Ionizing radiation is a useful tool for cancer therapy. In hematopoietic cell lines such as malignant lymphoma and leukemia cells, apoptotic cell death is prone to be induced by ionizing radiation. In these cell lines, ionizing radiation is known to easily promote apoptotic signaling such as cytochrome c release from mitochondria, followed by activation of caspase and caspase-activated DNase. However, in solid tumor cell lines derived from adenocarcinoma, squamous cell carcinoma or melanoma, the constitutive or inducible antiapoptotic proteins such as Bcl-2 family and the inhibitor of apoptosis protein (IAP) family proteins seem to inhibit this apoptotic signaling. Survivin is a IAP family, which is intensively expressed in the G2/M phase and phosphorylated by cell division cycle 2 (CDC2), and known to have strongly antiapoptotic activity through interaction with Smac/DIABLO which acts as a proapoptotic protein. Its expression is prominently upregulated in most human cancer cells, but is undetectable or very low in normal tissues. Therefore, survivin seems to be an ideal target for radiosensitization in solid tumor cells. In this study, I firstly examined whether dominant negative vectors encoding survivin mutants, T34A (phosphorylation site by CDC2) and D53A (binding domain for smac/DIABLO) abrogate the function of constitutive survivin and facilitate radiation-induced apoptotic cell death in tumor cells. In first experiment, we prepared two adenoviral vectors for pAd-T34A and pAd-D53A to investigate the mechanism of radioresistance of solid tumor cells. When T34A and D53A were overexpressed in human lung carcinoma A549 and human cervical carcinoma HeLa cells, radiation-induced apoptosis was significantly enhanced. Furthermore, we examined the binding capability of survivin with Smac/DIABLO in the cells overexpressing these mutants. Coimmunoprecipitation analysis revealed that mutant forms of survivin, D53A and T34A, could bind to Smac/DIABLO but with much less affinity compared to the authentic form. These results suggest that radiation-induced apoptosis of tumor cells is increased by inhibition of the interaction between survivin and Smac/DIABLO through overexpression of T34A and D53A. These results indicated that radiation-induced apoptosis of tumor cells is increased by inhibition between survivin and Smac/DIABLO through overexpression of T34A and D53A, suggesting that survivin may be effective for therapeutic treatment in radioresistant solid tumors. Recently, it has been reported that apoptosis in human leukemia Molt-4 cells is remarkably inhibited if antioxidants such as N-acetyl-L-cystein (NAC) and Trolox are added to the medium within several minutes after X irradiation. This suggests that the secondary production of ROS occurs as a late event after irradiation and that these secondary ROS play an important role in radiation-induced apoptotic signaling. Mitochondria in mammalian cells are well-known to play an important role in the intrinsic pathway of genotoxic-agent-induced apoptosis by releasing cytochrome c into cytosol and to be a major source of reactive oxygen species (ROS). The second aim of this study was to examine whether mitochondrial ROS are involved in radiation-induced apoptotic signaling in A549 cells. Post-irradiation treatment with NAC inhibited cytochrome c release from mitochondria but did not affect expression levels of Bcl-2, Bcl-XL and Bax, suggesting that the late production of ROS triggered cytochrome c release. This treatment also inhibited radiation-induced apoptosis in T34A- and D53A-survivin-overexpressed