Direct analysis of growth factor requirements for isolated human fetal hepatocytes.
Direct analysis of growth factor requirements for isolated human fetal hepatocytes.
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直接分析分离的人胎儿肝细胞的生长因子需求。
DOI：
10.1007/bf02620987
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
McKeehan,WL
中科院分区：
文献类型：
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作者：
Hoshi,H;Kan,M;McKeehan,WL
从人胎肝中分离肝细胞，以分析生长因子和激素对人肝细胞增殖和功能的直接影响。机械破碎，然后解离胎肝组织与胶原酶/分散酶混合物导致肝细胞的高产率和活力。在无精氨酸、补充鸟氨酸的培养基中选择肝细胞，并通过形态学、白蛋白产生和鸟氨酸摄取到细胞蛋白中来定义。筛选了20多种生长因子、激素、促有丝分裂剂和粗器官及细胞提取物对肝细胞生长的刺激作用，结果显示EGF、胰岛素、地塞米松和集中在牛神经提取物和肝癌细胞条件培养基中的因子支持肝细胞在胶原包被基质上的附着、维持和生长。选择并定义为分化肝细胞的细胞群体具有约4个累积群体倍增的增殖潜力。EGF和胰岛素在没有其他激素和生长因子的情况下协同刺激DNA合成。虽然神经提取物增加肝细胞数量，没有影响的DNA合成的神经提取物或纯化的肝素结合生长因子从神经提取物可以证明在定义的激素，肝癌条件培养基或血清的存在或不存在。在所有条件下，肝癌细胞条件培养基对肝细胞数量和DNA合成的影响最大。透析血清蛋白（1 mg/ml）在高10倍的蛋白浓度下具有与肝癌细胞条件培养基（100 μg/ml）相似的效果。结果表明，肝癌细胞条件培养基可能是一个浓缩的和不太复杂的来源比血清的纯化和表征额外的正常肝细胞生长因子。
Hepatocytes were isolated from human fetal liver in order to analyze the direct effects of growth factors and hormones on human hepatocyte proliferation and function. Mechanical fragmentation and then dissociation of fetal liver tissue with a collagenase/dispase mixture resulted in high yield and viability of hepatocytes. Hepatocytes were selected in arginine-free, ornithine-supplemented medium and defined by morphology, albumin production and ornithine uptake into cellular protein. A screen of over twenty growth factors, hormones, mitogenic agents and crude organ and cell extracts for effect on the stimulation of hepatocyte growth revealed that EGF, insulin, dexamethasone, and factors concentrated in bovine neural extract and hepatoma cell-conditioned medium supported attachment, maintenance and growth of hepatocytes on a collagen-coated substratum. The population of cells selected and defined as differentiated hepatocytes had a proliferative potential of about 4 cumulative population doublings. EGF and insulin synergistically stimulated DNA synthesis in the absence of other hormones and growth factors. Although neural extracts enhanced hepatocyte number, no effect on DNA synthesis of neural extracts or purified heparin-binding growth factors from neural extracts could be demonstrated in the absence or presence of defined hormones, hepatoma-conditioned medium or serum. Hepatoma cell-conditioned medium had the largest impact on both hepatocyte cell number and DNA synthesis under all conditions. Dialyzed serum protein (1 mg/ml) at 10 times higher protein concentration had a similar effect to hepatoma cell-conditioned medium (100 μg/ml). The results suggest that hepatoma cell conditioned medium may be a concentrated and less complicated source than serum for purification and characterization of additional normal hepatocyte growth factors.