Restitution of single-cell defects in the mouse colon epithelium differs from that of cultured cells
Restitution of single-cell defects in the mouse colon epithelium differs from that of cultured cells
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DOI：
10.1152/ajpregu.00470.2005
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发表时间：
2006-06-01
影响因子：
2.8
通讯作者：
Gitter, AH
中科院分区：
文献类型：
--
作者：
Günzel, D;Florian, P;Gitter, AH
结肠上皮的完整性至关重要，并且由于小缺陷不断发生，因此快速修复（恢复）至关重要。为了探讨修复机制，通过离子导入Ca 2+在小鼠结肠表面上皮细胞中诱导单细胞损伤。使用共聚焦激光扫描显微镜（CLSM）和电生理技术的功能密封监测所产生的缺陷的关闭。缓解被评价为诱导泄漏的电导指数下降的时间常数τ，在对照条件下总计为0.28 min。4分钟后，泄漏完全密封。因此，修复比先前研究的HT-29/B6细胞快得多（tau = 5.73 min）。在培养的细胞中，细胞松弛素D延迟恢复天然结肠上皮细胞（tau = 0.69分钟），表明参与愈合过程中的肌动蛋白，但是，没有积累的肌动蛋白周围的病变被检测到。单独或与TNF-α组合的IFN-γ与上皮细胞的长期孵育分别将tau增加至0.49和0.59分钟。与培养的细胞相反，单独的TNF-α不影响恢复。短暂（< 10 min）暴露于溶血性E. coli O 4培养物不影响上皮的形态，但延迟恢复。在CLSM研究中，在损伤诱导开始后4分钟，缺陷仍然清晰可见。非溶血性E. coliO 4突变体则没有这种效应。总之，天然结肠中的单细胞缺陷导致功能性渗漏，其密封速度比细胞培养快。促炎细胞因子和致病菌延迟恢复。这表明非常小的病变在肠道致病过程开始时起着关键作用。
Integrity of colon epithelium is of crucial importance and, as small defects occur constantly, rapid repair (restitution) is essential. To investigate the mechanism of restitution, single-cell lesions were induced in mouse colonic surface epithelia by iontophoretic injection of Ca2+. Closure of the resulting defects was monitored using confocal laser scanning microscopy (CLSM), and functional sealing by electrophysiological techniques. Restitution was evaluated as the time constant tau of the exponential decrease in conductance of an induced leak and amounted to 0.28 min under control conditions. After 4 min, the leak was completely sealed. Repair was thus considerably faster than in previously investigated HT-29/B6 cells (tau = 5.73 min). As in cultured cells, cytochalasin D delayed restitution in native colon epithelia (tau = 0.69 min), indicating the involvement of actin in the healing process; however, no accumulation of actin surrounding the lesion was detected. Long-term incubation of epithelia with IFN-gamma alone or in combination with TNF-alpha increased tau to 0.49 and 0.59 min, respectively. In contrast to cultured cells, TNF-alpha alone did not affect restitution. A brief (< 10 min) exposure to the sterile filtered supernatant of hemolytic E. coli O4 cultures did not affect the morphology of the epithelium, but delayed restitution. In CLSM studies, defects were still clearly visible 4 min after the onset of lesion induction. The supernatant of a nonhemolytic E. coli O4 mutant did not exhibit this effect. In conclusion, single-cell defects in native colon cause functional leaks that seal faster than in cell cultures. Proinflammatory cytokines and pathogenic bacteria delay restitution. This suggests a key role of very small lesions at the onset of pathogenic processes in the intestine.