Early gene expression analyzed by cDNA microarray and RT-PCR in osteoblasts cultured with water-soluble and low molecular chitooligosaccharide.
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 >/=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 can be controlled by water-soluble chitosan.
Ohara
Naoko
N
Division of Cariology, Nagasaki University Graduate School of Biomedical Sciences, 852-8588, Nagasaki, Japan.
Hayashi
Yoshihiko
Y
Yamada
Shizuka
S
Kim
Se-Kwon
SK
Matsunaga
Tsunenori
T
Yanagiguchi
Kajiro
K
Ikeda
Takeshi
T
D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D019002:CD56 Antigen / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D002686:Chitin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D048271:Chitosan; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008970:Molecular Weight; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D010012:Osteogenesis / Q000187:drug effects; D012516:Osteosarcoma / Q000378:metabolism* / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012995:Solubility; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D016212:Transforming Growth Factor beta; D014867:Water / Q000737:chemistry*
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D019002:CD56 Antigen; D016212:Transforming Growth Factor beta; D014867:Water; D002686:Chitin; D048271:Chitosan; D010959:Tissue Plasminogen Activator