Recombinant human stem cell factor stimulates differentiation of mast cells from dispersed human fetal liver cells.
Recombinant human stem cell factor stimulates differentiation of mast cells from dispersed human fetal liver cells.
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DOI：
10.1182/blood.v80.12.3009.bloodjournal80123009
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发表时间：
1992-12
期刊：
影响因子：
20.3
通讯作者：
A. Irani;G. Nilsson;U. Miettinen;S. Craig;L. Ashman;T. Ishizaka;K. Zsebo;L. Schwartz
中科院分区：
文献类型：
--
作者：
A. Irani;G. Nilsson;U. Miettinen;S. Craig;L. Ashman;T. Ishizaka;K. Zsebo;L. Schwartz
我们先前已经显示了类胰蛋白酶+人肥大细胞的体外发育，这些肥大细胞来自与鼠3 T3成纤维细胞共培养的胎肝细胞。在这项研究中，重组人干细胞因子（rhuSCF），配体的c-kit原癌基因产物称为试剂盒，刺激的生长和分化，主要是肥大细胞从分散的胎肝细胞，而重组人白细胞介素-3（rhuIL-3）刺激嗜碱性粒细胞的分化沿着其他细胞类型。在rhuSCF或rhuIL-3的存在下，开始培养胎肝细胞并维持长达6周。细胞离心涂片中的异染细胞主要根据类胰蛋白酶表达被鉴定为肥大细胞，并通过免疫组织化学使用抗类胰蛋白酶和糜蛋白酶的单克隆抗体被鉴定为MCT或MCTC，而嗜碱性粒细胞是异染的、多形的，并且缺乏这些蛋白酶。用放射免疫法测定类胰蛋白酶和组胺水平，用肽水解法测定类胰蛋白酶和糜蛋白酶活性，用流式细胞仪用单克隆抗体YB5.B8测定细胞表面Kit。肥大细胞的主要存在仅发生在补充rhuSCF的培养物中。肥大细胞的百分比和总数随着rhuSCF浓度的增加而增加，并在55 ng/mL时达到平台。在此浓度的rhuSCF下，肥大细胞在第7天首次出现;到第42天，存在106%的起始细胞数，其中85%为类胰蛋白酶+，31%为弱糜蛋白酶+。这些肥大细胞出现不成熟的超微结构的标准，大多数细胞是单核细胞，但有些细胞核深深分裂叶。类胰蛋白酶+肥大细胞在rhuSCF培养第21天和第28天的DNA合成通过溴脱氧尿苷掺入来证明。组胺（1.2 pg/肥大细胞）和类胰蛋白酶（0.9 pg/肥大细胞）的计算水平与先前在与鼠3 T3成纤维细胞共培养实验中测定的水平相似。糜酶活性在大多数细胞提取物中检测不到。在第0天，4%至20%的胎肝细胞表达细胞表面Kit。在rhuSCF存在下，Kit+细胞的百分比和总数以及每个细胞的Kit表观浓度沿着类胰蛋白酶+细胞的数量而增加。在rhuIL-3、甲苯胺蓝+、类胰蛋白酶-存在下，第14天首先出现最多细胞（11% +/-2.5%）。这些甲苯胺蓝+细胞的百分比然后在第21天和第35天下降到约6%，而阳性细胞的总数下降超过10倍。在rhuIL-3存在下的Kit+细胞从第3天的9%下降到第35天的2%。（400字处截断摘要）
We have previously shown the development in vitro of tryptase+ human mast cells from fetal liver cells cocultured with murine 3T3 fibroblasts. In this study, recombinant human stem cell factor (rhuSCF), the ligand for the c-kit proto-oncogene product called Kit, stimulated the growth and differentiation primarily of mast cells from dispersed fetal liver cells, whereas recombinant human interleukin-3 (rhuIL-3) stimulated the differentiation of basophils along with other cell types. Cultures of fetal liver cells were initiated and maintained in the presence of rhuSCF or rhuIL-3 for up to 6 weeks. Metachromatic cells in cytospins were identified as mast cells primarily on the basis of tryptase expression, and as MCT or MCTC by immunohistochemistry using monoclonal antibodies against tryptase and chymase, whereas basophils were metachromatic, polymorphonuclear, and lacked these proteases. Levels of tryptase and histamine were measured by radioimmunoassay, tryptase and chymase activities by peptide hydrolysis, and cell surface Kit by flow cytometry with the monoclonal antibody YB5.B8. The predominant presence of mast cells occurred only in the cultures supplemented with rhuSCF. The percentage and total number of mast cells increased over time with increasing concentrations of rhuSCF and reached a plateau at 55 ng/mL. At this concentration of rhuSCF, mast cells first appeared by day 7; by day 42, 106% of the starting number of cells were present and 85% of these were tryptase+, 31% being weakly chymase+. These mast cells appeared immature by ultrastructural criteria; most cells were mononuclear, but some had nuclei with deeply divided lobes. DNA synthesis in tryptase+ mast cells at days 21 and 28 of culture with rhuSCF was demonstrated by incorporation of bromodeoxyuridine. Calculated levels of histamine (1.2 pg/mast cell) and tryptase (0.9 pg/mast cell) were similar to those determined previously in coculture experiments with murine 3T3 fibroblasts. Chymase activity was undetectable in most cell extracts. On day 0, 4% to 20% of fetal liver cells expressed cell surface Kit. In the presence of rhuSCF, the percentages and total numbers of Kit+ cells and the apparent concentration of Kit per cell increased along with the number of tryptase+ cells. In the presence of rhuIL-3, toluidine blue+, tryptase- cells first and maximally appeared at day 14 (11% +/- 2.5%). The percentage of these toluidine blue+ cells then declined to about 6% by days 21 and 35, while the total number of positive cells declined over 10-fold. Kit+ cells in the presence of rhuIL-3 declined from 9% on day 3 to 2% on day 35.(ABSTRACT TRUNCATED AT 400 WORDS)