| 研究課題/領域番号 |
16K09805
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| 研究機関 | 長崎大学 |
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研究代表者 |
ログノビッチ タチアナ 長崎大学, 原爆後障害医療研究所, 助教 (30423643)
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| 研究分担者 |
中沢 由華 名古屋大学, 環境医学研究所, 助教 (00533902)
サエンコ ウラジミール 長崎大学, 原爆後障害医療研究所, 准教授 (30343346)
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| 研究期間 (年度) |
2016-04-01 – 2019-03-31
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| キーワード | mtDNA / MiSeq NGS / mtDNA deletions |
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| 研究実績の概要 |
The aim of our study is, using next-generation sequencing, to evaluate the mitochondrial DNA (mtDNA) deletion profile as a potential individual molecular marker of radiation exposure in radiation-induced and sporadic PTCs and in exposed to ionizing radiation (IR) human primary thyrocytes.
MtDNA is recognized to evolve 10-100 times faster than nuclear DNA due to the aggressive environment reach in oxygen species specific to mitochondrion, increased infidelity of mtDNA polymerase γ, slippage of mitochondrion systems of DNA repair, particulars of mtDNA structure, spatial proximity to mitochondrial membrane, and peculiarities of mtDNA replication and transcription. In our previous work we showed that in contrast to sporadic PTCs, a significant correlation between the prevalence of large-scale mtDNA deletions and relative mtDNA content was found in tumor tissues of radiation-associated PTCs. Additionally, using primary cultures of human thyrocytes exposed to IR (from 0.5 to 5 Gy), we found a significant dose-dependent increase of mtDNA deletions, while mtDNA content did not change. Hence, IR can generate large-scale mtDNA deletions in thyrocytes after exposure, however we could not determine the particularities of deletions caused by IR.
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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
Due to the availability of NGS, it was possible to detect sequence of each particular deletion of mtDNA arising as a result of irradiation.
Using MiSeq (Illumina) we already compared the mtDNA large-scale deletions in N (normal) and T (tumor) counterparts of radiation-induced PTCs from Belarus. In T counterparts we found significantly more deletions then in N counterparts.
During the previous research period we prepared many primary cultures of thyrocytes, exposed them to 0.01, 0.025, 0.05 and 0.1 Gy (low-dose IR) and 0.25, 0.5, 1, 2 and 5 Gy, allowed to recover for 48h before DNA extractions. Using 2 sets of overlapping primers and Prime STAR GXL DNA polymerase (Takara Bio) we will perform PCR to reach amplicon size of 9kb. We purified PCR products using NucleoSpin Gel and PCR Clean-up (Takara Bio) before preparing library for sequencing using MiSeq (Illumina).
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| 今後の研究の推進方策 |
We plan to perform a comprehensive analysis of thyroid mtDNA deletions using MiSeq NGS (Illumina). Using the advantage and accuracy of NGS, we expect to discover radiation-specific mtDNA deletions in primary thyrocytes exposed to different doses (including low doses) of IR, and by comparing the patterns (size, localization in the mtDNA genome and sequence around breakpoints) of deletions in radiation-induced (post-Chernobyl) and sporadic PTCs in different age groups.
1.NGS allows to detect all types of deletions in whole mitochondrial genome with high accuracy and precision.
2.Comparison of mtDNA deletion in radiation-induced and sporadic PTCs in all age groups will allow to determine radiation-specific mtDNA deletions, which will be confirmed in irradiated primary thyrocytes.
3.Together this information is expected to make possible to use mtDNA deletions as individual molecular marker specific to radiation exposure.
4.Additional insights into the etiology of Fukushima PTCs will be obtained.
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| 次年度使用額が生じた理由 |
The fund-consuming parts of the study during the last year included preparing primary cultures of thyrocytes, exposed them to IR, extraction the DNA and PCR amplification. All reagents for these methods were not expensive, that’s budget was not fully used.
Next plan
In this year we plan to use almost all budget for performing NGS using MiSeq (Illumina).
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