Department of Dermatology, Harvard Medical School, Boston, MA, USA.
Wellman Center for Photomedicine, Boston, MA, USA.
Sci Rep. 2017 Apr 3;8:45789. doi: 10.1038/srep45789.
A model of neutrophil migration across epithelia is desirable to interrogate the underlying mechanisms of neutrophilic breach of mucosal barriers. A co-culture system consisting of a polarized mucosal epithelium and human neutrophils can provide a versatile model of trans-epithelial migration in vitro, but observations are typically limited to quantification of migrated neutrophils by myeloperoxidase correlation, a destructive assay that precludes direct longitudinal study. Our laboratory has recently developed a new isotropic 1-μm resolution optical imaging technique termed micro-optical coherence tomography (μOCT) that enables 4D (x,y,z,t) visualization of neutrophils in the co-culture environment. By applying μOCT to the trans-epithelial migration model, we can robustly monitor the spatial distribution as well as the quantity of neutrophils chemotactically crossing the epithelial boundary over time. Here, we demonstrate the imaging and quantitative migration results of our system as applied to neutrophils migrating across intestinal epithelia in response to a chemoattractant. We also demonstrate that perturbation of a key molecular event known to be critical for effective neutrophil trans-epithelial migration (CD18 engagement) substantially impacts this process both qualitatively and quantitatively.
需要建立一个中性粒细胞穿过上皮细胞的模型来探究中性粒细胞突破黏膜屏障的潜在机制。由极化的黏膜上皮细胞和人中性粒细胞组成的共培养系统可以提供体外跨上皮细胞迁移的多功能模型，但观察结果通常仅限于通过髓过氧化物酶相关性来定量迁移的中性粒细胞，这种破坏性测定方法排除了直接的纵向研究。我们实验室最近开发了一种新的各向同性 1μm 分辨率光学成像技术，称为微光学相干断层扫描（μOCT），可以实现共培养环境中中性粒细胞的 4D（x，y，z，t）可视化。通过将 μOCT 应用于跨上皮细胞迁移模型，我们可以稳健地监测中性粒细胞在趋化刺激下穿过上皮边界的空间分布和数量随时间的变化。在这里，我们展示了我们的系统在应用于响应趋化因子穿过肠上皮的中性粒细胞时的成像和定量迁移结果。我们还证明了，已知对有效中性粒细胞跨上皮迁移至关重要的关键分子事件（CD18 结合）的干扰会显著影响该过程的质量和数量。
