ABSTRACT
Gingival epithelial cells and fibroblasts play important roles and have a harmonious relationship under normal and disease conditions, but the precise differences between theses cells remain unknown. To study the differences in gene expression between human gingival epithelial cells (HGE) and human gingival fibroblasts (HGF), mRNA was recovered from primary cultured cells and analyzed using cDNA microarray technology. The cDNA retro-transcribed from equal quantities of mRNA was labeled with the fluorescent dyes Cy5 and Cy3. The mixed probes were then hybridized with 7276 genes on the DNA microarray, after which fluorescence signals were scanned and further analyzed using GeneSpring software. Of the 7276 genes screened, 469 showed expression levels that were more than 2-fold greater in HGE than in HGF, while 293 showed expression levels that were more than 2-fold greater in HGF than in HGE. To confirm the reliability of the microarray results, keratin K5 and desmocolin, and vimentin and gp130, which showed higher mRNA levels in HGE and HGF, respectively, were selected and their mRNA levels were further analyzed by RT-PCR. The results of RT-PCR correlated well with those of microarray analysis. The present findings using a DNA microarray to detect differences in the gene expression profiles of HGE and HGF may be beneficial for genetic diagnosis of periodontal tissue metabolism and periodontal diseases.
Subject(s)
Fibroblasts/metabolism , Gene Expression Profiling , Gene Expression Regulation/genetics , Gingiva/metabolism , Antigens, CD/genetics , Cells, Cultured , Cytokine Receptor gp130 , Desmocollins , Desmosomes/genetics , Epithelial Cells/metabolism , Fluorescent Dyes , Gingiva/cytology , Humans , Keratin-5 , Keratins/genetics , Membrane Glycoproteins/genetics , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , Receptors, Cytokine/genetics , Receptors, Oncostatin M , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Vimentin/geneticsABSTRACT
Chronic caffeine exposure during pregnancy has an effect on fetal growth; however, the adverse effects of caffeine on embryogenesis are not well understood and controversial. We used cDNA microarray technology to determine whether caffeine alters gene expressions in a human cytotrophoblast-like cell line, BeWo. We found that the expression of the B-cell CLL/lymphoma 2 (Bcl-2) gene in BeWo cells was down-regulated by caffeine, suggesting that chronic exposure during the gestational period could exert an influence on embryogenesis. We then focused on the Bcl-2- and Bcl-2-associated X protein gene, Bax, to study the responsive gene expression in BeWo cells as well as placentas of pregnant rats fed a diet supplemented with caffeine (2 mg/100 g body weight) during gestation, and analyzed the gene expressions using LightCycler-based quantitative real-time polymerase chain reaction assays. We found a significantly decreased level of Bcl-2 mRNA expression, which demonstrated the influence of caffeine on placental function.
Subject(s)
Caffeine/pharmacology , Gene Expression Regulation/drug effects , Placenta/drug effects , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger/metabolism , Animals , Base Sequence , Cell Line , DNA Primers , Female , Placenta/metabolism , Pregnancy , RNA, Messenger/genetics , RatsABSTRACT
Defensins are a family of cationic antimicrobial peptides that participate in host defense. Human beta-defensin (hBD)-2 has a potent bactericidal activity against a wide spectrum of microorganisms. Because human gingival epithelium is constantly exposed to a variety of microbial challenges, it is considered that hBD-2 has an important role in the protective mechanisms against oral bacterial infection. However, little is known about the production of hBD-2 in tissues of the oral cavity. Six rat monoclonal antibodies (MAbs) raised against chemically synthesized hBD-2 have been characterized. Rat MAbs were specific for the conformational epitopes on hBD-2, but not to hBD-1. To identify the epitope on hBD-2, a series of six overlapping peptides covering the hBD-2 whole sequence were synthesized and the immunoreactivities of six MAbs were examined. The FCPRRYK domain in hBD-2 was recognized by all six MAbs and suggested to be an epitope region. By immunocytochemistry, hBD-2 was localized focally in the epidermis of the human gingival tissue using the MAbs. The MAbs specifically recognized against hBD-2 will be a useful tool to study the functional role of antimicrobial agents and an important asset in the imaging of oral infection processes.
