Transient Cell Membrane Disruptions induce Calcium Waves in Corneal Keratocytes
Transient Cell Membrane Disruptions induce Calcium Waves in Corneal Keratocytes
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DOI：
10.1038/s41598-020-59570-7
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发表时间：
2020-02-18
影响因子：
4.6
通讯作者：
Watsky, Mitchell A.
中科院分区：
文献类型：
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作者：
Chen, Zhong;Lu, Xiaowen;Watsky, Mitchell A.
本研究的目的是确定单个角化细胞的短暂性细胞膜破坏（TPMDs）是否可以触发邻近角化细胞的信号事件。从人角膜（HCSC）和小鼠角膜（MCSC）中培养基质细胞。利用多光子显微镜在Cal-520-AM负载细胞中产生TPMDs。tpmd诱导的钙增加（Ca-i(++)）在含Ca++和不含Ca++的含有thapsigarin、ryanodine、BAPTA-AM、18- α -甘草次酸（18 α -GA）、apyrase、BCTC、AMG 9810或AMTB的溶液中测量。荧光强度记录为响应的细胞数量和荧光随时间曲线下的面积。测定了离体角膜邻近细胞的最大应答距离。采用免疫荧光染色法检测HCSC和MCSC中的Connexin 43蛋白，并采用角膜摩擦法确认机械操作后是否发生TPMDs。我们的研究结果表明，无论是在培养角膜还是离体角膜中，单细胞TPMDs都会在邻近的角化细胞中产生Ca++波。Ca++的来源包括细胞内和细胞外，信号可以通过ATP和/或间隙连接介导，并且依赖于物种。基质摩擦证实TPMDs确实发生在机械操作之后。角膜上皮TPMDs及其相关信号事件可能在轻微或严重角膜创伤后常见。
The purpose of this study was to determine if transient cell membrane disruptions (TPMDs) in single keratocytes can trigger signaling events in neighboring keratocytes. Stromal cells were cultured from human corneas (HCSC) and mouse corneas (MCSC). TPMDs were produced using a multiphoton microscope in Cal-520-AM loaded cells. TPMD-induced calcium increases (Ca-i(++)) were measured in Ca++-containing and Ca++-free solutions containing thapsigargin, ryanodine, BAPTA-AM, 18-alpha -glycyrrhetinic acid (18 alpha -GA), apyrase, BCTC, AMG 9810, or AMTB. Fluorescence intensity was recorded as the number of cells responding and the area under the fluorescence versus time curve. The maximum distance of responding neighboring cells in ex vivo human corneas was measured. Connexin 43 protein in HCSC and MCSC was examined using immunofluorescence staining, and corneal rubbing was applied to confirm whether TPMDs occur following mechanical manipulation. Our results demonstrate that single cell TPMDs result in Ca++ waves in neighboring keratocytes both in culture and within ex vivo corneas. The source of Ca++ is both intra-and extra-cellular, and the signal can be mediated by ATP and/or gap junctions, and is species dependent. Stromal rubbing confirmed that TPMDs do occur following mechanical manipulation. Keratocyte TPMDs and their associated signaling events are likely common occurrences following minor or major corneal trauma.