A549 cells. Experiments using DCFDA (a classical ROS fluorescence probe) and MitoAR (a novel mitochondrial ROS probe) demonstrated that intracellular and mitochondrial ROS were enhanced 6 h after X irradiation. Furthermore, the ROS production ability of mitochondria isolated from A549 cells was evaluated by ESR spectroscopy combined with a spin-trapping reagent (CYPMPO). When isolated mitochondria were incubated with NADH, succinate and CYPMPO, an ESR spectrum due to CYPMPO–OOH was detected. This NADH/succinate-dependent ROS production from mitochondria of irradiated cells was significantly increased in comparison with that of unirradiated cells. Oxymetry using litium 5,9,14,23,27,32,36-octa-n-butoxy-2,3-naphthalocyanine (LiNc-BuO) and ESR spectroscopy showed that oxygen consumption in irradiated A549 cells, which was inhibited by rotenone, a mitochondrial complex I inhibitor, was increased in comparison with that of unirradiated cells. These results indicate that ionizing radiation enhances the ROS production from mitochondria to trigger cytochrome c release in A549 cells. In summary, present experiments clearly demonstrated that abrogation of the function of constitutive survivin facilitate radiation-induced apoptotic cell death in radioresistant solid tumor cells such as A549 and HeLa cells and radiation-induced secondary ROS from mitochondria play as an essential role in cytochrome c release from mitochondria in their apoptotic signal pathways. / Hokkaido University (北海道大学) / 博士 / 獣医学

To identify the action of BK in the enteric nervous system (ENS), the effects of bradykinin (BK) on the enteric neuron and glial cells were examined by focusing on the interaction between them, using primary culture of myenteric plexus cells from the rat intestine. Furthermore, the impacts of lipopolysaccharide (LPS) or inflammatory cytokines, elevated in serum of inflammatory bowel disease (IBD) patients, on the interaction were investigated. 1. The immunocytochemical analysis using anti-protein gene product (PGP9.5) and anti-S100 antibodies showed that cells in primary culture of myenteric plexus were composed of neurons (30%) and enteric glial cells (68%). The processes of these neurons connected with other neurons and formed neural networks. 2. In both primary cultured myenteric plexus and whole mount preparation of myenteric plexus, less than 10% of neurons showed an immunoreactive (IR) positive to calbindin, a marker of intrinsic primary afferent neuron. BK increased [Ca2+]i to the same extent in both calbindin IR-positive or -negative neurons. 3. BK evoked [Ca2+]i rise in neurons in a dose-dependent manner. The number of neurons responded to BK were increased by increasing concentrations of BK. 4. Both B1 and B2 receptor mRNAs were detected in cultured myenteric plexus cells. The neural [Ca2+]i increase by BK was abolished by a B2 receptor antagonist HOE140, but not affected by a B1 receptor antagonist Lys-des-Ag9-HOE140. A B1 receptor agonist des-Arg9-BK failed to cause a [Ca2+]i response. 5. Double immunostaining using antibodies against B2 receptors together with PGP9.5 or S100 indicated that B2 receptors were expressed in both enteric neurons and glial cells. 6. The neural [Ca2+]i response to BK was attenuated by removal of external Ca2+ or by Cd2+, a blocking agent of voltage dependent Ca2+ channels. In the neurons depleted of Ca2+ from internal stores by thapsigargin, [Ca2+] response to BK became smaller. 