Isseroff R R, Martinez D T, Ziboh V A
J Invest Dermatol. 1985 Aug;85(2):131-4. doi: 10.1111/1523-1747.ep12276536.
The availability of methods for the in vitro cultivation of keratinocytes has spawned numerous studies utilizing these systems to analyze epidermal biochemical pathways, e.g., eicosanoid production. To determine whether these culture systems are indeed valid models for studies of eicosanoid products, we analyzed the fatty acid (FA) composition, especially of eicosanoid precursors linoleic acid (LA) and arachidonic acid (AA), of cultured and noncultured mouse keratinocytes. Neonatal mouse epidermal keratinocytes were cultivated in Dulbecco's modification of Eagle's medium + 10% fetal calf serum (FCS). Lipids of the cultivated cells, as well as noncultivated keratinocytes and whole epidermis were extracted in CHCl3:MeOH (2:1) and lipid classes separated by thin-layer chromatography. The FA composition of the total lipid extract as well as of the phospholipid class was determined by gas-liquid chromatography of FA methyl esters. There was a gradual decrease in the LA levels in the cultured cells; by day 5 of culture the cells demonstrated a 4-fold (p less than 0.001) decrease in LA as compared to either noncultured cells or whole epidermis. Levels of AA, on the other hand, remained unchanged during culture. Analysis of the FCS used in the culture medium revealed that the level of LA was 4-fold lower than that of normal mouse serum. Since LA is an essential FA which is not synthesized by the cell, the decreased LA in cultured cells probably results from the paucity of this FA in the FCS-containing culture medium. These studies indicate that keratinocytes cultivated in FCS-containing medium demonstrate profound alterations in levels of LA. Hence, in vitro keratinocyte studies dependent on cellular polyunsaturated FA substrates should be interpreted with caution. The relationship of altered cellular levels of LA on keratinocyte differentiation remains to be determined.
角质形成细胞体外培养方法的出现催生了大量利用这些系统分析表皮生化途径（如类花生酸生成）的研究。为了确定这些培养系统是否确实是研究类花生酸产物的有效模型，我们分析了培养的和未培养的小鼠角质形成细胞的脂肪酸（FA）组成，尤其是类花生酸前体亚油酸（LA）和花生四烯酸（AA）的组成。新生小鼠表皮角质形成细胞在杜尔贝科改良的伊格尔培养基+10%胎牛血清（FCS）中培养。培养细胞、未培养的角质形成细胞和整个表皮的脂质用氯仿：甲醇（2:1）提取，脂质类别通过薄层色谱法分离。总脂质提取物以及磷脂类别的FA组成通过FA甲酯的气液色谱法测定。培养细胞中LA水平逐渐下降；培养第5天时，与未培养细胞或整个表皮相比，细胞中LA下降了4倍（p<0.001）。另一方面，AA水平在培养过程中保持不变。对培养基中使用的FCS分析显示，LA水平比正常小鼠血清低4倍。由于LA是细胞不能合成的必需脂肪酸，培养细胞中LA的减少可能是由于含FCS的培养基中这种脂肪酸含量不足。这些研究表明，在含FCS培养基中培养的角质形成细胞LA水平有显著变化。因此，依赖细胞多不饱和FA底物的体外角质形成细胞研究应谨慎解释。LA细胞水平改变与角质形成细胞分化的关系仍有待确定。
