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将来の新エネルギーを創出すると期待されている核融合技術には，燃料となるトリチウムが大量に使用され，放射線作業従事者のトリチウムによる被曝が懸念される．また福島第一原子力発電所の廃炉作業の工程では，発生しているトリチウム汚染水対策による，作業従事者の健康問題が懸念されている．トリチウム被曝の形態は，低線量・低線量率の内部被曝が想定されるが，経口・吸入・皮膚吸収により体内に取り込まれたトリチウム水は，全身均一に分布することから影響は小さくないと考えられる．さらに有機結合型トリチウムは生体構成分子として体内に蓄積され，長期被曝を生じるので，トリチウムの化学形の考慮は重要となる．トリチウムの生体影響を検討するために，生物学的効果をX線やγ線と比較する必要があり，トリチウムの生物学的効果比（RBE）の研究が多くされてきた．本総説では，確率的影響に分類される放射線発がんや，発がんの原因となる突然変異など生物効果を指標にして得られたRBE について紹介する．加えて，近年開発されてきた，低線量・低線量率被曝の生物影響を検出可能な遺伝子改変マウスを使った動物実験系の原理と，トリチウム実験の概要についても紹介する．, Nuclear fusion technology is expected to create new energy in the future. However, nuclear fusion requires a large amount of tritium as a fuel, leading to concern about the exposure of radiation workers to tritium beta radiation. Furthermore, countermeasures for tritium-polluted water produced in decommissioning of the reactor at Fukushima Daiichi Nuclear Power Station may potentially cause health problems in radiation workers. Although, internal exposure to tritium at a low dose/low dose rate can be assumed, biological effect of tritium exposure is not negligible, because tritiated water (HTO) intake to the body via the mouth/inhalation/skin would lead to homogeneous distribution throughout the whole body. Furthermore, organically-bound tritium (OBT) stays in the body as parts of the molecules that comprise living organisms resulting in long-term exposure, and the chemical form of tritium should be considered. To evaluate the biological effect of tritium, the effect should be compared with that of other radiation types. Many studies have examined the relative biological effectiveness (RBE) of tritium. Hence, we report the RBE, which was obtained with radiation carcinogenesis classified as a stochastic effect, and serves as a reference for cancer risk. We also introduce the outline of the tritium experiment and the principle of a recently developed animal experimental system using transgenic mouse to detect the biological influence of radiation exposure at a low dose/low dose rate.

赤色蛍光強発現遺伝子導入マウス(tdTomatoマウス)は再生医療研究のバイオイメージングにおいて有望視されている実験動物である.特に,このマウスから採取した組織再生性細胞や組織片をヌードマウスなどの重症複合免疫不全マウスに移植してその組織再生能力を評価する実験系においては,移植後の細胞や組織片の体内動態をその赤色強蛍光を追跡することによりリアルタイムに観察できると期待されている.しかし,このマウスと既存の緑色蛍光強発現遺伝子導入マウス(EGFPマウス)とのバイオイメージングに関する有用性を比較した例はない.今回,我々はtdTomatoマウスの成体においてIVIS^ Lumina Imaging Systemを用いて蛍光シグナル強度を測定し, EGFPマウスと比較検討した.さらに同一試料における各蛍光タンパクのmRNA発現量を計測し,蛍光イメージングの結果と比較した.またtdTomatoマウスについては,生後7日齢を用いて全身の凍結連続切片を作製し,各臓器における組織レベルでの蛍光発現の詳細な検索を行い,蛍光イメージングならびにmRNA発現量との比較を行った.その結果, tdTomatoマウスより摘出されたほぼ全ての臓器において, IVIS^ Lumina Imaging Systemによる高い蛍光シグナル強度が認められたが, EGFPマウスでは肝臓,脾臓,腎臓において顕著な蛍光発現が認められず, EGFPの発現に臓器特異性が存在する可能性が示唆された.また各臓器のtdTomato mRNA発現量もIVIS^ Lumina Imaging Systemにより観察された蛍光シグナル強度と同様の傾向を示した.加えて, tdTomatoマウスの組織切片による観察では,ほとんどの臓器の細胞で蛍光発現を確認できた.しかしながら,蛍光イメージング, mRNA発現量,組織切片による観察結果において,同様の傾向を示さない臓器も認められたことから,今後,このような研究や検証には切片法を含めた複数の手法を用いる必要が示唆された.以上の結果から総合して判断すると, tdTomatoマウスは,既存のEGFPマウスと比較して,その移植実験への応用性の点で有用性が高いことが示唆された., The red fluorescent transgenic mouse has given us a promising insight into regenerative medicine. Each organ of this animal has already been investigated by the method of using a fluorescent imaging system, but not by histological methods. First of all, we compared all the organs of the red fluorescent transgenic mouse with previous studies using enhanced green fluorescent protein (EGFP) mice by the IVIS Luminal Imaging. Next the expression levels of each organs mRNA was measured by RT-PCR. Further, the histological sections of the red fluorescent transgenic mice (7 days after-birth) were taken by light microscope. The results of the three methods were compared and the following findings were made ; We found a discrepancy between the fluorescent imaging method which was used in past reports and the expression levels of mRNA and multiple discrepancies between these and the histological observations. Explaining the discrepancies was beyond the scope of this experiment. We thought that two or more methods including the histological examination were reguired by future research and verification. The red fluorescent transgenic mouse was more useful than the EGFP mouse in the fluorescent expression of the hepar, spleen and ren. All of the organs in the red fluorescent transgenic mouse have a strong fluorescent expression. We hope that these red fluorescent transgenic mice are used in the research of regenerative medicine and the metastic route of the tumor stem cell.

