Department of Otorhinolaryngology, Jikei University School of Medicine, 3-25-8 Nishi-Shinbashi, Minato-ku, Tokyo, 105-8461, Japan.
Sci Rep. 2020 Oct 8;10(1):16853. doi: 10.1038/s41598-020-73817-3.
The proliferation and differentiation of cultured epithelial cells may be modified by Rho-associated kinase (ROCK) inhibition and extracellular Ca concentration. However, it was not known whether a combination would influence the behavior of cultured epithelial cells through changes in the phosphorylation of non-muscle myosin light chain II (MLC). Here we show that the combination of ROCK inhibition with Ca elevation regulated the phosphorylation of MLC and improved both cell expansion and cell-cell adhesion during the culture of human nasal mucosal epithelial cell sheets. During explant culture, Ca enhanced the adhesion of nasal mucosal tissue, while ROCK inhibition downregulated MLC phosphorylation and promoted cell proliferation. During cell sheet culture, an elevation of extracellular Ca promoted MLC phosphorylation and formation of cell-cell junctions, allowing the harvesting of cell sheets without collapse. Moreover, an in vitro grafting assay revealed that ROCK inhibition increased the expansion of cell sheets three-fold (an effect maintained when Ca was also elevated), implying better wound healing potential. We suggest that combining ROCK inhibition with elevation of Ca could facilitate the fabrication of many types of cell graft.
培养的上皮细胞的增殖和分化可以通过 Rho 相关激酶 (ROCK) 抑制和细胞外 Ca 浓度来调节。然而，尚不清楚通过改变非肌肉肌球蛋白轻链 II (MLC) 的磷酸化，组合是否会通过改变非肌肉肌球蛋白轻链 II (MLC) 的磷酸化来影响培养的上皮细胞的行为。在这里，我们表明 ROCK 抑制与 Ca 升高的组合调节 MLC 的磷酸化，并在人鼻黏膜上皮细胞片的培养过程中改善细胞扩张和细胞间黏附。在组织块培养过程中，Ca 增强了鼻黏膜组织的黏附性，而 ROCK 抑制下调了 MLC 的磷酸化并促进了细胞增殖。在细胞片培养过程中，细胞外 Ca 的升高促进了 MLC 的磷酸化和细胞间连接的形成，使细胞片在不塌陷的情况下被收获。此外，体外移植实验表明，ROCK 抑制使细胞片的扩张增加了三倍（当 Ca 也升高时，这种作用得以维持），这意味着具有更好的伤口愈合潜力。我们认为，将 ROCK 抑制与 Ca 升高相结合可以促进多种类型的细胞移植物的制造。
