Medicine
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Chapters
Summary June 9th, 2021
Please note that all translations are automatically generated.
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这项研究旨在开发一种新的人体组织性视网膜培养（HORC）模型，防止在外植过程中损害视网膜的完整性。这是通过培养视网膜与过度的四肢和底层视网膜色素上皮-胆碱（RPE-胆碱）和硬膜。
Transcript
该技术保留了所有视网膜层和细胞类型原位，使其在临床上与动物和体外模型相比更加相关。此协议的主要优点是，它最大限度地减少了组织处理过程中视网膜完整性中断，这在比较健康和患病视网膜时至关重要。视网膜疾病可以通过在特定条件下培养组织来模仿。
这允许测试新的药物疗法。解剖和样品采集过程很复杂。因此，视觉演示对于帮助读者理解书面协议并成功执行程序至关重要。
演示这个程序的将是来自我实验室的二年级博士生郭查里斯。准备含有杜尔贝科改性鹰中/营养混合物F-12和抗生素和抗菌混合物的培养基。首先，将500微升的介质物放到24井的盘子中，在37摄氏度和5%的二氧化碳的加湿孵化器中平衡。
在外生提取之前准备好介质，避免在随后添加视网膜时脱落。要提取视网膜外植物，首先将眼罩放在培养皿上，虹膜和镜头朝上，ONH 与培养皿接触。用钳子将眼罩稳定在豪华巴士上，沿着利姆布斯的外缘进行小切口，从而分离虹膜和镜头。
小心地取出虹膜和透镜，确保避免干扰视网膜。使用明亮的白光源识别 ONH，并在四个象限处向 ONH 旋转培养皿，以便于处理。小心地铺开和压平眼杯。
将钳子涂抹在硬膜上，而不是视网膜上，以避免破坏视网膜的完整性。从孵化器中取出包含预制介质的板。在没有视网膜褶皱的区域，在视网膜上放置手术颤音，然后用力压入硬盘，当您穿过培养皿时，应会发出裂开的声音。
旋转肌酸，以确保硬化已经完全穿透，使视网膜除植物现在从样品的其余部分分离。将钳子涂抹在硬囊上，将三明治视网膜除植物转移到培养介质中。在加湿的 5% 二氧化碳孵化器中，在 37 摄氏度下培养收集的三明治视网膜外植，持续长达 72 小时。
血氧林和欧辛染色表明，三明治视网膜外植保持完整性和独特的跛脚菌结构，从GCL到 ONL，内核和外核层具有紧凑的核。在同一样本的边缘发现视网膜的完整性中断，视网膜已脱离底层RPE胆碱和硬膜，显示在 INL 和 ONL 中视网膜厚度降低和核损失。在基础条件下培养的视网膜外植中，没有发现标志着细胞凋亡的 TUNEL 阳性细胞核，即使在 72 小时后也显示出持续的细胞活力。
GFAP 标签仅限于 GCL 和 IPL，没有胶质纤维化，这是病理学的标志，将被确定为 GFAP 在 ONL 中的扩展表达。维门汀从结节细胞层到外核层都得到了高度的表达，在正常的视网膜组织中可以看到，它显示了穆勒细胞的完整性。Luminex磁性检测表明，IL-18、VEGF、IL-6和IL-8水平在24小时后显著高于高葡萄糖和促炎细胞因子中三明治视网膜除菌培养物的基线。
72小时后，IL-18 和 VEGF 水平仍显著提高，但在 IL-6 和 IL-8 级别中未发现显著差异。执行此程序时，留下足够的残留玻璃，这将权衡视网膜下来，使其不脱离RP胆囊，并漂浮在文化媒体。培养视网膜可以使用免疫造血化学和组织学进一步描述，而释放到培养介质中的炎症细胞因子可以使用Luminex磁性检测来测量。
与动物和体外模型相比，该模型在临床上更易翻译，更适合于测试视网膜疾病新疗法的疗效。
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