7. Indomethacin, a non-selective cyclooxygenase (COX) inhibitor, significantly suppressed the neural [Ca2+]i response to BK. Pretreatment with prostaglandin E2 (PGE2), but not PGI2, significantly potentiated the [Ca2+]i response to BK. Neither PGE2 nor PGI2 affected the resting [Ca2+]i level. An EP1 receptor antagonist SC19220 suppressed the neural response to BK. Unlike PGE2, an EP3 receptor agonist sulprostone did not potentiate the neural response to BK. 8. The release of PGE2 from cultured myenteric plexus cells was increased by BK in a dose-dependent manner, which was inhibited by indomethacin. 9. The [Ca2+]i response to BK in neurons cultured at the high density was significantly larger than that at the low density. The response to BK in neurons was greatly diminished by reducing the number of enteric glial cells in culture. Indomethacin inhibited the response to BK in neurons cultured at the high density, but not the low density. Under the culture conditions of lowering the number of enteric glial cells, indomethacin failed to inhibit the neural response to BK. 10. In cultured myenteric neurons, BK evoked a slow and sustained depolarization and action potential discharges superimposed on it in some neurons. BK-induced depolarization and action potentials were suppressed by indomethacin. The current intensity required for evoking action potentials was decreased by BK, without changing membrane potential threshold of evoking action potentials. 11. BK evoked dose-dependent increases of [Ca2+]i in enteric glial cells, which were hardly diminished by the removal of external Ca2+. In glial cells depleted Ca2+ from internal stores by thapsigargin, BK failed to evoke [Ca2+]i increase. BK-induced glial [Ca2+]i increase was abolished by a phospholipase C inhibitor U73122. 12. [Ca2+]i responses to BK in enteric neurons and glial cells were potentiated by treatment with LPS or IL-1β, but not with TNF-α Similar potentiation was induced by LPS or IL-1β in the pure culture of glial cells. These augmenting effects of LPS on responses to BK were suppressed by pretreatment with an interleukin-1 receptor antagonist IL-1ra. 13. In cultured myenteric plexus cells, the augmenting effects of LPS and IL-1β on neural [Ca2+]i responses to BK were suppressed by reducing the number of enteric glial cells in culture. 14. LPS increased IL-1β secretion either from cultured myenteric plexus cells containing neurons and glial cells or from pure culture of glial cells. 15. IL-1β augmented BK-induced [Ca2+]i increases in both enteric neurons and glial cells in exposure time-dependent manner. In untreated neurons and glial cells, BK-induced [Ca2+]i increase was not affected by a B1 receptor antagonist des-Arg9-HOE140, but abolished by a B2 receptor antagonist HOE140. After cultured with IL-1β, the augmented [Ca2+]i response to BK in both cell types were partly inhibited by the B1 receptor antagonist. The B2 receptor antagonist completely abolished the responses to BK in neurons but not in enteric glial cells. 16. In glial cells cultured without IL-1β, two different B1 receptor agonists, Lys-des-Arg9-BK and BK fragment 1-8, evoked tiny changes in [Ca2+]i. After culture with IL-1β, substantial [Ca2+]i increase occurred with both B1 receptor agonists in enteric glial cells but not neurons. 17. IL-1β potentiated the expression level of mRNA of B1 receptor but not B2 receptor in cultured myenteric plexus cells. The signal intensity of B1 receptor IR increased in enteric glial cells immunoreactive to S100. 18. The EP1 receptor antagonist SC19220 suppressed the IL-1β-enhancing neural response to BK. The amount of PGE2 release by BK from myenteric plexus cells was increased by IL-1β. The B1 receptor agonist produced a significant release of PGE2 from IL-1β-treated myenteric plexus cells but not from untreated cells. 