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Immunologia e infezioni

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

Published: January 19, 2024
doi:
10.3791/66132
, , ,
1Alkek Center for Metagenomics and Microbiome Research, Department of Molecular Virology and Microbiology,Baylor College of Medicine

Summary

该协议描述了一种在病毒感染的人肠道类器官中进行钙成像的方法，并提供了一种分析方法。

Abstract

钙信号传导是几乎每个组织不可或缺的调节剂。在肠上皮细胞内，钙参与分泌活性、肌动蛋白动力学、炎症反应、干细胞增殖和许多其他未表征的细胞功能的调节。因此，绘制肠上皮内的钙信号动力学可以深入了解稳态细胞过程，并揭示对各种刺激的独特反应。人肠道类器官 （HIO） 是一种用于研究肠上皮的高通量人源模型，因此代表了研究钙动力学的有用系统。本文描述了一种使用基因编码钙指示剂 （GECI） 稳定转导 HIO 的方案，进行实时荧光显微镜检查，并分析成像数据以有意义地表征钙信号。作为代表性示例，用慢病毒转导三维 HIO 以稳定表达 GCaMP6s，这是一种基于绿色荧光蛋白的胞质 GECI。然后将工程化的HIO分散到单细胞悬浮液中，并作为单层接种。分化后，HIO单层感染轮状病毒和/或用已知刺激钙反应的药物治疗。装有温控加湿活体成像室的落射荧光显微镜可以对受感染或药物治疗的单层进行长期成像。成像后，使用免费提供的分析软件ImageJ分析采集的图像。总体而言，这项工作建立了一个适应性强的管道，用于表征HIO中的细胞信号转导。

Introduction

钙是一种广泛保守的第二信使，在调节细胞生理学中起着关键作用1。鉴于其电荷强、体积小、在生理条件下溶解度高，钙是蛋白质构象的理想操纵剂。这使得钙成为将电化学信号转导为酶促、转录或转录后改变的有力手段。内质网 （ER） 和质膜上严格的钙浓度梯度产生了高驱动力，允许胞质钙浓度快速变化。多种机制，包括缓冲和主动运输，都紧紧地维持了这种梯度。虽然这种维持对于正常的细胞功能是必要的，但这种维持在能量上是昂贵的，使其在压力状态下特别容易受到 影响 2.

因此，胞质溶胶内钙的失调是多种细胞应激的近乎普遍的信号。代谢紊乱、毒素、病原体、机械损伤和遗传扰动都会破坏钙信号传导。无论刺激如何，在全细胞水平上，胞质钙的持续、不受控制的升高都会促进细胞凋亡并最终导致坏死 3,4。然而，较低振幅或较高频率的胞质钙水平的改变具有不同的影响2。同样，钙波动的结果可能因发生钙波动的空间微域而异5.因此，监测钙水平可以深入了解动态信号转导过程，但这需要具有相对较高的时间和空间分辨率的采样。

基因编码钙指示剂 （GECI） 是在活细胞系统中连续采样的有力工具6。一些使用最广泛的 GECI 是基于 GFP 的钙反应性荧光蛋白，称为 GCaMPs7。经典 GCaMP 是三个不同蛋白质结构域的融合：环状置换 GFP （cpGFP）、钙调蛋白和 M136。钙调蛋白结构域在结合钙时发生构象变化，使其与 M13 相互作用。钙调蛋白-M13 相互作用诱导 cpGFP 的构象变化，从而在激发时增加其荧光发射。因此，钙浓度的增加与GCaMP荧光强度的增加有关。这些传感器可以是胞质的，也可以是靶向特定的细胞器8。

与大多数组织类似，钙调节胃肠上皮内的各种功能。肠上皮是营养和液体吸收不可或缺的一部分，但也必须形成紧密的屏障和免疫界面，以避免病原体入侵或毒性侮辱。钙依赖性途径几乎影响所有这些重要功能9,10,11。然而，肠上皮内的钙信号传导仍然是一个未被充分探索的前沿领域，具有作为治疗靶点的潜力。虽然在体内监测肠上皮内的钙动力学继续面临挑战，但人肠道类器官 （HIO） 为实验提供了适应性强的离体系统12。HIO 是源自人类肠道干细胞的 3 维 （3D） 球体，在分化后概括了天然肠上皮细胞的大部分细胞多样性 12。

该协议描述了设计表达GECI的HIO的综合方法，然后将工程HIO制备为用于活细胞钙成像的单层。它提供了病毒感染作为破坏钙信号传导的病理操作的一个例子，并提供了一种分析方法来量化这些变化。

