Department of Wound Repair and Dermatologic Surgery, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, China.
Hubei Clinical Medical Research Center of Cord Blood Hematopoietic Stem Cells, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, China.
Chin Med J (Engl). 2022 Jan 12;135(3):324-332. doi: 10.1097/CM9.0000000000001936.
Sweat secreted by eccrine sweat glands is transported to the skin surface through the lumen. The eccrine sweat gland develops from the initial solid bud to the final gland structure with a lumen, but how the lumen is formed and the mechanism of lumen formation have not yet been fully elucidated. This study aimed to investigate the mechanism of lumen formation of eccrine gland organoids (EGOs).
Human eccrine sweat glands were isolated from the skin for tissue culture, and the primary cultured cells were collected and cultured in Matrigel for 14 days in vitro. EGOs at different development days were collected for hematoxylin and eosin (H&E) staining to observe morphological changes and for immunofluorescence staining of proliferation marker Ki67, cellular motility marker filamentous actin (F-actin), and autophagy marker LC3B. Western blotting was used to detect the expression of Ki67, F-actin, and LC3B. Moreover, apoptosis was detected using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay kit, and the expression of poly (ADP-ribose) polymerase and Caspase-3 was detected by Western blot. In addition, 3-methyladenine (3MA) was used as an autophagy inhibitor to detect whether the formation of sweat glands can be effectively inhibited.
The results showed that a single gland cell proliferated rapidly and formed EGOs on day 4. The earliest lumen formation was observed on day 6. From day 8 to day 14, the rate of lumen formation in EGOs increased significantly. The immunofluorescence and Western blot analyses showed that the expression of Ki67 gradually decreased with the increase in days, while the F-actin expression level did not change. Notably, the expression of autophagy marker LC3B was detected in the interior cells of EGOs as the apoptosis signal of EGOs was negative. Compared with the control group, the autophagy inhibitor 3MA can effectively limit the formation rate of the lumen and reduce the inner diameter of EGOs.
Using our model of eccrine gland 3D-reconstruction in Matrigel, we determined that autophagy rather than apoptosis plays a role in the lumen formation of EGOs.
外泌汗腺分泌的汗液通过腔道运输到皮肤表面。外泌汗腺从初始的实心芽发育到最终具有腔道的腺体结构，但腔道是如何形成的以及腔道形成的机制尚未完全阐明。本研究旨在探讨外泌腺类器官（EGO）腔道形成的机制。
从皮肤中分离出人外泌汗腺进行组织培养，收集原代培养的细胞，在 Matrigel 中培养 14 天。收集不同发育天数的 EGO 进行苏木精和伊红（H&E）染色，观察形态变化，并用增殖标志物 Ki67、细胞运动标志物丝状肌动蛋白（F-actin）和自噬标志物 LC3B 进行免疫荧光染色。Western blot 检测 Ki67、F-actin 和 LC3B 的表达。此外，使用末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）凋亡检测试剂盒检测细胞凋亡，Western blot 检测多聚（ADP-核糖）聚合酶和 Caspase-3 的表达。此外，使用 3-甲基腺嘌呤（3MA）作为自噬抑制剂，检测是否能有效抑制腺体的形成。
结果表明，单个腺细胞快速增殖，第 4 天形成 EGO。最早观察到第 6 天出现腔道形成。第 8 天至第 14 天，EGO 中腔道形成的速度明显增加。免疫荧光和 Western blot 分析表明，Ki67 的表达随天数的增加逐渐降低，而 F-actin 的表达水平没有变化。值得注意的是，在 EGO 内部细胞中检测到自噬标志物 LC3B，因为 EGO 的凋亡信号为阴性。与对照组相比，自噬抑制剂 3MA 可有效限制腔道形成率并减小 EGO 的内径。
利用我们在 Matrigel 中重建外泌腺 3D 结构的模型，我们确定自噬而非凋亡在 EGO 腔道形成中起作用。
Zotero 插件