19. Under the conditions of depletion of Ca2+ from internal stores by thapsigargin and the removal of external Ca2+, BK failed to evoke PGE2 release from cultured myenteric plexus cells. A23187, a Ca2+ ionophore, significantly increased PGE2 release, which was suppressed by a non-selective phospholipase A2 inhibitor aristolochic acid. 20. After cultured with IL-1β, either aristolichic acid or indomethacin suppressed the neural [Ca2+]i response to BK and BK-induced PGE2 release from myenteric plexus cells. A selective COX-2 inhibitor, nimesulide, significantly, but not completely, abolished the augmented [Ca2+]i responses to BK. This drug inhibited partly the BK-induced PGE2 release from myenteric plexus cells cultured with IL-1β but not without IL-1β. 21. IL-1β increased the intensity of COX-2 IR in all positive to S100 IR-positive, but not PGP9.5 IR-positive cells. These results shows that BK causes a [Ca2+]i increase and depolarization in rat myenteric neurons through the activation of B2 receptors, which was partly associated with PGE2 released from glial cells in response to BK. Furthermore, these results also indicate that LPS up-regulates B1 receptors and COX-2 in enteric glial cells via secretion of IL-1β in an autocrine fashion. The enhancement of PGE2 production results in the alteration of cross-talk of the neuron-glial interaction. Such functional changes of enteric glial cells may cause dysfunction in the gut of IBD patients. / Hokkaido University (北海道大学) / 博士 / 獣医学

There are many species of boreal or temperate deer (Lincoln 1992) and they are short-day breeders (Sadleir 1987, Lincoln 1985, Loudon and Brinklow 1992) mating in autumn and fawning in early summer (Kaji 1988, Koizumi 1991, Matsuura et al. 2004a). Their seasonal reproductive pattern ensures that fawns are born at a time of year when food is available for lactating and the weather is favorable for survival of offspring (Loudon and Brinklow 1992). Although most of parturition takes place during period of about one month (Guinness et al. 1978a, Koizumi 1991, Birgersson and Ekvall 1997, Bowyer et al. 1998), range of 83 to 135 days for birth date due to late parturitions were reported in sika deer (Koizumi 1991, Matsuura 2004). Late conception leads to late parturition (Matsuura et al. 2004a) and late parturition results in decline in reproductive success of females, defined as the number of fawns a mother reared to one year old over a specified period of time (red deer: Clutton-Brock et al. 1982), due to increase in mortality rate of fawn (Guinness et al. 1978b) or decrease in female conception rate in the following mating season (Clutton-Brock et al. 1983). Therefore, conceptions at the appropriate breeding season and subsequent maintenance of pregnancy throughout gestation are required for females to enhance their reproductive success. Since most of female conceive at the first estrus of the season (Matsuura et al. 2004a), the change in physiological condition from the anestrous season to the estrous season is important factors which responsible for determining the time of conception. However, there is no insight into mechanism for the successful conception and physiological factor influences to the conception date, and even basic information about the reproductive physiology such as ovarian dynamics and changes in hormones around the conception are not well known in sika deer. Therefore this study was conducted to clarify the