Blisters have previously been observed in keratinocyte cultures depleted of vitamin A, and in cultures of keratinocytes from patients with epidermolysis bullosa. We have found that blistering may occur in keratinocyte cultures from normal human epidermis, grown under standard conditions, and our aim was to further characterize the mechanism of blister formation. Keratinocytes were seeded at 10(5) cells per 35 mm collagen-coated dish with a 3T3 feeder layer. Blisters were macroscopic, fluid-filled structures which formed irrespective of donor site, or donor age, and were noted on various alternative substrates (collagen, 3T3 + plastic, plastic alone). Blistering commenced around day 12, prior to confluency, and new blisters were formed for up to 5 weeks post-plating. Maximal numbers (up to 70 per dish) were present around days 12 to 20. Cleavage occurred at the cell/collagen interface to form a blister roof composed of 6 to 9 cell layers. The lowest layer appeared metabolically active, but, in contrast to peri-blister regions, lacked hemidesmosomes. The central 2 to 3 layers contained membrane-coating granules and keratohyalin granules while the superficial strata resembled rudimentary corneocytes. Cultures supplemented with 10(-5) M vitamin A formed no blisters, which correlated with suppressed differentiation. Ouabain (10(-7) M) caused blister collapse and a reversible inhibition of new blister formation. We conclude that blisters are a consistent finding in keratinocyte cultures grown under standard conditions. Their formation may be associated with active transport and triggered during differentiation. Further examination of this phenomenon might shed light on whether differentiation itself has an influence on keratinocyte attachment to substrate.

译文

:以前在缺乏维生素A的角质形成细胞培养物中以及在大疱性表皮松解症患者的角质形成细胞培养物中观察到水泡。我们发现在正常条件下生长的正常人表皮的角质形成细胞培养物中可能会出现水泡,我们的目标是进一步表征水泡形成的机制。角质形成细胞以每35毫米带有3T3饲养层的胶原涂层培养皿中的10(5)个细胞接种。水泡是宏观的,充满液体的结构,与供体部位或供体年龄无关而形成,并且在各种替代性底物(胶原蛋白,3T3塑料,仅塑料)上被注意到。在融合前的第12天左右开始起泡,并在接种后长达5周的时间内形成新的水泡。在第12至20天左右出现最大数量(每个培养皿最多70个)。在细胞/胶原蛋白界面发生裂解,形成由6至9个细胞层组成的水泡屋顶。最低层表现出代谢活性,但与水泡周围区域相比,缺乏半桥粒。中央的2至3层包含膜包衣颗粒和角膜透明质酸颗粒,而表层则类似于原始角质细胞。补充有10(-5)M维生素A的培养物未形成水泡,这与抑制分化相关。瓦巴因(10(-7)M)引起水泡塌陷和新水泡形成的可逆抑制。我们得出结论,水泡是在标准条件下生长的角质形成细胞培养物中的一致发现。它们的形成可能与主动转运有关,并在分化过程中被触发。对该现象的进一步检查可能会揭示分化本身是否会影响角质形成细胞对基质的附着。

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