| 研究課題/領域番号 |
16K09805
|
|---|
| 研究機関 | 長崎大学 |
|---|
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, 特任研究員 (30423643)
|
|---|
| 研究分担者 |
中沢 由華 長崎大学, 原爆後障害医療研究所, 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)
|
|---|
| 研究期間 (年度) |
2016-04-01 – 2019-03-31
|
| キーワード | mtDNA / MiSeq NGS / mtDNA deletions |
|---|
| 研究実績の概要 |
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 ionizing radiation (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.
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.
|
| 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
During the 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).
|
| 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.
|
| 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.
|
| 次年度使用額の使用計画 |
In this year we plan to use almost all budget for performing NGS using MiSeq (Illumina).
|
| 備考 |
長崎大学原研医療
http://www-sdc.med.nagasaki-u.ac.jp/drms/
|
