Molecular Biology Of Mast Cell Growth & Differentiation

我们已经建立了人肥大细胞系(HUMC)，称为变态反应性疾病实验室(LAD)1和2，它们依赖于干细胞因子，具有成熟细胞的外观，并在Fc-γRI聚集后脱颗粒。LAD细胞系在KIT中似乎没有突变。在与Nilsson博士的合作中，我们还记录并表征了HMC-1细胞的两个亚系，它们在KIT中有不同的突变，在表型上也不同。总之，这些细胞系提供了一种研究HUMC生物学的手段，而不需要从血液或骨髓中的前体培养HUMC。 血小板生成素(TPO)是一种肝细胞源性生长因子，在特定的生长因子存在下，可促进血小板数量，促进巨核细胞(MK)生长，促进红细胞、单核细胞、肥大细胞和粒细胞的增殖。为了探索TPO单独支持肥大细胞增殖的能力，我们在为培养MK而开发的条件下，在rhTPO中培养CD34+细胞，并检测这些培养物中是否存在HuMCs。同样，我们将重组人TPO添加到在干细胞因子中培养的CD34+细胞中，已知这种因子可以促进HUMC的发育。在添加了重组人TPO的无血清培养液(SFM)中，2-3周的人CD34+细胞分化为MKs(85-90%)和低分化的HUMC(5-10%)。不依赖于SCF的HuMCs表达TPO(c-MPL)受体、类胰蛋白酶和糜酶，并能在单独使用rhTPO的SFM中存活，或在rhSCF中再培养时增殖。当rhTPO与10 ng/mlrhSCF联合使用时，可观察到少量的HUMC。而在含100 ng/mlrhSCF的HUMC中，加入50 ng/mlrhTPO后，HUMC数量明显减少，并出现单核细胞。在所有条件下，TPO的加入有利于KIT-Low HUMC的生长。因此，TPO对SCF依赖的HUMC的作用随细胞因子浓度的不同而不同，较高剂量的rhTPO有利于MK的增殖。

We have developed human mast cell (HuMC) lines termed Laboratory of Allergic Diseases(LAD) 1 and 2 which are dependent upon stem cell factor, have the appearance of mature cells, and degranulate following aggregation of Fc gamma RI on their surface. LAD cell lines do not appear to have mutations in KIT. In collaboration with Dr. Nilsson, we have also documented and characterized two sublines of HMC-1 cells which vary in mutations in KIT and vary in phenotype. Together, these cell lines provide a means to study the biology of HuMC without the need to culture HuMC from precursors in blood or marrow. Thrombopoietin (TPO) is a hepatocyte-derived growth factor known to promote platelet number, to have growth-promoting potential for megakaryocytes (MKs), and to promote erythrocyte, monocyte, mast cell, and granulocyte proliferation in the presence of specific growth factors. To explore the ability of TPO alone to support mast cell proliferation, we cultured CD34+ cells in rhTPO under conditions developed to culture MKs and examined these cultures for the presence of HuMCs. Similarly, we added rhTPO to CD34+ cells cultured in stem cell factor, known to promote HuMC development. Using serum-free media (SFM) supplemented with rhTPO, human CD34+ cells at 2-3 weeks differentiated into MKs (85-90%) and (5-10%) poorly differentiated HuMC. SCF-independent HuMCs expressed TPO (c-Mpl) receptors, tryptase and chymase; and were capable of surviving in SFM with rhTPO alone, or proliferating when recultured in rhSCF. Small numbers of HuMC were noted when rhTPO was combined with 10 ng/ml rhSCF. However, in HuMC cultures with 100 ng/ml rhSCF, the addition of 50 ng/ml rhTPO significantly reduced HuMC numbers, with the appearance of MKs. Under all conditions, the addition of TPO favored the growth of KIT-low HuMC. Thus, the effect of TPO on SCF-dependent HuMC varies depending on the concentration of cytokines, with higher doses of rhTPO favoring MK proliferation.