人体胎盘生理学综合研究的4血管取样方法<em>体内</em
Summary
我们提出了一种在体内研究人胎盘生理学的详细方法。该方法将来自胎盘和母胎侧的进出血管的血液采样与体积血流和胎盘组织取样的超声测量相结合。
Abstract
人胎盘在子宫内仍然无法进行研究。因此，目前对人类胎盘生理学的了解在很大程度上取决于动物研究，尽管胎盘解剖学中物种的多样性，血液动力学和妊娠持续时间。绝大多数人胎盘研究是离体灌注研究或体外滋养层研究。虽然体外研究和动物模型是必不可少的，但是将这些研究结果外推到体内的人类胎盘是不确定的。我们的目的是在体内研究人胎盘生理学，并提出该方法的详细方案。在计划剖宫产期间，利用子宫内膜的子宫静脉进入子宫静脉，我们从胎盘母体和胎侧的进出血管中收集血样。组合时从体积血流测量的血液样本中心测量，我们能够量化胎盘和胎儿摄取和任何化合物的释放。此外，来自相同母胎胎系的胎盘组织样本可以提供运动员密度和活性以及体内胎盘功能的其它方面的测量。通过这种4血管采样方法的综合使用，我们能够在正常和病理性怀孕中测试体内胎盘营养转移和代谢的一些现有概念。此外，该方法能够将胎盘分泌的物质鉴定为母体循环，这可能是对胎盘功能障碍生物标志物的检索的重要贡献。
Introduction
根据卫生，美国全国学院，胎盘是在人体1，2，3中的至少理解器官。 在体内不易对正在进行的怀孕造成不道德的风险进行人体胎盘的访问和研究是困难的。因此，人体胎盘功能的研究主要是基于体外和离体模型。多数在胎盘转运和代谢的体内研究以前已经在动物4,5被执行，6。然而，由于胎盘结构和功能在物种间有很大差异，所以必须谨慎地将动物的结果外推至人体。只有少数较小的人体内研究已经调查了在正常生理学下的胎盘和胎儿摄取和运输人的条件下，并且没有已经探索几种化合物7，8，9，10，11，12，13的集成传输。这些基础研究表明，人胎盘的体内研究是可行的，并且它们可以用于多种目的。首先，主要来自体外 ， 体外和动物研究的胎盘功能的现有概念可以在人的环境中进行测试，从而提供对人胎盘的新颖和更具体的洞察。其次，与异常胎儿生长，先兆子痫，母亲糖尿病，代谢综合征和其他母体代谢紊乱相关的功能障碍性胎盘的性质可以更好地表征。第三，人体内研究提供了开发诊断的机会tic和胎盘功能的预测工具。
在这个背景下，我们旨在建立一个全面的生理数据收集，以调查体内人胎盘功能。在计划剖宫产期间，我们利用腹腔内进入子宫静脉从胎盘的母胎和胎侧的进出血管收集血液样品（4血管采样方法）。这些样品用于计算营养物质和其他物质的成对动静脉浓度差异14 。此外，我们通过超声测量胎盘两侧的体积血流量。因此，可以量化任何化合物的胎盘和胎儿摄取。此外，能够确定由胎盘释放到母体和胎儿循环15，16，17的物质。结合时20d与母亲和儿童的临床参数，以及胎盘的分析和其他相关组织，这种方法具有令人兴奋的潜力，在同一个母亲，胎儿对胎盘功能的许多方面集成在体内 。
Protocol
该研究由奥斯陆大学医院数据保护官员和挪威南部卫生研究伦理学区域委员会2419/2011批准。所有参与者签署书面知情同意书。 准备工作注意：程序的时间表如图1所示 。 图1 ： 描述涉及4血管采样程序的时间和人员的流程?…
Representative Results
4血管采样方法适用于临床实践，我们成功获得了209例母亲/婴儿对的血液样本。其中128个我们也实现了测量体积血流量。在70个母胎胎系中获得了完整的4血管采样和母体和胎儿血管的良好质量流量测量（ 图3 ）。此外，我们迄今已收集了30名先兆子痫患者的血液和胎盘样品。之前我们已经发表了关于营养成分的人胎盘转移，以及血管活性胎盘因素释?…
Discussion
胎盘4血管采样方法有三个主要目的。首先，我们的葡萄糖和氨基酸研究证明，它可以用于研究胎盘在母体一侧如何摄取特定物质，并可能转移到脐带循环和胎儿。第二，该方法与研究胎盘产生的物质高度相关，并释放到母体或胎儿循环中，如黄体酮结果所示。第三，研究胎儿体内如何在快速生长和组织重塑过程中消除废物可能是有用的。
4血管方法的关键步…
Divulgations
The authors have nothing to disclose.
