Andersen Hanne Busk, Holm Mette, Hetland Thea Eline, Dahl Christine, Junker Steffen, Schiøtz Peter Oluf, Hoffmann Hans Jürgen
Department of Paediatrics, Aarhus University Hospital, DK-8200 Aarhus N, Denmark.
J Immunol Methods. 2008 Jul 31;336(2):166-74. doi: 10.1016/j.jim.2008.04.016. Epub 2008 May 16.
During the last two decades different scientific groups have investigated the phenotype and function of in vitro generated human mast cells (MC). The cells have been shown to display variable surface markers and functional characteristics. The phenotypic differences may reflect different culture conditions, protocols or the use of different progenitors. To investigate the significance of different progenitors, we have compared MC generated from CD133(+) progenitor cells from cord blood (CBMC) or peripheral blood (PBMC). The progenitors were cultured for 7 weeks in the presence of IL-6 and SCF, with addition of IL-3 the first 3 weeks, and FCS during week 7. The phenotype of the established MC was characterized by surface marker expression levels, metachromasia, histamine and tryptase contents and their function was evaluated by receptor-mediated release of histamine and PGD(2). The generated metachromatic (<99%) MC were 75% tryptase(+), regardless of the source of progenitor cell. Expression of c-kit/CD117, CD203c, and FcepsilonRI was comparable. The density of c-kit/CD117 receptors on CBMC was higher that of PBMC (p<0.001). The density of CD203c and FcepsilonRI was higher on PBMC (p<0.001). PBMC contained more histamine (p<0.001), expressed more FcepsilonRI (p<0.001) and released more histamine (p<0.001) and PGD(2) (p<0.001) upon ligation of FcepsilonRI, than CBMC. Culture with IL-4 increased expression of tryptase, FcepsilonRI, CD117 and CD203c, secretion of histamine and PGD(2) of PBMC, and histamine secretion of CBMC. Cord and peripheral blood may give rise to different types of MC. The question addressed should determine the progenitor cell and protocol to be used.
在过去二十年中，不同的科研团队对体外培养生成的人肥大细胞（MC）的表型和功能进行了研究。研究表明，这些细胞呈现出可变的表面标志物和功能特性。表型差异可能反映了不同的培养条件、方案或不同祖细胞的使用情况。为了研究不同祖细胞的意义，我们比较了由脐带血（CBMC）或外周血（PBMC）中的CD133(+)祖细胞生成的MC。将祖细胞在IL-6和SCF存在的情况下培养7周，前3周添加IL-3，第7周添加胎牛血清（FCS）。通过表面标志物表达水平、异染性、组胺和类胰蛋白酶含量来表征所生成MC的表型，并通过组胺和前列腺素D2（PGD2）的受体介导释放来评估其功能。无论祖细胞来源如何，所生成的异染性（<99%）MC中有75%是类胰蛋白酶阳性（+）。c-kit/CD117、CD203c和高亲和力IgE受体（FcepsilonRI）的表达相当。CBMC上c-kit/CD117受体的密度高于PBMC（p<0.001）。PBMC上CD203c和FcepsilonRI的密度更高（p<0.001）。与CBMC相比，PBMC含有更多的组胺（p<0.001），表达更多的FcepsilonRI（p<0.001），并且在FcepsilonRI被结扎后释放更多的组胺（p<0.001）和PGD2（p<0.001）。用IL-4培养可增加PBMC中类胰蛋白酶、FcepsilonRI、CD117和CD203c的表达，组胺和PGD2的分泌，以及CBMC中组胺的分泌。脐带血和外周血可能产生不同类型的MC。所探讨的问题应决定所使用的祖细胞和方案。
