Early gene expression analyzed by cDNA microarray and RT-PCR in osteoblasts cultured with water-soluble and low molecular chitooligosaccharide
Early gene expression analyzed by cDNA microarray and RT-PCR in osteoblasts cultured with water-soluble and low molecular chitooligosaccharide
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DOI：
10.1016/j.biomaterials.2003.08.022
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发表时间：
2004-05-01
期刊：
影响因子：
14
通讯作者：
Ikeda, T
中科院分区：
文献类型：
--
作者：
Ohara, N;Hayashi, Y;Ikeda, T
壳聚糖具有多种生物活性。然而，很少有人知道壳聚糖如何调节硬组织形成细胞。在添加10%FBS和0.005%壳寡糖的α-MEM中培养成骨细胞3天后，碱性磷酸酶（ALP）活性显著高于对照组（P < 0.05）。本研究旨在探讨水溶性壳寡糖对成骨细胞基因表达的影响。从分离的RNA合成cDNA探针并用荧光染料标记。与人1.0（R）cDNA芯片杂交，分析荧光信号。cDNA微阵列分析显示，与对照组相比，实验组中有16个基因在3天后以大于或等于1.5倍的信号比水平表达。RT-PCR分析表明，壳聚糖低聚物诱导两个基因，CD 56抗原和组织型纤溶酶原激活剂的表达增加。此外，BMP-2的mRNA的表达在培养3天后的实验组和对照组中几乎是相同的，但在用壳聚糖低聚物培养7天后略有增加。这些结果表明，超低浓度壳寡糖可通过mRNA水平调控成骨细胞的活性，而与细胞增殖和分化相关的基因可能受到水溶性壳寡糖的调控。(C)2003 Elsevier Ltd.保留所有权利。
Chitosan has a variety of biological activities. However, little is known about how chitosan modulates the hard tissue forming cells. When we cultured an osteoblastic cell line in alpha-MEM supplemented with 10% FBS and 0.005% chitooligosaccharide for 3 days, alkaline phosphatase (ALP) activity was significantly high compared with the control culture group (P < 0.05). This study was focused on gene expression in osteoblasts cultured with water-soluble chitooligosaccharide. cDNA probes were synthesized from isolated RNA and labeled with fluorescent dye. They were hybridized with Human 1.0(R) cDNA microarray, and fluorescent signal was analyzed. cDNA microarray analysis revealed that 16 genes were expressed at greater than or equal to 1.5-fold higher signal ratio levels in the experimental group compared with the control group after 3 days. RT-PCR analysis showed that chitosan oligomer induced an increase in the expression of two genes, CD56 antigen and tissue-type plasminogen activator. Furthermore, the expression of mRNAs for BMP-2 was almost identical in the experimental and control groups after 3 days of culture, but slightly increased after 7 days of culture with chitosan oligomer. These results suggest that a super-low concentration of chitooligosaccharide could modulate the activity of osteoblastic cells through mRNA levels and that the genes concerning cell proliferation and differentiation call be controlled by water-soluble chitosan. (C) 2003 Elsevier Ltd. All rights reserved.