| 研究課題/領域番号 |
15K08201
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| 研究機関 | お茶の水女子大学 |
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研究代表者 |
グホ サビン お茶の水女子大学, 理学部, 研究員 (30453179)
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| 研究分担者 |
和気 秀文 順天堂大学, スポーツ健康科学部, 教授 (50274957)
宮本 泰則 お茶の水女子大学, 基幹研究院, 准教授 (50272737)
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| 研究期間 (年度) |
2015-04-01 – 2018-03-31
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| キーワード | Microarray / NTS / gender / SHR / pathway / real-time PCR / injection / blood pressure |
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| 研究実績の概要 |
The goal of our research during FY2015 was to identify genes differentially expressed between the NTS of male and female Spontaneous Hypertensive Rats (SHRs). Total RNAs from the NTS of 6 male and 6 female SHRs (8 weeks old) were submitted to one color Sureprint Rat gene expression Microarray (Agilent). The resulting manually curated list of genes presenting a differential expression with a fold change ratio superior to 2 (1336 genes) was submitted to Expression Analysis Systemic Explorer (Huang DW et al. 2009) for Gene ontology “GO” terms enrichment and Pathways analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) or Panther Pathway databases. Interestingly, “Cytokine-cytokine receptor interaction” and “Apoptosis signaling” pathways, including genes previously identified as differentially expressed in the NTS of male SHRs and normotensive WKY (Wistar Kyoto), appeared among the list of pathways significantly associated with genes differentially expressed in the NTS of female and male SHRs. “Neuroactive Ligand-receptor interaction” is another significant pathway including genes potentially involved in the gender differences of the blood pressure neural regulation. So far, Trpv4, Prlr, Trr, Clic6, Kl and Chrm1 genes were selected for validation by quantitative real-time PCR. Additionally, the physiological role of Chrm1 (Cholinergic muscarinic receptor 1) and estrogen on arterial pressure (AP) control was also investigated after injection of carbachol and 17B estradiol, respectively, within the NTS of male and female SHRs.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
We were planning 1) to validate by real-time RT-PCR the differential expression of the candidate genes identified by microarray and 2) to investigate the expression level and localization of the corresponding proteins by using semi-quantitative western-blotting and immunofluorescence staining during the FY2015. We have started the validation of several candidate genes by using real-time RT-PCR experiments. However, we haven't completed this part yet since additional RNA samples from the NTS of male and female SHRs are needed to confirm the statistical significance of the differential gene expression. Also, we started to analyze the microarray data with an different software (Pathway Studio, Elsevier) and expect to find additional candidate genes that will need to be validated by real-time RT-PCR.
As for the investigation of the corresponding proteins expression, we have just set up the western-blotting and immunofluorescence/confocal techniques in my laboratory in Ochanomizu University and we plan to start the experiments during the beginning of FY2016. In another hand, some experiments planned for the FY2017 have been started during the FY2015 due to technical conveniences. Indeed, since the brain injection and AP monitoring system were already available in Juntendo University, we have started to investigate the cardiovascular effect of some candidate molecules (estrogen and Chrm1) by injecting them into the NTS and by monitoring cardiovascular parameters.
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| 今後の研究の推進方策 |
At the beginning of FY2016, the microarray data will be further analyzed with Pathway Studio software (Elsevier). New potential candidates are expected to be discovered, the differential expression of which will be confirmed by using quantitative RT-PCR experiment. The protein expression of the validated genes will be analyzed by using semi-quantitative western-blotting or Enzyme Immuno Assay experiments and immunofluorescence on free-floating brain sections. Then, I will investigate whether the gender specific genes found in the FY2015/2016 are altered by menopause and more specifically by sex hormones. Indeed, loss of estrogen after menopause is associated with an increase of AP in women, suggesting that sex hormones could play a role in the gender differences in AP control. However, the mechanisms responsible for the difference in AP control after menopause are not fully understood. I will characterize the gene and protein expression profiles of gender-specific molecules identified in FY2015/2016 in the NTS of post-menopausal female rats and post-menopausal female treated with estrogen. I will use ovariectomized female rats as a model of post-menopausal condition and estrogen will be chronically subcutaneously infused by osmotic minipumps in some of these animals. Expression level of candidate gender specific genes/proteins will be measured in the cardiovascular centers of intact females, age-matched ovariectomized females and age-matched estrogen-tested ovariectomized females by using quantitative real-time PCR and semi-quantitative western-blotting/Elisa respectively.
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| 次年度使用額が生じた理由 |
We have not performed the protein expression analysis yet as originally planned for FY 2015 so we haven’t bought the consumable for the western-blotting /immunofluorescence experiments yet.
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| 次年度使用額の使用計画 |
With the rest of money from FY2015 to be used in FY2016, we will buy the consumable for protein expression analysis by western-blotting/Enzyme immunoassay and immunofluorescence experiments (antibodies, reagents)
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