Pain Management Research Institute and Kolling Institute, University of Sydney at Royal North Shore Hospital, St. Leonards, New South Wales, Australia.
J Urol. 2013 Aug;190(2):737-45. doi: 10.1016/j.juro.2013.01.048. Epub 2013 Jan 23.
Recent evidence suggests that the urothelium functions as a sensory transducer of chemical, mechanical or thermal stimuli and signals to nerve terminals and other cells in the bladder wall. The cellular and molecular basis of neuro-urothelial communication is not easily studied in the intact bladder. This led us to establish a method of co-culturing dorsal root ganglion sensory neurons and bladder urothelial cells.
Sensory neurons and urothelial cells obtained from dorsal root ganglia and bladders dissected from adult female Sprague-Dawley® rats were isolated by enzyme treatment and mechanical dissociation. They were plated together or separately on collagen coated substrate and cultured in keratinocyte medium for 48 to 72 hours. Retrograde tracer labeling was performed to identify bladder afferents used for functional testing.
Neurite growth and complexity in neurons co-cultured with urothelial cells was increased relative to that in neuronal monocultures. The growth promoting effect of urothelial cells was reduced by the tyrosine kinase inhibitor K252a but upstream inhibition of nerve growth factor signaling with TrkA-Fc had no effect. Fura-2 calcium imaging of urothelial cells showed responses to adenosine triphosphate (100 μM) and activation of TRPV4 (4α-PDD, 10 μM) but not TRPV1 (capsaicin, 1 μM), TRPV3 (farnesyl pyrophosphate, 1 μM) or TRPA1 (mustard oil, 100 μM). In contrast, co-cultured neurons were activated by all agonists except farnesyl pyrophosphate.
Co-culturing provides a new methodology for investigating neuro-urothelial interactions in animal models of urological conditions. Results suggest that neuronal properties are maintained in the presence of urothelium and neurite growth is potentiated by a nerve growth factor independent mechanism.
最近的证据表明，尿路上皮作为化学、机械或热刺激的感觉转导器发挥作用，并向膀胱壁中的神经末梢和其他细胞发出信号。在完整的膀胱中，神经-尿路上皮通讯的细胞和分子基础不易研究。这促使我们建立了一种共培养背根神经节感觉神经元和膀胱尿路上皮细胞的方法。
通过酶处理和机械解离从成年雌性 Sprague-Dawley®大鼠的背根神经节和膀胱中分离感觉神经元和尿路上皮细胞。将它们一起或分别铺在胶原蛋白包被的基质上，并在角质形成细胞培养基中培养 48 至 72 小时。进行逆行示踪标记以鉴定用于功能测试的膀胱传入神经。
与神经元单培养相比，与尿路上皮细胞共培养的神经元的轴突生长和复杂性增加。尿路上皮细胞的促生长作用被酪氨酸激酶抑制剂 K252a 减弱，但神经生长因子信号的上游抑制 TrkA-Fc 没有影响。尿路上皮细胞的 Fura-2 钙成像显示对三磷酸腺苷（100μM）和 TRPV4（4α-PDD，10μM）的激活有反应，但对 TRPV1（辣椒素，1μM）、TRPV3（法呢基焦磷酸，1μM）或 TRPA1（芥末油，100μM）没有反应。相比之下，共培养的神经元除法呢基焦磷酸外，对所有激动剂均有反应。
共培养为研究动物模型中尿路上皮与神经相互作用提供了一种新方法。结果表明，神经元特性在存在尿路上皮的情况下得以维持，并且轴突生长通过一种独立于神经生长因子的机制得到增强。
