| 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, 学部教育研究協力員 (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, ヒューマンライフイノベーション研究所, 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 / cardiovascular centers / gender / SHR / RT-qPCR / ovariectomy / treatment / blood pressure |
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| 研究実績の概要 |
Data from microarray experiments performed in FY2015 on NTS (Nucleus of Solitarii Tract) and two additional cardiovascular centers (hypothalamus and ventral medulla) from female and male SHRs (Spontaneous Hypertensive Rats) were analyzed with David and Pathway Studio softwares. Specific pathways were identified as predominantly enriched in the list of genes differentially expressed between males and females tissues, among them, cytokine-cytokine receptor interaction, cell adhesion molecules and neuroactive ligands in the NTS, Arachidonic acid metabolism in the hypothalamus and ventral medulla and long term potentiation in the ventral medulla. New candidates were selected for validation and RT-qPCR experiments confirmed the gender-specific differential expression of CDH1 and TRPV4 in the NTS, CDH1, Col1A1 and Col3A1 in the hypothalamus, GRIA2 in ventral medulla of SHRs. The protein expression of GRIA2, a subunit of the ionotropic AMPA type of glutamate receptors, was confirmed in the ventral medulla neurons by immunohistochemistry. The injection of glutamate (non selective agonist for GRIA2) was performed into the ventral medulla and induced changes in arterial pressure (AP) parameters. To analyze the role of sex hormones on gender differences in gene expression, twelve female SHRs were ovariectomized and estrogen was chronically subcutaneously infused by osmotic minipumps in six of them for one month. Vaginal smears confirmed that ovariectomy and estrogen infusion were both successful. Brain tissues (cardiovascular centers) were extracted for gene expression analysis.
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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
The goals of our research during FY2016 was to 1) analyze microarray data with additional softwares 2) complete the validation of gender specific gene expression in SHRs 3) assess whether these validated genes are influenced by estrogen infusion in a model of post-menopausal rat.
These goals were, for the most part, achieved and additional experiments were performed. The validation of gene expression changes took longer than expected because of high variability between rats. In fact, a search for the most reliable reference genes for RT-qPCR had to be undergone and additional tissues were collected from new groups of SHRs to increase the biological replicates number. Also, we strengthened our study by analyzing microarray results from other cardiovascular centers and assessing the expression profile of the validated genes in WKY rats, a normotensive strain that does not exhibit gender difference in AP level at similar age. Thus, experiments of ovariectomy and estrogen infusion were slightly delayed, however, the surgery and treatment were successful, brain tissues are now ready for processing. As for the protein expression assessment, we could confirm the presence of GRIA2 in the rat ventral medulla neurons by immunofluorescence. However, the differential expression between males and females could not be concluded. The effect of glutamate (a non-selective agonist for GRIA2) on AP parameters was confirmed after its microinjection in the ventral medulla of SHRs, however, the comparison between males and females is difficult to quantify due to a different basal AP.
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| 今後の研究の推進方策 |
During FY 2017, I will complete the validation of microarray results and the characterization of gender specific gene expression profile in the NTS and other cardiovascular centers of SHRs. Some newly validated candidate genes such as TRPV4 (Transient receptor potential cation channel subfamily V member 4), known for inducing hypotension, remain to be tested for their gender-specific expression in the cardiovascular centers of WKY. The location and expression level of the corresponding proteins will be assessed by immunohistochemistry and/or western botting when possible. The sensitivity to estrogen of the validated genes expression will be tested in the cardiovascular centers of ovariectomized female SHRs treated with estrogen. The functional role of the validated genes will be investigated by injection of target molecules into the cardiovascular centers. The study of the functional role of TRPV4 will be investigated in priority by injecting its agonist (4apDD) and antagonist (RN1734) into the NTS of male and female rats. The relationship between the basal level of cardiovascular parameters and gene expression of validated molecules is expected to help elucidating parts of the mechanisms of primary hypotension in females.
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| 次年度使用額が生じた理由 |
Our original plan has been slightly delayed due to the difficulty to validate microarray results with RT-qPCR methods. Thus, we haven't performed all the expected experiments related to the validation of candidate genes at the protein expression level yet and we haven't purchased all the material necessary for western-blotting and immunohistochemistry yet.
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| 次年度使用額の使用計画 |
With the rest of money from FY2016 to be used in FY2017, we will purchase western-blotting and immunohistochemistry consumable for candidate protein expression analysis in male and female rats.Target molecules to be microinjected into the brain cardiovascular centers for validated genes functional role study will also be purchased.
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