Arrowsmith Sarah, Keov Peter, Muttenthaler Markus, Gruber Christian W
Harris-Wellbeing Preterm Birth Research Centre, Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool;
School of Biomedical Sciences, The University of Queensland.
J Vis Exp. 2018 Jan 26(131):56639. doi: 10.3791/56639.
Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We describe methods to measure ex vivo myometrium contractility. This assay can be used to investigate factors and molecules involved in the modulation of myometrial contraction and to determine their excitatory or inhibitory actions, and hence their therapeutic potential in vivo. Biopsies are obtained from women undergoing cesarean section delivery with informed consent. Fine strips of myometrium are dissected, clipped and attached to a force transducer within 1 mL organ baths superfused with physiological saline solution at 37 °C. Strips develop spontaneous contractions within 2-3 h under set tension and remain stable for many hours (>6 h). Strips can also be stimulated to contract such as by the endogenous hormones, oxytocin and vasopressin, which cause concentration-dependent modulation of contraction frequency, force and duration, to more closely resemble contractions in labor. Hence, the effect of known and novel drug leads can be tested on spontaneous and agonist-induced contractions. This protocol specifically details how this assay can be used to determine the potency of known and novel agents by measuring their effects on various parameters of human myometrial contraction. We use the oxytocin- and V1a receptor antagonists, atosiban and SR49059 as examples of known compounds which inhibit oxytocin- and vasopressin-induced contractions, and demonstrate how this method can be used to complement and validate pharmacological data obtained from cell-based assays to aid drug development. The effects of novel agonists in comparison to oxytocin and vasopressin can also be characterized. Whilst we use the example of the oxytocin/ vasopressin system, this method can also be used to study other receptors and ion channels that play a role in uterine contraction and relaxation to advance the understanding of human uterine physiology and pathophysiology.
新型药物化合物或生化探针的发现与特性研究依赖于强大且与生理相关的检测系统。我们描述了测量离体子宫肌层收缩性的方法。该检测可用于研究参与子宫肌层收缩调节的因素和分子，并确定它们的兴奋或抑制作用，从而确定其在体内的治疗潜力。在获得知情同意后，从接受剖宫产的女性身上获取活检组织。将子宫肌层的细条剥离、剪断，并在37℃下用生理盐水灌注的1 mL器官浴槽中连接到力传感器上。肌条在设定张力下2 - 3小时内会出现自发收缩，并在数小时（>6小时）内保持稳定。肌条也可被刺激收缩，如通过内源性激素催产素和血管加压素，它们会引起收缩频率、力量和持续时间的浓度依赖性调节，更类似于分娩时的收缩。因此，可以测试已知和新型药物先导物对自发和激动剂诱导收缩的影响。本方案详细说明了如何通过测量已知和新型药物对人子宫肌层收缩各种参数的影响来确定其效力。我们以催产素和V1a受体拮抗剂阿托西班和SR49059为例，它们是抑制催产素和血管加压素诱导收缩的已知化合物，并展示了该方法如何用于补充和验证从基于细胞的检测中获得的药理学数据，以辅助药物开发。与催产素和血管加压素相比，新型激动剂的作用也可以得到表征。虽然我们以催产素/血管加压素系统为例，但该方法也可用于研究在子宫收缩和舒张中起作用的其他受体和离子通道，以推进对人类子宫生理学和病理生理学的理解。
