Stenn K S, Core N G
In Vitro Cell Dev Biol. 1986 Apr;22(4):217-22. doi: 10.1007/BF02623306.
To understand the earliest phases of epidermal cell spreading we have sought a defined in vitro system. We studied the divalent cation dependence of guinea pig epidermal cell spreading in media containing varying concentrations of cations. No spreading occurred in calcium-magnesium-free Dulbecco's modified Eagle's medium (CMF-DME) in the presence of cation-free fetal bovine serum; however, significant spreading occurred if the medium was supplemented with Mg++ plus Ca++ or Mg++ alone. Supplementing with Ca++ alone led to much less spreading. These cations in CMF-DME did not support spreading in the absence of serum or the presence of serum albumin. Assaying cell spreading in a simple salt solution consisting of NaCl, KCl, Tris buffer, pH 7.4 plus dialyzed serum and a series of divalent cation supplements (Ca++, Mg++, Mn++, Co++, Zn++, Ni++), showed that only Mg++ and Mn++, and to a lesser extent, Ca++, supported cell spreading. In contrast to Mg++, however, Mn++ could support spreading in the absence of whole serum if serum albumin were present. Although Mn++ plus serum albumin supported more rapid spreading at lower cation concentrations than Mg++ plus serum, equal concentrations of Ca++ completely blocked the Mn++ effect. In contrast to the increasing cell spreading, which occurred in Mg++-containing medium with time, cell death occurred in Mn++-containing medium by 24 h. Consonant with studies from other laboratories, human foreskin fibroblasts spread in Mn++-containing salt solution in the absence of protein supplements. These experiments indicate for epidermal cell spreading that Mg++ is the important cation in tissue culture media, that under proper cation conditions epidermal cells do not need a specific spreading protein (i.e. a protein that has been demonstrated to support cell spreading), that Mn++ and Mg++-induced spreading seem to represent different mechanisms, that fibroblastic and epidermal cells have different cation requirements for in vitro spreading, and that the crucial role cations play in cell spreading remains to be elucidated.
为了解表皮细胞铺展的最早阶段，我们一直在寻找一种明确的体外系统。我们研究了豚鼠表皮细胞在含有不同阳离子浓度的培养基中铺展对二价阳离子的依赖性。在无钙镁的杜尔贝科改良伊格尔培养基（CMF-DME）中，加入无阳离子的胎牛血清时，细胞不会铺展；然而，如果培养基补充Mg++加Ca++或仅补充Mg++，则会出现明显的铺展。仅补充Ca++时，铺展程度要小得多。在无血清或存在血清白蛋白的情况下，CMF-DME中的这些阳离子不支持细胞铺展。在由NaCl、KCl、pH 7.4的Tris缓冲液、透析血清和一系列二价阳离子补充剂（Ca++、Mg++、Mn++、Co++、Zn++、Ni++）组成的简单盐溶液中检测细胞铺展，结果表明只有Mg++和Mn++，以及程度较小的Ca++支持细胞铺展。然而，与Mg++不同的是，如果存在血清白蛋白，Mn++可以在无全血清的情况下支持细胞铺展。尽管Mn++加血清白蛋白在较低阳离子浓度下比Mg++加血清支持更快的铺展，但相同浓度的Ca++完全阻断了Mn++的作用。与含Mg++的培养基中细胞铺展随时间增加相反，含Mn++的培养基中细胞在24小时内死亡。与其他实验室的研究一致，人包皮成纤维细胞在无蛋白质补充剂的含Mn++的盐溶液中铺展。这些实验表明，对于表皮细胞铺展来说，Mg++是组织培养基中的重要阳离子，在适当的阳离子条件下，表皮细胞不需要特定的铺展蛋白（即已被证明支持细胞铺展的蛋白），Mn++和Mg++诱导的铺展似乎代表不同的机制，成纤维细胞和表皮细胞在体外铺展时有不同的阳离子需求，阳离子在细胞铺展中所起的关键作用仍有待阐明。