characteristics of reproductive physiology of sika deer with special interest in around conception and gestation period by revealing the changes in reproductive organs and steroid hormones. There are two approaches to study about the issue; using carcasses and using live animals. Carcasses are available from nuisance control, sport hunting and hunting for the research purpose, and provide information on morphology and histology of reproductive organs in which animals are needed to be killed. Although abundant carcasses can be obtained, information from carcasses reflects the condition only at the time they were sampled. On the other hand, studying live animals provides the temporal changes in reproductive physiology in animals. However, number of captive deer available for physiological study is limited due to lack of institution holding animals, insufficient equipment, and financial problem in Japan. Therefore, combination of these two approaches is essential for further improvement for understanding reproductive physiology of sika deer. Carcasses provide the physiological data such as occurrence of ovulation (Suzuki and Ohtaishi 1993) and steroidogenic ability of corpus luteum (CL; Matsuura et al. 2004c). In the study using carcasses, the reproductive status must be estimated from the condition of the female, and when they are pregnant, gestational ages are estimate from fetal weight in sika deer (Suzuki et al. 1996). However, present method of gestational age estimation will contain an intrinsic error at early gestational stage. The accurate estimation of early gestational age of the samples contributes to revealing characteristics of reproductive physiology around early pregnancy. Study using live animals is advantageous in understanding temporal changes in the individuals and noninvasive examination, such as behavioral observation and fecal progesterone analysis (Matsuura et al. 2004a), provided the important insight in sika deer. However, data of noninvasive examination are limited to indirect changes in reproductive physiology. To know the characteristics around the first estrus and conception more in detail, invasive methods as it has be done in other cervid species, such as blood collection for hormone assay (Plotka et al. 1977, Kelly et al. 1982, Adam et al. 1985, Garcia et al. 2003) and transrectal ultrasonography for follicular and luteal dynamics (Asher et al. 1997, McCorkell et al. 2004, 2006, 2007) of captive animals under restrain or immobilization are also needed. In sika deer, there is a unique reproductive characteristics observed from early pregnancy. It is multiple CLs formation in spite of having singleton (Yamauchi et al. 1984, Suzuki et al. 1992, Suzuki and Ohtaishi 1993). Since both CLs have ability to synthesis progesterone, it is assumed that forming surplus CL have an important role for establishment and maintenance of pregnancy (Matsuura et al. 2004c). However, significance and function of surplus CL is not obvious, and even the origin and the timing of formation of surplus CL are not known. For understanding the conception and maintenance of pregnancy in this species, these questions must be revealed. In present thesis, the author focused on conception and pregnancy period in sika deer. In chapter 1, the temporal ovarian dynamics and changes in steroid hormones were investigated from anestrous to estrous season to know the characteristics during the seasonal transition and to discuss the factor influence to the successful conception. In chapter 2, the temporal ovarian dynamics and changes in steroid hormone during conception and early gestation were investigated and the significance of multiple CLs was discussed. In chapter 3, description of fetal development and estimation of fetal age during early pregnancy was reported. In chapter 4, distribution of steroid hormone receptors in uteri derived from the wild deer were examined to know the steroid hormone action site at the each stage of pregnancy. / 受胎日の遅延はシカ類の繁殖成功度に悪影響を与える。