Grabbe J, Welker P, Rosenbach T, Nürnberg W, Krüger-Krasagakes S, Artuc M, Fiebiger E, Henz B M
Department of Dermatology, Humboldt University, Berlin, Germany.
J Invest Dermatol. 1996 Aug;107(2):219-24. doi: 10.1111/1523-1747.ep12329664.
Stem cell factor, a recently discovered growth factor for hematopoietic stem cells, mast cells, and melanocytes, was initially reported to be produced by fibroblasts. In this study, we investigated the secretion of this factor from human HaCaT cells during in vitro culture and compared it to synthesis by cells in the skin. Release of stem cell factor from freshly cultured keratinocytes was comparable to that of HaCaT cells and was nearly half that produced by fibroblasts and umbilical vein endothelial cells. No stem cell factor was detectable in culture supernatants of melanocytes. HaCaT cells underwent spontaneous differentiation after a period of proliferation until confluency. Depending on duration of culture, they released increasing amounts of stem cell factor (approximately 150 pg/10(6) cells on day 3 (proliferating cells) vs approximately 450 pg/10(6) cells on day 14 (differentiating cells) measured by enzyme-linked immunosorbent assay. Stimulation for 24 h with the calcium ionophore A 23187 (10(-6) to 10(-8) M) further enhanced release. Western blot analysis of HaCaT cell lysates with a stem cell factor antibody revealed two proteins with the known molecular weights of membrane-bound and soluble stem cell factor. By semiquantitative reverse transcriptase polymerase chain reaction, full-length as well as spliced type stem cell factor mRNA was found to be increased in differentiating versus proliferating HaCaT cells. Keratinocytes are thus potentially important sources of stem cell factor in human skin, and HaCaT cells provide a useful model for further studies of stem cell factor from keratinocytes.
干细胞因子是一种最近发现的针对造血干细胞、肥大细胞和黑素细胞的生长因子，最初报道称其由成纤维细胞产生。在本研究中，我们调查了人HaCaT细胞在体外培养期间该因子的分泌情况，并将其与皮肤细胞的合成情况进行比较。新鲜培养的角质形成细胞释放的干细胞因子与HaCaT细胞相当，几乎是成纤维细胞和脐静脉内皮细胞产生量的一半。在黑素细胞的培养上清液中未检测到干细胞因子。HaCaT细胞在增殖一段时间直至汇合后会自发分化。根据培养时间的不同，它们释放出越来越多的干细胞因子（通过酶联免疫吸附测定法测量，第3天（增殖细胞）约为150 pg/10⁶个细胞，第14天（分化细胞）约为450 pg/10⁶个细胞）。用钙离子载体A 23187（10⁻⁶至10⁻⁸ M）刺激24小时可进一步增强释放。用干细胞因子抗体对HaCaT细胞裂解物进行蛋白质印迹分析，发现了两种具有已知分子量的膜结合型和可溶性干细胞因子的蛋白质。通过半定量逆转录聚合酶链反应发现，与增殖的HaCaT细胞相比，分化的HaCaT细胞中全长以及剪接型干细胞因子mRNA均增加。因此，角质形成细胞可能是人类皮肤中干细胞因子的重要潜在来源，而HaCaT细胞为进一步研究角质形成细胞的干细胞因子提供了有用的模型。
