
| Light source | Sutter Instruments | Lambda XL | |
| Manifold pump tubing | Fisherbrand | 14-190-510 | ID 1.52 mm |
| Manifold pump tubing | Fisherbrand | 14-190-533 | ID 2.79 mm |
| MgCl2 | Sigma | M9272 | |
| Mice | Jackson Lab | 664 | 2-4 months old female C57BL/6J |
| Microforge | Narishige | MF-830 | |
| Micromanipulator | Sutter Instruments | MP-285 | |
| Microscope | Olympus | BX51W | |
| Mounting media with DAPI | Invitrogen | S36964 | Slowfade Diamond Antifade with DAPI |
| NaCl | Sigma | S7653 | |
| pClamp software | Molecular Devices | Version 10.4 | Patch-clamp electrophysiology data acquisition and analysis software |
| Peristaltic pump | Gilson | Minipuls 3 | |
| Piezoelectric actuator | Thorlabs | PAS005 | |
| Pipette holder | World Precision Instruments | ||
| Pipette puller | Narishige | PP-830 | |
| Quick exchange heated base with perfusion and adapter ring kit | Warner Instruments | QE-1 | Quick exchange platform fits 35 mm dish |
| Rhodamine-phalloidin | Invitrogen | R415 | |
| Sigma-Plot | Systat Software Inc | Version 14.0 | Scientific graphing and data analysis software |
| Silicone elastomer | Dow | Sylgard 184 | |
| Single channel open-loop piezo controller | Thorlabs | MDT694B | |
| Square grid holder pad | Ted Pella | 10520 | |
| Suture | AD Surgical | S-S618R13 | 6-0 Sylk |
| Teflon mounting rod | Custom made | Use to mount the piezoelectric actuator in the micromanipulator | |
| Tubing | Fisher Scientific | 14171129 | Tygon S3 ID 1/16 IN, OD 1/8 IN |
| USB Digital I/O device | National Instruments | NI USB-6501 |
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Diesen Artikel zitieren
Carattino, M. D., Ruiz, W. G., Apodaca, G. Ex Vivo Analysis of Mechanically Activated Ca2+ Transients in Urothelial Cells. J. Vis. Exp. (187), e64532, doi:10.3791/64532 (2022).