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Abstract
Introduction
Protocol
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Medicina

使用氟磷钙标签和纳米粒子跟踪分析估计人类泌尿纳米晶体

Published: February 09, 2021
doi:
10.3791/62192
, ,
1Department of Urology,University of Alabama at Birmingham

Summary

这项研究的目的是确定纳米粒子跟踪分析（NTA）能否检测和量化含有健康成年人纳米晶体的尿钙。目前的研究结果表明，NTA可能是估计肾结石疾病期间泌尿纳米晶体的潜在工具。

Abstract

肾结石在全世界成人和儿童中越来越普遍。最常见的肾结石类型由草酸钙（CaOx）晶体组成。当尿液中富含矿物质（如钙、草酸盐、磷酸盐）并形成肾结石之前时，就会发生结晶。评估石器晶体的标准方法包括显微镜、过滤和离心。然而，这些方法主要检测微晶体，而不是纳米晶体。纳米晶体被建议对肾脏上皮细胞的危害大于体外微晶体。在这里，我们描述了纳米粒子跟踪分析（NTA）检测人类泌尿纳米晶体的能力。健康成年人在饮用草酸盐负荷以刺激泌尿纳米晶体之前，先吃受控的草酸盐饮食。尿在草酸盐负荷前后收集了24小时。用乙醇加工和清洗样品以净化样品。泌尿纳米晶体沾染了钙结合氟磷，氟-4 AM。染色后，使用 NTA 确定纳米晶体的大小和计数。这项研究的结果表明，NTA可以有效地检测健康成年人的纳米晶体。这些发现表明，NTA可能是肾结石患者纳米晶体的有价值的早期检测方法。

Introduction

当尿液中富含矿物质时，尿晶体就会形成。这可能发生在健康的人，但更常见的个人肾结石1。泌尿晶体的存在和积累会增加患肾结石的风险。具体来说，当晶体与兰德尔的斑块结合，核化，积累，并随着时间的推移生长2，3，4时，就会发生这种情况。晶体在肾结石形成之前和晶体的评估可能具有预测价值的肾结石前3，5。具体来说，结晶体已被建议有用，以预测在含有结石6，7的草酸钙病史患者的结石复发的风险。

据报道，晶体对肾上皮和循环免疫细胞功能8、9、10、11、12、13产生负面影响。此前有报道称，与健康个体相比，从草酸钙（CaOx）肾结石前体中循环的单核细胞抑制了细胞生物能学。此外，CaOx晶体可减少细胞生物能学，并破坏单核细胞8中的红氧平衡。食用富含草酸盐的膳食可能导致结晶体，导致肾管损伤，并改变保护肾结石形成的尿大分子的生产和功能。多项研究已证明，尿晶体的形状和大小可能因尿液17、18、19的pH值和温度而异。此外，尿蛋白已被证明可以调节晶体行为20。道登等人19日提出，结晶体分析可能有助于肾结石病患者的管理，并评估他们对治疗的反应。目前可用于评估晶体存在的一些常规方法包括极化显微镜21、22、电子显微镜23、粒子计数器3、尿液过滤24、蒸发3、5或离心21。这些研究为肾结石领域提供了宝贵的见解。然而，这些方法的局限性是无法可视化和量化大小小于 1μm 的晶体。这种大小的晶体可能通过附着在兰德尔的斑块上来影响CaOx石的生长。

与较大的微晶体25相比，纳米晶体已证明对肾脏细胞造成广泛损伤。纳米晶体的存在已经报告在尿液中使用纳米粒子分析仪26，27。最近的研究已经使用荧光标记的双磷酸盐探针（亚龙-氟辛/阿伦德龙酸盐-Cy5）来检查纳米晶体使用纳米级流细胞测量28。这种染料的限制是，它不是具体的，将结合几乎所有类型的石头，除了半胱氨酸。因此，准确评估个体中纳米晶体的存在可能是诊断结晶体和/或预测结石风险的有效工具。本研究的目的是利用纳米粒子跟踪分析（NTA）检测和量化含有纳米晶体的钙（大小为 <1微米）。为此，NTA 技术与含钙氟磷、氟-4 AM 相结合，用于检测和量化健康成年人尿液中含有纳米晶体的钙。

Protocol

这项工作中概述的所有实验都得到了阿拉巴马大学伯明翰分校（UAB）机构审查委员会的批准。健康成人（33.6±3.3岁;n=10）如果有正常的血液综合代谢板，非烟草使用者，非怀孕，体重指数在20-30公斤/米2之间，并且没有慢性疾病或急性疾病，则报名参加研究。健康参与者在研究开始前签署了书面知情同意书。 1. 临床协议和尿液收集 让参与者在收集尿液（24 小时?…

Representative Results

这项研究的结果表明，NTA可以有效地检测人类尿液中含有尿纳米晶体的钙的平均大小和浓度。这是通过使用氟磷、氟-4 AM 和纳米粒子跟踪分析实现的。Fluo-4 AM 能够与 CaOx 和 CaP 晶体结合。如图3A所示，CaOx晶体的大小确定在50-270纳米之间，平均浓度为1.26 x 109颗粒/mL。CaP晶体的大小在30-225纳米之间，平均浓度为2.22×109粒子/mL（图3B）。为了…

Discussion

NTA在本研究中使用钙结合探针Flo-4 AM来评估人类尿液中的纳米晶体。没有标准方法可以检测尿液中的纳米晶体。一些研究小组已经检测出尿液中的纳米晶体，并依靠使用广泛的协议或方法，这些协议或方法限制了他们量化样本的能力。这项研究展示了一种特定而敏感的方法，用于检测参与饮食喂养研究的人类尿液中含有纳米晶体的钙，该研究包括摄入高…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者感谢所有研究参与者和 UAB CCTS 生物营养核心和 UAB 高分辨率成像服务中心的贡献。这项工作得到了国家卫生研究院DK106284和DK123542（TM）和UL1TR003096（国家推进转化科学中心）的支持。

Materials

Benchtop Centrifuge Jouan Centrifuge CR3-12
Calcium Oxalate monohydrate Synthesized in the lab as previously described29. Store at RT; Stock 10 mM
Calcium Phosphate crystals (hydroxyapatite nanopowder) Sigma 677418 Store at RT; Stock 10 mM
Ethanol Fischer Scientific AC615095000 Store at RT; Stock 100%
Fluo-4 AM* AAT Bioquest, Inc. 20550 Store at Freezer (-20°C); Stock 5 mM
Gold Nanoparticles Sigma 742031 Store at 2-8°C
NanoSight Instrument Malvern Instruments, UK NS300
Syringe pump Harvard Apparatus 98-4730
Virkon Disinfectant LanXESS Energizing Company, Germany LSP
*Fluorescence dyes are light sensitive; stock and aliquots should be stored in the dark at -20°C.
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Tags

Keywords: Urinary NanocrystalsCalcium Fluorophore LabelingNanoparticle Tracking AnalysisKidney Stone FormationCrystal UreaOxalate LoadUrine PHUrine VolumeCalcium OxalateCalcium PhosphateFluo-4 AMGold NanoparticlesNanoparticle Tracking Analysis (NTA)
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Kumar, P., Bell, A., Mitchell, T. Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis. J. Vis. Exp. (168), e62192, doi:10.3791/62192 (2021).
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