Hepatocellular carcinoma (HCC) is one of the major causes of cancer- death in Japan. Majority of HCC patients are due to chronic hepatitis B or C infection. Interferon is a principal anti-viral agent for chronic viral hepatitis which has variety of biological functions. We particularly focused on the anti-proliferative function of interferon and sought to determine whether the temporal administration of IFN-β prevents hepatocarcinogenesis in a mouse model where HCC develops without necroinflammation. Hepatocarcinogenic mice that are transgenic for the hepatitis B virus X gene (HBx-Tg) were treated with IFN-β or saline (control) for 3 months, from 3 to 6 months of age, and the incidence of HCC was determined at 18 months of age. The incidence of HCC was significantly lower in the IFN-β-treated mice than the controls (0 vs 50%, P

Tissue inhibitor of metalloproteinase-1 (TIMP-1) has been shown to be increased in liver fibrosis development, however, the direct role of TIMP-1 has not been defined. To elucidate the role of TIMP-1 in liver fibrogenesis, we developed liver-targeted TIMP-1 transgenic mice under the control of albumin promoter/enhancer. A model of CCl^4 -induced hepatic fibrosis was used to assess the extent of fibrosis develtpment in TIMP-1 transgenic (TIMP-Tg) mice and control hybrid (Cont) mice. Without any treatment, overexpression of TIMP-1 in the liver did not induce liver fibrosis. After four-week treatment with CCl^4, however, TIMP-Tg-mice revealed a seven-fold increase in liver fibrosis compared to the Cont--mice by densitometric analysis. The hepatic collagen and hydroxyproline content and serum fibrosis markers, such as 7S-collagen, P-Ⅲ-N-P, were also significantly increased in the TIMP-Tg-mice. Furthermore, immunohistochemical analysis showed that collagen-Ⅰ and collagen-Ⅳ accumulations were markedly increased along the sinusoidal lining in the liver of the TIMP-Tg-mice with a pattern similar to that of α-smooth muscle actin (α-SMA) positive cells. Activated hepatic stellate cells (HSC) from TIMP-Tg-mice displayed markedly increased RNA expression of pro-collagen-Ⅰ and collagen-Ⅳ in response to exogenous recombinant TIMP-1 (rTIMP-1). These results suggest that TIMP-1 does not by itself result in liver fibrogeneis, but strongly promotes liver fibrosis development through HSC stimulation.

Article, 信州医学雑誌 38(1): 3-11(1990)

We have demonstrated that type 5 Adenylyl Cyclase (AC5), a major cardiac AC isoform, plays a major role in determining the magnitude of cardiac response to autonomic regulation under microgravity using transgenic mouse models with either disrupted or overexpressed AC5 in the heart (Space Utiliz Res 23, 267-268, 2007). To test the hypothesis that specific AC5 stimulator (NKH477) may be useful for the treatment of autonomic dysfunction after spaceflight, we implanted osmotic mini-pump (1 mg/kg/day) in wild-type mice and analyzed heart rate variability during parabolic flight. The standard deviation of normal R-R intervals, a marker of total autonomic variability, was increased under microgravity in each group. However, the magnitude of the increase was much smaller in NKH477 group. We also examined the ratio between Low Frequency (LF) and High Frequency (HF), a marker of sympathetic activity, and normalized HF, a marker of parasympathetic activity, and found that these parameters in NKH477 group were similar to those in cardiac-specific AC5 overexpression mice we have previously examined. These results indicate that NKH477 could be able to stabilize the changes in autonomic indexes in response to microgravity, suggesting that it may be a useful for the treatment of autonomic dysfunction after spaceflight., 資料番号: AA0063706072

Bone metabolism is performed by bone-degrading osteoclasts and bone-building osteoblasts on bone surface. The osteocyte is a third cell type in bone that is derived from osteoblast lineage cells and embedded in the mineralized matrix. In order to delineate the in vivo function of the osteocyte, we have developed a transgenic mouse model that enables targeted ablation of osteocytes following a single injection of diphtheria toxin. Under normal loading condition, ablation of osteocytes caused aberrant activation of osteoclastic bone resorption with elevated expression of RANKL (Receptor Activator of NF-KB Ligand). In contrast, during unloading by tail suspension, the osteocyte-depleted mice were protected from disuse-induced bone atrophy. These data suggest that osteocytes play a pivotal role in sensing and/or transducing mechanical signals to adapt the structure and function of bone according to changing mechanical environments., 資料番号: AA0063706060

We used transgenic mouse models with either disrupted (AC5KO) or overexpressed AC5 in the heart (AC5TG), and analyzed Heart Rate Variability (HRV) in pre-microgravity level flight, under microgravity, and in post-microgravity level flight during parabolic flight. Heart rate under microgravity became lower than that in pre-microgravity level flight in Wild Type (WT) and AC5TG while it was unchanged in AC5KO. The ratio between Low Frequency (LF) and High Frequency (HF) in HRV analysis became significantly lower under microgravity in WT and AC5TG while there was no such a decrease in AC5KO. Normalized HF became significantly greater in WT under microgravity, and became even greater in AC5TG. In contrast, there was no such increase in AC5KO under microgravity. Accordingly, changes in autonomic indexes in response to microgravity were augmented in AC5TG while attenuated in AC5KO, suggesting that the amount of AC5 plays a major role in determining the magnitude of cardiac responses, at least, to microgravity., 資料番号: AA0063349075