Spanakis S G, Petridou S, Masur S K
Department of Ophthalmology, Mount Sinai School of Medicine of the City University of New York, NY 10029-6574, USA.
Invest Ophthalmol Vis Sci. 1998 Jul;39(8):1320-8.
Within the corneal stroma, keratocytes communicate through gap junctions. These plasma membrane channels, which connect the cytoplasm of adjacent cells, are composed of connexins. In a cell culture model, an investigation was conducted to determine whether connexin-based gap junction intercellular communication is present in fibroblasts and myofibroblasts, both of which replace keratocytes after wounding.
Fibroblasts and myofibroblasts were grown according to preestablished methods. Phenotype was determined by immunocytochemistry. A gap junction-permeant dye, Lucifer yellow or Cascade blue, and nonpermeant 10-kDa Texas red-dextran were used. Tracer fluorescent dyes were introduced by scrape-loading or by microinjection, and their diffusion into adjacent cells was recorded photographically. Inhibition of gap junction dye transfer was elicited by treatment with 18-alpha-glycyrrhetinic acid (AGA).
In confluent fibroblast or myofibroblast cultures, the scrape-loaded dextran probe remained within wounded cells, whereas the Lucifer yellow or Cascade blue dye diffused into adjacent intact cells. Similarly, in nonconfluent fibroblast and myofibroblast cultures, microinjected Lucifer yellow rapidly diffused from the microinjected cell to adjacent cells. Treatment with 2 microM AGA, an uncoupling agent, blocked the spread of Lucifer yellow in fibroblast and myofibroblast cultures.
Cultured fibroblasts and myofibroblasts have functional gap junctions as has previously been demonstrated for keratocytes in vivo. Thus, fibroblasts and myofibroblasts have the ability to establish and maintain intercellular communication with themselves and with nonactivated keratocytes. This property may be critical in the wound-healing process, especially in the avascular corneal environment.
在角膜基质中，角膜细胞通过缝隙连接进行通讯。这些连接相邻细胞胞质的质膜通道由连接蛋白组成。在细胞培养模型中，进行了一项研究以确定基于连接蛋白的缝隙连接细胞间通讯是否存在于成纤维细胞和肌成纤维细胞中，这两种细胞在受伤后都会取代角膜细胞。
成纤维细胞和肌成纤维细胞按照既定方法培养。通过免疫细胞化学确定细胞表型。使用了一种可透过缝隙连接的染料，荧光素黄或级联蓝，以及不可透过的10 kDa德克萨斯红 - 葡聚糖。通过刮擦加载或显微注射引入示踪荧光染料，并通过摄影记录它们向相邻细胞的扩散。用18-α-甘草次酸（AGA）处理可抑制缝隙连接染料转移。
在汇合的成纤维细胞或肌成纤维细胞培养物中，刮擦加载的葡聚糖探针保留在受伤细胞内，而荧光素黄或级联蓝染料扩散到相邻的完整细胞中。同样，在未汇合的成纤维细胞和肌成纤维细胞培养物中，显微注射的荧光素黄迅速从显微注射的细胞扩散到相邻细胞。用2 microM AGA（一种解偶联剂）处理可阻断荧光素黄在成纤维细胞和肌成纤维细胞培养物中的扩散。
培养的成纤维细胞和肌成纤维细胞具有功能性缝隙连接，这与之前在体内角膜细胞中所证明的情况相同。因此，成纤维细胞和肌成纤维细胞有能力与自身以及未活化的角膜细胞建立并维持细胞间通讯。这种特性在伤口愈合过程中可能至关重要，尤其是在无血管的角膜环境中。
