| 研究課題/領域番号 |
17K13013
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| 研究機関 | 東京大学 |
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研究代表者 |
モンターニュ ケヴィン 東京大学, 大学院工学系研究科(工学部), 特任講師 (50606118)
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| 研究期間 (年度) |
2017-04-01 – 2020-03-31
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| キーワード | exosomes / cartilage / cell signalling / hydrostatic pressure |
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| 研究実績の概要 |
Exosomes are small vesicles released by most cells and have recently attracted much focus as a very promising tool for regenerative medicine. Those vesicles have been shown to contain various proteins, messenger RNAs and micro-RNAs, and play a crucial role in cell signaling. Hydrostatic pressure (HP) is one of the main mechanical stimuli sensed by cartilage cells during joint loading in vivo and is known to affect the differentiation of cartilage, moderate pressure promoting differentiation, and excessive pressure leading to de-differentiation. The purpose of the project is to understand how HP affects the production of cartilage exosomes and to test whether those findings can be applied to tissue engineering and the prevention of cartilage degradation in osteoarthritis.
During the first year of the project, we have optimized the method for extracting exosomes by a precipitation method, and have shown that HP applied for up to 24h does not affect the quantity of exosomes released in the medium by ATDC5 cells.
During the second year, the effects of exosomes isolated from differentiated ATDC5 cells on un-differentiated ATDC5 cells were investigated. RNA was extracted and characterization of the micro-RNAs contained in exosomes isolated from pressurized cells is now under way.
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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 objective of the first year was to produce and purify exosomes from cartilage cells under physiological and excessive hydrostatic pressure and start their characterization. As originally expected, we have managed to purify exosomes from the culture medium of cells submitted to high hydrostatic pressure. Those were quantified and it was shown that pressure applied for up to 24h did not affect their quantity.
During the second year of the project, exosomes were purified from ATDC5 cells differentiated into chondrocytes and their effect on differentiation was assessed. No significant effect, however, has been observed on ATDC5 differentiation. Identification of micro-RNAs contained in exosomes isolated from cells submitted to high pressure is now under way.
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| 今後の研究の推進方策 |
The objective of the coming year is to identify the RNA species present in exosomes produced under hydrostatic pressure. RNAs have now been isolated from ATDC5 cells submitted to high pressures and microarray analysis is under way. microRNAs will first be identified; messenger RNAs will be investigated if necessary. Gene expression analysis will be performed to confirm the microarray results. Furthermore, exosomes produced under moderate and high pressures will be isolated and their effects on chondrogenic differentiation will be investigated in vitro.
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| 次年度使用額が生じた理由 |
During the second year, microarray analysis was also to be carried out but, in order to increase the reliability of the data, the RNA isolation process had to be improved to enhance the purity of the isolated RNA. RNAs have now been isolated from ATDC5 cells submitted to high pressures and microarray analysis is under way. During the final year, microRNAs will first be identified; messenger RNAs will be investigated if necessary. Gene expression analysis will be performed to confirm the microarray results. Furthermore, exosomes produced under moderate and high pressures will be isolated and their effects on chondrogenic differentiation will be investigated in vitro.
We expect that the improved quality of the RNA will lead to more reliable microarray results that yield meaningful insights into the mechanisms of pressure mechanotransduction, osteoarthritis and cartilage physiology.
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