そのため、適切な時期に受胎し、妊娠を維持することは繁殖成功を高める重要な要因となる。そこで本研究では、受胎時期と妊娠維持の時期に注目した。ニホンジカにおいては、卵巣動態やホルモン濃度の変化などの基本的な繁殖生理学的情報が欠けているため、どのような生理的要因が受胎時期に影響を与えているかは不明である。そこで本研究では、受胎時期と妊娠維持の時期周辺の基礎的な生理状態および生理機構の特徴について検討した。第1章では、非発情期から発情期への移行期における卵巣動態とホルモン濃度変化の特徴を明らかにすることを目的とした。8頭の飼育個体を用い、卵胞や黄体の動態、ステロイドホルモン濃度の変化を初回発情周期中とその前後の期間で比較した。発情開始以前から初回発情までは7個体を、初回発情から発情周期3～6回分の期間は3個体を実験に用いた。2～3日間隔で経直腸による超音波画像診断と血中プロジェステロンおよびエストラジオール17.濃度測定を行った。その結果、全ての個体において初回の発情以前に発情を伴わない排卵がみられた。また、それに続く一過性の黄体形成と、低濃度のプロジェステロン濃度の上昇が1回以上観察された。プロジェステロンは発情を引き起こす因子であるため、一過性のプロジェステロンによる感作は、発情期の開始に寄与していると推察された。また、発情周期中には主に2ないし3回の卵胞発育波が観察された。さらに、発情時には子宮頸管粘液の結晶化が観察され、発情の指標となることが示唆された。第2章では、受胎時期と妊娠初期における卵巣動態とホルモン濃度変化の特徴を明らかにし、妊娠しているニホンジカにおいて特徴的に見られる複数の黄体の起源と形成時期を明らかにし、その存在意義を検討することを目的とした。6頭の飼育個体を用いて受胎から妊娠初期までの卵胞と黄体の動態およびステロイドホルモン濃度の変化を2～3日間隔で調べた。その結果、初回の発情で妊娠した個体3頭において2つの黄体が形成された。2つ目の黄体は、発情に伴う排卵の後に出現した最初の卵胞発育波の主席卵胞が起源であり、これがエストロジェンの上昇と共に排卵し、黄体を形成した。一方、2回目の発情で妊娠した個体３頭では、発情後の主席卵胞は排卵せず、黄体は1つしか形成されなかった。そのため、2つ目の黄体は発情期初回の発情で受胎した場合に形成されることが示唆された。また、黄体の数によってプロジェステロン濃度に差が認められなかったため、2つ目の黄体は1つ目の黄体のみでは不十分なプロジェステロンの分泌を補助することで、初回発情での妊娠の維持に寄与していると推察された。第3章では、妊娠初期における胎子の成長を明らかにし、正確な胎齢推定を可能にすることを目的とした。5頭の飼育個体を用い、妊娠59～61日まで2～3日間隔で経直腸超音波画像診断により胎子の成長を観察した。妊娠20～26日までに、全ての個体において胎子が確認された。胎子の直頭殿長、曲頭殿長、頭長、胸深、心拍数を計測した結果、両頭殿長の対数を取った値とその他の計測値は直線的な増加を示した。胎齢推定式を算出した結果、直頭殿長が最も早期から計測可能で、高い相関を示すことが明らかとなった。したがって、直頭殿長を計測することで、正確に妊娠初期の胎齢が推定できることが判明した。第4章では、妊娠期間中の子宮におけるプロジェステロンとエストロジェンの作用部位の変化を推定することを目的とした。複数黄体を持つ24の死体から得られた子宮組織を用いて、発情直後から妊娠末期までのエストロジェン受容体. (ER.)とプロジェステロン受容体(PR)の発現を免疫組織化学的に調べた。胎齢は第3章の結果と既存の方法をもとに推定した。その結果、ER.およびPRが妊娠25日まで確認され、他の家畜反芻動物と比較すると、受胎後も遅い時期まで発現していることが明らかとなった。したがって、ニホンジカでは妊娠25日程度まで、プロジェステロンのみならずエストロジェンが作用していることが示唆された。本研究では、非発情期から発情期、受胎から妊娠初期までの卵胞や黄体の動態と血中ステロイドホルモン変化、さらに妊娠個体の子宮におけるステロイドホルモン受容体の分布から、これまで情報の少なかったニホンジカの発情期への移行期、受胎時期や妊娠初期における繁殖生理学的特徴を明らかにした。また、妊娠初期の正確な胎齢推定の方法を確立したことにより、今後野外で得られたサンプルの有効活用を可能にした。 / Hokkaido University (北海道大学) / 博士 / 獣医学

神経伝播を示すウイルスは末梢神経系から中枢神経系へと神経を伝播し、致死的な脳炎・脳症を惹起する。神経伝播を示すウイルスとしては、狂犬病ウイルス、単純ヘルペスウイルス、ボルナ病ウイルス、オーエスキー病ウイルス、豚血球凝集性脳脊髄炎ウイルスなどが挙げられる。これらのウイルスの神経細胞内での感染および増殖機構を明らかにすることは、ウイルスの性状を理解する上でも、これらのウイルス感染による疾患の予防・治療法を開発する上でも重要である。脊髄背根神経節（DRG）は中枢神経系へのウイルスの侵入門戸またはウイルス増殖の場として重要な役割を果たしており、DRG 細胞はこれまでに神経親和性ウイルスの感染および伝播メカニズムを解明するための実験に利用されてきた。当研究室では、これまでに新生マウスDRG 初代培養細胞を用いたin vitro の実験系で高病原性トリインフルエンザウイルスの神経伝播の証明を行い、さらに細胞骨格阻害剤を用いてこの伝播が微小管依存性軸索輸送とは異なることを報告している。本研究では神経伝播を示すウイルスとして、豚血球凝集性脳脊髄炎ウイルス（HEV）、オーエスキー病ウイルス（PRV）、狂犬病ウイルス（RV）を選択し、これらの感染および増殖について新生マウスDRG 初代培養細胞を用いたin vitro 実験系で検索した。第1 章では、HEV の神経細胞における感染および増殖と細胞骨格との関連について、微小管依存性に細胞内輸送をされることが証明されているPRV と比較検討した。まず、新生マウスのDRG 初代培養細胞にHEV またはPRV を接種し、その感染性を免疫蛍光染色により検討した。その結果DRG 神経細胞では、HEV およびPRV 共にウイルス抗原が認められた。一方、非神経細胞（シュワン細胞および線維芽細胞）においては、ウイルス抗原がPRVでは認められたが、HEV では全く認められなかった。また、微小管または中間径フィラメントの選択的阻害により、DRG 神経細胞におけるHEV およびPRV の感染は有意に抑制された。これらの成績から、PRV と比較してHEV の感染はより強い神経細胞特異性を有すること、および神経細胞での感染はPRV と同様に微小管と中間径フィラメントに依存することが明らかとなった。第2 章では、非常に強い神経親和性を有し、末梢神経から中枢神経系へと神経伝播することが報告されているRV を用いて、同様の実験を行った。RV の検出にはnucleoprotein（N 蛋白）に対するモノクローナル抗体を用いた。RV 抗原は神経細胞と非神経細胞のいずれにおいても検出されたが、神経細胞の抗原陽性細胞数はHEV やPRV と比較して低値であった。また、微小管、中間径フィラメント、マイクロフィラメントの選択的阻害により、RV 抗原陽性細胞数に有意な変化は認められなかった。以上の成績より、RV 抗原陽性細胞数の低値は、神経細胞内での増殖速度もしくは伝達速度の遅さによるものと推察され、これが動物およびヒトの狂犬病における潜伏期間の長さと関連している可能性が考えられた。RV の感染および輸送が微小管およびアクチンフィラメントに依存することが過去に報告されており、今回の成績と異なる理由については、ウイルス粒子の輸送およびパッケージングにはこれらの細胞骨格成分が関与するが、ウイルスN 蛋白は微小管およびアクチンフィラメントとは独立した機構により合成される可能性が推察された。また、RV の非神経細胞への感染が認められたことから、これらの細胞がRV の神経細胞内での感染および増殖の支持細胞として関与している可能性と、シュワン細胞における感染がRV の神経伝播に関与する可能性が考えられた。以上の結果は神経親和性ウイルスの感染および伝播様式の多様性を示唆しており、これらのウイルス感染症の予防・治療法を開発する上で重要な知見と考えられた。 / Hokkaido University (北海道大学) / 博士 / 獣医学

