Blat C, Villaudy J, Harel L
Institut Curie Pavillon Trouillet Rossignol, Paris, France.
Exp Cell Res. 1994 Nov;215(1):114-8. doi: 10.1006/excr.1994.1322.
Up until now the phenomenon of density dependent inhibition of growth has remained unexplained; one hypothesis suggests that autocrine growth inhibitory molecules are secreted in the medium of dense cultures and inhibit cell growth. From medium conditioned by mouse fibroblasts we purified a 45-kDa inhibitory factor which is an insulin-like growth factor binding protein (IGFBP-3). Based on different results we assumed that IGFBP-3 is a bifunctional molecule and has inhibitory function independent of its known function of binding IGF. In the present publication we attempted to verify whether IGFBP-3 is involved in DDI of growth. IGFBP-3 was secreted by mouse embryo fibroblasts (MEF). Its concentration in the medium increased with the cell density of the culture and was large at saturation density when DNA synthesis was minimum. Medium conditioned (CM) by dense culture was inhibitory compared to fresh medium and this inhibition disappeared when CM was preincubated with anti-IGFBP-3 IgG. Addition of FGFb to MEF dense cultures increased but transiently DNA synthesis which decreased as soon as 24 h after growth factor addition. By contrast accumulation of IGFBP-3 in the medium increased with time and was large at the time when DNA synthesis was minimum. Our results suggest that the rapid decrease of DNA synthesis in stimulated dense culture was the result of both depletion of the medium (particularly of FGFb) and the increase in concentration of inhibitory molecules like IGFBP-3. Addition of FGFb and preincubation of CM with anti-IGFBP-3 IgG were able to greatly reduce the inhibition.
到目前为止，生长的密度依赖性抑制现象仍未得到解释；一种假说是，自分泌生长抑制分子在高密度培养的培养基中分泌并抑制细胞生长。从小鼠成纤维细胞条件培养基中，我们纯化出一种45 kDa的抑制因子，它是一种胰岛素样生长因子结合蛋白（IGFBP - 3）。基于不同的结果，我们推测IGFBP - 3是一种双功能分子，具有独立于其已知的结合IGF功能的抑制功能。在本出版物中，我们试图验证IGFBP - 3是否参与生长的密度依赖性抑制（DDI）。IGFBP - 3由小鼠胚胎成纤维细胞（MEF）分泌。其在培养基中的浓度随着培养物的细胞密度增加而增加，并且在饱和密度时达到最大值，此时DNA合成最低。与新鲜培养基相比，高密度培养条件下的条件培养基（CM）具有抑制作用，而当CM与抗IGFBP - 3 IgG预孵育时，这种抑制作用消失。向MEF高密度培养物中添加碱性成纤维细胞生长因子（FGFb）可增加但短暂地促进DNA合成，在添加生长因子后24小时内DNA合成即开始下降。相比之下，培养基中IGFBP - 3的积累随时间增加，并且在DNA合成最低时达到最大值。我们的结果表明，在受刺激的高密度培养中DNA合成的快速下降是培养基耗尽（特别是FGFb）和抑制分子如IGFBP - 3浓度增加的共同结果。添加FGFb以及将CM与抗IGFBP - 3 IgG预孵育能够大大降低这种抑制作用。
