Kuntz Sabine, Kunz Clemens, Rudloff Silvia
Institute of Nutritional Science, University of Giessen, Wilhelmstrasse 20, Giessen 35392, Germany.
Br J Nutr. 2009 May;101(9):1306-15. doi: 10.1017/S0007114508079622. Epub 2008 Dec 15.
Oligosaccharides are present in human milk in large amounts and in a high variety. We have previously shown that these oligosaccharides are strong inhibitors of proliferation and inducers of differentiation in intestinal cell lines. To elucidate the molecular mechanism, we investigated the influence on cell cycle events via flow cytometry and expression levels by using quantitative real-time RT-PCR. Human intestinal cells, i.e. HT-29, HIEC and Caco-2 cells, were exposed to neutral or acidic human milk oligosaccharides. Both fractions induced a concentration-dependent G2/M arrest. Cell cycle analysis for HT-29 revealed 37 % of cells in G1 and 35 % in G2/M (neutral oligosaccharides) and incubation with acidic oligosaccharides led to 42 % cells in G1 and 40 % in G2/M. In control experiments without oligosaccharides we found 71 % of cells to be in G1 and 17 % in G2/M. This G2/M arrest was associated with changes in mRNA expression of cyclin A and B. A G2/M arrest with concomitant alterations of cell cycle gene expression could also be shown for HIEC and Caco-2 cells. Analysing the expression of cyclin-dependent kinase inhibitors p21(cip1) and p27(kip1) and the tumour suppressor p53 we observed that the expression of p21(cip1) was p53-independent and necessary for arresting cells in the G2/M phase, while p27(kip1) was associated with differentiation effects. Both neutral and acidic human milk oligosaccharides were able to induce epidermal growth factor receptor, extracellular signal-regulated kinase 1/2 and p38 phosphorylation. These results suggest that oligosaccharides from human milk inhibited intestinal cell proliferation and altered cell cycle dynamics by affecting corresponding regulator genes and mitogen-activated protein kinase signalling.
低聚糖大量且多样地存在于人乳中。我们之前已经表明，这些低聚糖是肠道细胞系中增殖的强效抑制剂和分化的诱导剂。为了阐明分子机制，我们通过流式细胞术研究了对细胞周期事件的影响，并使用定量实时逆转录聚合酶链反应研究了表达水平。将人肠道细胞，即HT-29、HIEC和Caco-2细胞暴露于中性或酸性人乳低聚糖中。这两种组分均诱导浓度依赖性的G2/M期阻滞。对HT-29细胞的细胞周期分析显示，G1期有37%的细胞，G2/M期有35%的细胞（中性低聚糖），而用酸性低聚糖孵育导致G1期有42%的细胞，G2/M期有40%的细胞。在没有低聚糖的对照实验中，我们发现71%的细胞处于G1期，17%的细胞处于G2/M期。这种G2/M期阻滞与细胞周期蛋白A和B的mRNA表达变化有关。HIEC和Caco-2细胞也表现出G2/M期阻滞以及细胞周期基因表达的相应改变。分析细胞周期蛋白依赖性激酶抑制剂p21（cip1）和p27（kip1）以及肿瘤抑制因子p53的表达，我们观察到p21（cip1）的表达不依赖于p53，并且是将细胞阻滞在G2/M期所必需的，而p27（kip1）与分化效应有关。中性和酸性人乳低聚糖均能够诱导表皮生长因子受体、细胞外信号调节激酶1/2和p38磷酸化。这些结果表明，人乳中的低聚糖通过影响相应的调节基因和丝裂原活化蛋白激酶信号传导来抑制肠道细胞增殖并改变细胞周期动力学。