The senescence-accelerated mouse (SAM) was developed through selective breeding of the AKR/J strain based on a graded score for senescence, life span, and pathological phenotypes. There are nine senescence-prone (SAMP) strains and three senescence-resistant (SAMR) strains. SAMP strains have a shortened life span and show early manifestations of senescence, such as various skin lesions and increased lordokyphosis, after a period of normal development. Among SAMR strains, SAMR1 is long-lived, showing resistance to early senescence, and is used as a control. Among SAMP strains, SAMP6 is considered to be a model of senile osteoporosis with slow bone loss after 4 months of age. Recently, it was reported that SAMP6 exhibited increased expression of S100β in the brain compared to SAMR1, suggesting that SAMP6 is also useful as a model of age-related diseases related to central nervous system alterations. I performed a battery of behavioral analyses using 1- (juvenile stage), 4.6- (adult stage), and 8.12-month-old (old stage) SAMP6 and age-matched SAMR1 to investigate the age-related changes in behavioral features and the mechanisms involved. The battery of behavioral analyses revealed innate behavioral alterations in SAMP6, including higher motor activity, lower anxiety, increased short-term memory, a motor coordination deficit, and antidepressant activity. The higher motor activity of SAMP6 was observed until the adult stage, and then the motor activity began to decline, and lower motor activity was observed at the old stage, indicating that the motor activity of SAMP6 exhibited the same pattern of age-related changes as seen in the bone mass of SAMP6. The marked motor coordination deficit of SAMP6 was observed at the juvenile and old stages, whereas amelioration in the motor coordination deficit was seen in the adult stage, suggesting that the motor coordination of SAMP6 exhibited a pattern of age-related changes similar to that of the bone mass of SAMP6. On the other hand, the differences in anxiety and antidepressant activity between SAMP6 and SAMR1 decreased gradually with age, indicating that the lower anxiety and antidepressant activity of SAMP6 showed another pattern of age-related change. No apparent age-related change was observed in the increased short-term memory of SAMP6. Accordingly, the behavioral features of SAMP6 were divided into three categories based on the pattern of age-related changes: (1) accelerated-senescence-like behaviors; (2) behaviors with age-related changes; and (3) behaviors with no age-related changes. The expression of tyrosine hydroxylase, an enzyme involved in the biosynthesis of dopamine, and its phosphorylated form was increased in the striatum and nucleus accumbens (NAc) of juvenile SAMP6, suggesting an increase in the concentration of dopamine in the juvenile SAMP6 brain. This was thought to be one of the innate alterations related to the higher motor activity of SAMP6. Increased expression of D1 in the striatum, an over-activated D1 signal cascade, and an increased dopamine concentration in the NAc were seen in adult SAMP6, which seemed to explain the higher activity of adult SAMP6. An