Levine J F, Stockdale F E
J Cell Biol. 1985 May;100(5):1415-22. doi: 10.1083/jcb.100.5.1415.
Mammary epithelium differentiates in a stromal milieu of adipocytes and fibroblasts. To investigate cell-cell interactions that may influence mammary epithelial cell differentiation, we developed a co-culture system of murine mammary epithelium and adipocytes and other fibroblasts. Insofar as caseins are specific molecular markers of mammary epithelial differentiation, rat anti-mouse casein monoclonal antibodies were raised against the three major mouse casein components to study this interaction. Mammary epithelium from mid-pregnant mice was plated on confluent irradiated monolayers of 3T3-L1 cells, a subclone of the Swiss 3T3 cell line that differentiates into adipocytes in monolayer culture and other cell monolayers (3T3-C2 cells, Swiss 3T3 cells, and human foreskin fibroblasts). Casein was synthesized by mammary epithelium only in the presence of co-cultured cells and the lactogenic hormone combination of insulin, hydrocortisone, and prolactin. Synthesis and accumulation of alpha-, beta-, and gamma-mouse casein within the epithelium was shown by immunohistochemical staining of cultured cells with anti-casein monoclonal antibodies, and the specificity of the immunohistochemical reaction was demonstrated using immunoblots. A competitive immunoassay was used to measure the amount of casein secreted into the culture medium. In a 24-h period, mammary epithelium co-cultured with 3T3-L1 cells secreted 12-20 micrograms beta-casein per culture dish. There was evidence of specificity in the cell-cell interaction that mediates hormone-dependent casein synthesis. Swiss 3T3 cells, newborn foreskin fibroblasts, substrate-attached material ("extracellular matrix"), and tissue culture plastic did not support casein synthesis, whereas monolayers of 3T3-L1 and 3T3-C2 cells, a subclone of Swiss 3T3 cells that does not undergo adipocyte differentiation, did. We conclude that interaction between mammary epithelium and other specific nonepithelial cells markedly influences the acquisition of hormone sensitivity of the epithelium and hormone-dependent differentiation.
乳腺上皮在脂肪细胞和成纤维细胞构成的基质环境中分化。为了研究可能影响乳腺上皮细胞分化的细胞间相互作用，我们建立了小鼠乳腺上皮细胞与脂肪细胞及其他成纤维细胞的共培养体系。鉴于酪蛋白是乳腺上皮分化的特异性分子标志物，我们制备了针对小鼠三种主要酪蛋白成分的大鼠抗小鼠酪蛋白单克隆抗体，以研究这种相互作用。将妊娠中期小鼠的乳腺上皮细胞接种在汇合的经辐照的3T3-L1细胞单层上，3T3-L1细胞是瑞士3T3细胞系的一个亚克隆，在单层培养中可分化为脂肪细胞，以及其他细胞单层（3T3-C2细胞、瑞士3T3细胞和人包皮成纤维细胞）。酪蛋白仅在与共培养细胞以及胰岛素、氢化可的松和催乳素的泌乳激素组合存在的情况下，由乳腺上皮合成。用抗酪蛋白单克隆抗体对培养细胞进行免疫组织化学染色，显示上皮细胞内α-、β-和γ-小鼠酪蛋白的合成与积累，并用免疫印迹法证明了免疫组织化学反应的特异性。采用竞争性免疫测定法测量分泌到培养基中的酪蛋白量。在24小时内，与3T3-L1细胞共培养的乳腺上皮细胞每个培养皿分泌12 - 20微克β-酪蛋白。有证据表明介导激素依赖性酪蛋白合成的细胞间相互作用具有特异性。瑞士3T3细胞、新生儿包皮成纤维细胞、底物附着物质（“细胞外基质”）和组织培养塑料不支持酪蛋白合成，而3T3-L1细胞和3T3-C2细胞单层（瑞士3T3细胞的一个不发生脂肪细胞分化的亚克隆）则支持。我们得出结论，乳腺上皮细胞与其他特定的非上皮细胞之间的相互作用显著影响上皮细胞激素敏感性的获得和激素依赖性分化。
