Kim, Youn-Jung
(Cellular and Molecular Toxicology Laboratory, Korea Institute of Science & Technology)
,
Kim, Mi-Soon
(Cellular and Molecular Toxicology Laboratory, Korea Institute of Science & Technology)
,
Jeon, Hee-Kyung
(Cellular and Molecular Toxicology Laboratory, Korea Institute of Science & Technology)
,
Ryu, Jae-Chun
(Cellular and Molecular Toxicology Laboratory, Korea Institute of Science & Technology)
Methylmercury (MeHg) is known to have devastating effects on the mammalian nervous system. In order to characterize the mechanism of MeHg-induced neurotoxicity, we investigated the analysis of transcriptional profiles on human 8k cDNA microarray by treatment of $1.4{\mu}M$ MeHg at 3, 12, ...
Methylmercury (MeHg) is known to have devastating effects on the mammalian nervous system. In order to characterize the mechanism of MeHg-induced neurotoxicity, we investigated the analysis of transcriptional profiles on human 8k cDNA microarray by treatment of $1.4{\mu}M$ MeHg at 3, 12, 24 and 48h in human neuroblastoma SH-SY5Y cell line. Some of the identified genes by MeHg treatment were significant at early time points (3h), while that of others was at late time points (48h). The early response genes that may represent those involved directly in the MeHg response included pantothenate kinase 3, a kinase (PRKA) anchor protein (yotiao) 9, neurotrophic tyrosine kinase, receptor, type 2 gene, associated with NMDA receptor activity regulation or perturbations of central nervous system homeostasis. Also, when SH-SY5Y cells were subjected to a longer exposure (48h), a relative increase was noted in a gene, glutamine-fructose-6-phosphate transaminase 1, reported that overexpression of this gene may lead to the increased resistance to MeHg. To confirm the alteration of these genes in cultured neurons, we then applied real time-RT PCR with SYBR green. Thus, this result suggests that a neurotoxic effect of the MeHg might be ascribed that MeHg alters neuronal receptor regulation or homeostasis of neuronal cells in the early phase. However, in the late phase, it protects cells from neurotoxic effects of MeHg.
Methylmercury (MeHg) is known to have devastating effects on the mammalian nervous system. In order to characterize the mechanism of MeHg-induced neurotoxicity, we investigated the analysis of transcriptional profiles on human 8k cDNA microarray by treatment of $1.4{\mu}M$ MeHg at 3, 12, 24 and 48h in human neuroblastoma SH-SY5Y cell line. Some of the identified genes by MeHg treatment were significant at early time points (3h), while that of others was at late time points (48h). The early response genes that may represent those involved directly in the MeHg response included pantothenate kinase 3, a kinase (PRKA) anchor protein (yotiao) 9, neurotrophic tyrosine kinase, receptor, type 2 gene, associated with NMDA receptor activity regulation or perturbations of central nervous system homeostasis. Also, when SH-SY5Y cells were subjected to a longer exposure (48h), a relative increase was noted in a gene, glutamine-fructose-6-phosphate transaminase 1, reported that overexpression of this gene may lead to the increased resistance to MeHg. To confirm the alteration of these genes in cultured neurons, we then applied real time-RT PCR with SYBR green. Thus, this result suggests that a neurotoxic effect of the MeHg might be ascribed that MeHg alters neuronal receptor regulation or homeostasis of neuronal cells in the early phase. However, in the late phase, it protects cells from neurotoxic effects of MeHg.
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제안 방법
Cluster analysis of MeHg-induced expression profiles of human neuroblastoma SH-SY5Y cells. Cells were treated with 1.4 µM MeHg and then harvested at the indicated times for microanay experiments and the expression data were then analyzed by regression models as described in Materials and Methods. Representative clusters of genes were shown in the clustergram for their temporal pattern of expression.
5-fold difference in the expression level with 5% of q vahie cutoff. For the selected genes, the patterns of gene expression were log transformed, centered by median, and subjected to cluster analyses by centered correlation and average linkage as the similarity/distance metric, using the hierarchical cluster algorithm in Cluster and TreeView software suite.
The real time RT-PCR was performed by using a SYBR supermix kit (Bio-Rad, USA), and running for 40-45 cycles at 95℃ for 20 s and 60℃ for 1 min. The PCR efficiency was examined by serially diluting the template cDNA and the melting curve data were collected to check the PCR specificity. Each cDNA sample was triplicated and the corresponding no-RT mRNA sample was included as a negative control.
대상 데이터
SH-SY5Y cell line was purchased from American Type Culture Collection (CRL-2266) and maintained in a humidified atmosphere of 5% CO2 and 95% air at 37℃. The culture medium was 90% culture medium (50% F-12 and 50% MEM; Gibco, USA) supplemented with 10% fet시 bovine serum (FBS; Gibco, USA) plus penicillin, streptomycin and non-essential amino acid.
데이터처리
Results are shown as means ± SD from three independent experiments. Statistical significance was assessed by a One-way analysis of variance (ANOVA), *P<0.05.
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