Protocol

本方案和代表性实验中使用的所有人类肠道类器官（HIO）均来源于德克萨斯医学中心消化疾病类肠核心获得和维护的人体组织。所有样本均根据贝勒医学院机构审查委员会批准的方案收集。 1.材料和试剂的制备 对于类器官维持，收集细胞培养处理的 24 孔板、基底膜基质 （BMM）、15 mL 锥形管和 1.5 mL 锥形管。为了制备不含生长因子 （CMGF-） 的完整培?…

Representative Results

图 1A 显示了包含 3 维人肠道类器官的 BMM 圆顶，这些类器官已被转导以稳定表达 GCaMP6。 图1B 显示了在接种后24、48和72小时重新铺板为单层的相同类器官系。为了验证GCaMP6s的功能，每2秒用荧光显微镜对单层进行成像4分钟，并在~20秒后向培养基中加入100nM ADP。图 1C绘制了每次曝光时在488nm通道上检测到的荧光强度。迹线显示基线荧…

Discussion

胞质 Ca2+ 水平的改变可能是上皮内病理的原因和结果 10,16,17。胞质钙的增加可以通过激活钙依赖性氯离子通道直接驱动分泌TMEM16A18,19。响应于 Ca2+ 的 TMEM16A 激活允许氯化物的顶端流出，建立促进液体分泌的渗透梯度20,21。?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了美国国立卫生研究院 （NIH） 的 R01DK115507 和 R01AI158683 （PI： J. M. Hyser） 的资助。受训者支持由 NIH 资助 F30DK131828 （PI： J.T. Gebert）、F31DK132942 （PI： F. J. Scribano） 和 F32DK130288 （PI： K.A. Engevik） 提供。我们要感谢德克萨斯医学中心消化系统疾病肠类核心提供类器官维持培养基。

Materials

Advanced DMEM F12 Gibco 12634028
[Leu15]-Gastrin I Sigma-Aldrich G9145
0.05% Trypsin EDTA  Gibco  25300054
0.05% Trypsin EDTA  Gibco  25300054
1.5mL microcentrifuge tubes Fisherbrand 5408137
15mL conical tubes Thermofisher Scientific 0553859A
16% formaldehyde Thermofisher Scientific 28906
1M HEPES Gibco 15630080
1M HEPES Gibco 15630080
1X PBS Corning  21-040-CV
25 gauge needle Thermofisher Scientific 1482113D
A-83-01 Tocris 2939
ADP Sigma-Aldrich  A2754
Advanced DMEM F12 Gibco 12634028
Antibiotic-antimycocytic  Gibco 15240062
Antibiotic-antimycotic  Gibco 15240062
B27 Supplement Gibco 17504-044
Bovine serum albumin FisherScientific  BP1600100
CellView Cell Culture Slide, PS, 75/25 MM, Glass Bottom, 10 compartments Greiner 543979
Collagen IV Sigma Aldrich C5533
DAPI Thermofisher Scientific D1306
EDTA Corning 46-034-CI
Fetal bovine serum  Corning  35010CV
Fetal bovine serum  Corning  35010CV
Fluorobrite Gibco A1896701
GlutaMAX  Gibco  35050079
GlutaMAX  Gibco  35050079
Human epidermal growth factor ProteinTech HZ-1326
Lentivirus VectorBuilder (variable)
Matrigel BD Biosceicen 356231/CB40230C
N2 Supplement Gibco 17502-048
N-acetylcysteine Sigma-Aldrich A9165-5G
NH4Cl Sigma-Aldrich  A9434
Nicotinamide Sigma-Aldrich N0636
Nunc Cell Culture Treated 24-well Plates Thermofisher Scientific 142475
Polybrene MilliporeSigma TR1003G
SB202190 Sigma-Aldrich S70767
Triton X-100 Fisher BioReagents BP151100
TrypLE Express Enzyme, no phenol red Thermofisher Scientific 12604013
Trypsin Worthington Biochemical NC9811754
Y-27632 Tocris 1254
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Tags

Keywords: Calcium ImagingVirus-infectedHuman Intestinal OrganoidGenetically Encoded Calcium IndicatorsRotavirusParacrine Purinergic SignalingCalcium SignalingIntestinal EpitheliumLive ImagingGCaMP6sHIO Monolayers
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Gebert, J. T., Scribano, F. J., Engevik, K. A., Hyser, J. M. Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators. J. Vis. Exp. (203), e66132, doi:10.3791/66132 (2024).
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