Schörkhuber M, Karner-Hanusch J, Sedivy R, Ellinger A, Armbruster C, Schulte-Hermann R, Marian B
Institute of Tumor Biology-Cancer Research, University of Vienna, Austria.
Cell Biol Toxicol. 1998 Jun;14(3):211-23. doi: 10.1023/a:1007418811589.
Primary cultures of normal colonic epithelial cells from both humans (HCEC) and rats (RCEC) have been established using coculture with colon fibroblasts isolated from rat term embryos. While no other factors we have analyzed had any effect on the survival of epithelial cells, which is normally 3-4 days, coculture with viable fibroblasts extended this period to at least 2 weeks. The effects depended on early passages and low seeding densities of the fibroblasts and on direct cell-cell contact. We have obtained cultures of epithelial cells expressing keratin, laminin, and uvomorulin, displaying a polygonal, epithelial morphology and forming microvilli. DNA synthesis as measured by BrdU uptake into DNA varied widely between colonies of the same culture depending on cell morphology: flat colonies of RCECs contained 5.7% +/- 0.56% BrdU-positive cells, while the proportion in dense three-dimensional colonies reached 50.3% +/- 2.6%. In HCECs the growth fraction was lower, but showed the same distribution between classes of colonies. In the presence of rat embryonic colon fibroblasts, growth factors exerted survival activity on colonic epithelial cells. Consecutive addition of insulin and epidermal growth factor/fibroblast growth factor (EGF/FGF) increased colony number (15.0 +/- 1.0 and 23.0 +/- 2.0 colonies/well respectively; p < or = 0.05 increased above control) and size (1022 +/- 155 and 1207 +/- 158 cells/colony respectively; p < or = 0.05 increased above control) compared to serum-free control medium and basic MEM without growth factors. BrdU labeling index was not increased, however: EGF/FGF actually decreased BrdU labeling from 33.2% +/- 3.9% in controls to 21.3% +/- 3.8% in the EGF/FGF group (p < or = 0.05) owing to the high proportion of flat colonies consisting of resting cells. The newly established culture model can now be used to investigate growth control mechanisms in colonic mucosa and the effects of toxic and/or tumor-promoting substances on these mechanisms.
通过与从大鼠足月胚胎中分离出的结肠成纤维细胞共培养，已建立了人类正常结肠上皮细胞（HCEC）和大鼠正常结肠上皮细胞（RCEC）的原代培养体系。虽然我们分析的其他因素对上皮细胞的存活均无影响（上皮细胞正常存活时间为3 - 4天），但与活的成纤维细胞共培养可将这一时期延长至至少2周。这些作用取决于成纤维细胞的早期传代和低密度接种，以及细胞间的直接接触。我们已获得表达角蛋白、层粘连蛋白和uvomorulin的上皮细胞培养物，这些细胞呈现多边形的上皮形态并形成微绒毛。通过将BrdU掺入DNA来测量的DNA合成在同一培养物的不同集落间差异很大，这取决于细胞形态：RCEC的扁平集落中含有5.7%±0.56%的BrdU阳性细胞，而密集的三维集落中的比例则达到50.3%±2.6%。在HCEC中生长分数较低，但在不同类型集落间显示出相同的分布。在存在大鼠胚胎结肠成纤维细胞的情况下，生长因子对结肠上皮细胞具有存活活性。与无血清对照培养基和不含生长因子的基础MEM相比，连续添加胰岛素和表皮生长因子/成纤维细胞生长因子（EGF/FGF）可增加集落数量（分别为15.0±1.0和23.0±2.0个集落/孔；p≤0.05，高于对照）和大小（分别为1022±155和1207±158个细胞/集落；p≤0.05，高于对照）。然而，BrdU标记指数并未增加：由于由静止细胞组成的扁平集落比例较高，EGF/FGF实际上使BrdU标记从对照中的33.2%±3.9%降至EGF/FGF组中的21.3%±3.8%（p≤0.05）。新建立的培养模型现在可用于研究结肠黏膜中的生长控制机制以及有毒和/或促肿瘤物质对这些机制的影响。