Acknowledgements
首先，我们衷心感谢参与这个项目的母亲。接下来，我们承认协助和促进抽样程序的所有人员，麻醉医师，护士麻醉师和外科护士。如果没有挪威东南部挪威地区卫生局和挪威妇女健康问题咨询组，奥斯陆大学以及奥斯陆大学医院提供的当地资金，该项目就不可能实现。
Materials
| Maternal body composition | |||
| Impedance scale | Tanita | or similar | |
| Ultrasound measurements | |||
| Sequoia 512 ultrasound machine | Acuson | equipped with a curved transducer with colour and pulsewave Doppler (frequency bandwidth 2-6 MHz) | |
| Blood samples | |||
| Arerial cannula | BD Medical | 682245 | or similar |
| 20cc Eccentric Luer Tip Syringe without Needle | Termo | SS-20ES | or similar. 3 needed. |
| 10cc Eccentric Luer Tip Syringe without Needle | Termo | SS-10ES | or similar. 9 needed. |
| 5cc 6% Luer Syringe without Needle | Termo | SS-05S1 | or similar. 2 needed. |
| Arterial blood gas syringe | Radiometer Medical | or similar. 4 needed. | |
| Sterile winged needle connected to flexible tubing, 21 gauge | Greiner Bio-One | 450081 | (intended for single use).3 needed. |
| Vacutainer tube 6 mL EDTA | Greiner Bio-One | 456043 | or similar. Label with sample site. 10 needed. |
| Vacutainer tube 5 ml LH Lithium Heparin Separator | Greiner Bio-One | 456305 | or similar. Label with sample site. 5 needed. |
| Vacutainer tube 6 mL Serum Clot Activator | Greiner Bio-One | 456089 | or similar. Label with sample site. 5 needed. |
| Vacutainer tube 3 ml 9NC Coagulation sodium citrate 3,2% | Greiner Bio-One | 454334 | or similar. Label with sample site. 5 needed. |
| Cryogenic vials, 2.0 mL | Corning | 430488 | or similar. Label with sample site, serum/type of plasma and ID. 90 needed. |
| Marked trays to transport the syringes | to transport the blood samples in the operation theatre | ||
| Rocker | for gentle mixing of the samples | ||
| Ice | in styrofoam box | ||
| Liquid nitrogen | in appropriate container | ||
| Placenta samples | |||
| Metal tray | |||
| Ice | in styrofoam box | ||
| Calibrated scale | |||
| Metal ruler | |||
| 1 M Phosphate buffered saline | Sigma | D1408 | or similar. Dilute 10 M to 1M before use |
| RNA stabilization solution | Sigma | R0901-500ML | or similar |
| Optimal Cutting Temperature (O.C.T.) compound | vwr | 361603E | or similar |
| Cryogenic vials, 2.0 mL | Corning | 430488 | or similar. Label with sample site. content and ID. 10 needed. |
| Centrifuge tubes, conical bottom 50 mL | Greiner Bio-One | 227,285 | or similar. Label with "RNA later", sample site and ID. 2 needed. |
| Liquid nitrogen | in appropriate container | ||
| Fetal body composition | |||
| Calibrated scale | |||
| Measuring tape |
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Citer Cet Article
Holme, A. M., Holm, M. B., Roland, M. C. P., Horne, H., Michelsen, T. M., Haugen, G., Henriksen, T. The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo. J. Vis. Exp. (126), e55847, doi:10.3791/55847 (2017).