Research Institute, Morinaga & Co. Ltd., 2-1-1, Shimosueyoshi, Tsurumi-ku, Yokohama, 230-8504, Japan,
Cytotechnology. 2003 Nov;43(1-3):135-44. doi: 10.1023/b:cyto.0000039897.78630.7a.
We established a real-time quantitative RT-PCR assay that permits rapid and sensitive screening of foods that increase the human beta-defensin-2 (hBD-2) mRNA level in human foreskin keratinocyte (HFK) cells. The range of hBD-2 mRNA concentrations suitable for the assay was between 8 x 10(-11) M (39-cycle amplification) and 8 x 10(-18) M (13-cycle amplification) as calibrated with standard hBD-2 cDNA. With this assay system, it was found that the stimulation of HFK cells by the addition of yeast powder at 5 g l(-1) to the culture medium resulted in about 40 times increase in hBD-2 mRNA level, though stimulation with Escherichia coli attained the same level of induction. The active component of yeast was insoluble in water. Simultaneous co-stimulation of HFK cells with E. coli and grains, such as amaranth, millet, soybean and sesame, boosted hBD-2 mRNA induction significantly (6.1, 2.5, 3.3, and 3.3 times, respectively) above the level attained with E. coli alone. The results of successive fractionations of amaranth grain powder by ether extraction and amylase digestion showed that the boosting activity of amaranth grain resided in its insoluble fraction. Significant boosting of hBD-2 mRNA induction in epithelial cells with foods opens a new possibility of developing functional foods that can protect the human body against microbial infection at the oral cavity, skin, and respiratory tract among others.
我们建立了一种实时定量 RT-PCR 检测方法，该方法可以快速灵敏地筛选出能够增加人包皮角质形成细胞（HFK）中人类防御素-2（hBD-2）mRNA 水平的食物。该方法检测 hBD-2 mRNA 的浓度范围为 8×10(-11) M（39 个循环扩增）至 8×10(-18) M（13 个循环扩增），通过标准 hBD-2 cDNA 进行校准。利用该检测系统，我们发现酵母粉添加到培养基中浓度为 5 g l(-1)时，可使 HFK 细胞中 hBD-2 mRNA 水平增加约 40 倍，而大肠杆菌的刺激也能达到相同的诱导水平。酵母的活性成分不溶于水。同时刺激 HFK 细胞与大肠杆菌和谷物，如苋菜籽、小米、大豆和芝麻，可显著增强 hBD-2 mRNA 的诱导（分别增加 6.1、2.5、3.3 和 3.3 倍）高于单独用大肠杆菌刺激的水平。对苋菜籽粉进行连续乙醚萃取和淀粉酶消化的分步处理结果表明，苋菜籽的增强活性存在于不溶性部分。食物对上皮细胞 hBD-2 mRNA 诱导的显著增强，为开发功能性食品以保护人体免受口腔、皮肤和呼吸道等部位的微生物感染提供了新的可能性。
