| Project/Area Number |
16K14764
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Multi-year Fund |
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| Research Field |
Morphology/Structure
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| Research Institution | Kindai University (2017-2019)
University of Tsukuba (2016) |
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Principal Investigator |
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| Project Period (FY) |
2016-04-01 – 2020-03-31
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| Project Status |
Completed (Fiscal Year 2019)
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| Budget Amount *help |
¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
Fiscal Year 2018: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2017: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2016: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
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| Keywords | 細胞分散 / プロテアーゼ / 消化酵素 / 酸性プロテアーゼ / 細胞培養 / ペプシン / 大腸菌発現系 / トランスクリプトーム解析 / RNA解析 |
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| Outline of Final Research Achievements |
Analysis of the rather rare and minor cells are generally difficult, as they are apt to be masked and hidden by the others, i.e., the majority of the cells. Conventional methods of dissociation of the cells via digestion of live cells with collagenases are not suitable for the minority cells, because the nature of the cells may be altered easily by the environmental changes during the enzymatic digestion. Here, tissue cells are dissociated via digestion with an acidic enzyme after fixation of the tissues. I found acidic conditions that are practically affordably for fixation and digestion without any degradation of RNA. In addition, a fluorescent protein was verified to be available as a reporter molecule in the downstream experiments performed in the acidic conditions. However, degradation of cells by proteases was inevitable in the conditions strict enough to dissociate cells, at least so far. Further experiments are necessary to establish a gold standard method.
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| Academic Significance and Societal Importance of the Research Achievements |
私生物のからだを構成する細胞は、同じように見えてもそれぞれに個性をもっている。個々の細胞を他の細胞から引き離して、発現するRNAを調べることで初期のがん細胞をはじめ細胞の振る舞い、いわば細胞の個性を知ることができる。本研究では、細胞の固定前後にRNA分解が起こらない酸性条件を特定した。その条件下ではたらく酸性プロテアーゼにより細胞の分散を試みたが細胞の損傷を抑えて機能する最適条件は今後の研究に委ねられる。
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