apparent decrease in the sensitivity of D1 of old SAMP6 compared to adult SAMP6 was observed, which was thought to be involved in the decreased motor activity of old SAMP6. These results suggest that an age-related alteration in the D1 sensitivity of SAMP6 is one of the mechanisms altering motor activity, one of the accelerated-senescence-like behaviors observed in this strain. On the other hand, the increased D3 expression in the cerebellum of adult SAMP6 was thought to be one of the mechanisms related to the motor coordination deficit, another accelerated-senescence-like behavior observed in this strain. However, further examinations of the D3 expression levels in juvenile and old SAMP6 cerebellum are needed to evaluate whether the altered D3 expression is involved in the accelerated-senescence-like alteration of this behavior. The serotonin system was studied to examine the mechanism of the lower anxiety and antidepressant activity, behaviors with age-related changes, of SAMP6. The expression of tryptophan hydroxylase, a serotonin biosynthesis enzyme, and its phosphorylated form were increased in the brainstem of juvenile SAMP6, suggesting an increase in the serotonin concentration in the juvenile SAMP6 brain. This was thought to be one of the innate mechanisms related to the lower anxiety and antidepressant activity of SAMP6. Serotonin concentrations were increased the cortex and NAc of adult SAMP6, which likely explained these behavioral patterns in adult SAMP6. However, further examination of the serotonin concentration of juvenile and old SAMP6 brains is needed to evaluate whether the altered serotonin concentration is involved in the age-related change of these behaviors. The dopamine and serotonin systems and N-methyl-D-aspartate (NMDA) receptors were studied to examine the mechanisms for increased short-term memory, a behavior with no age-related changes, of SAMP6. As mentioned above, the increased dopamine and serotonin concentrations in the juvenile SAMP6 brain were also thought to be one of the innate changes related to the increased short-term memory of SAMP6. In addition, expression of the NMDA receptor subunit 2B (NR2B) was increased in the forebrain of adult SAMP6, and this appeared to be involved in the increased short-term memory of adult SAMP6. Further examination of the mechanisms involved in this behavioral property of old SAMP6 is needed. In this study, a battery of behavioral analyses using animals at three different ages showed various behavioral alterations with aging. In addition, biochemical and pharmacological approaches revealed the involvement of several different mechanisms in the behavioral alterations. These results suggest that the higher brain functions are controlled by variable thresholds of the respective neurons and complex neuronal networks. Studies using SAMP6 might elucidate the influences of aging on higher brain functions and related mechanisms, resulting in the specification of the signal cascades that activate higher brain function and the development of new drugs that act on these cascades. These could increase the healthy longevity and quality of life of humans. / Hokkaido University (北海道大学) / 博士 / 獣医学

Hokkaido University (北海道大学) / 博士 / 獣医学

Autoimmune glomerulonephritis, a complication of systemic autoimmune disease, is caused by renal glomerular deposition of immune complexes circulating through the body. Previous studies have reported that the telomeric region of chromosome 1 in some disease model mice contains several loci that are associated with autoimmune glomerulonephritis susceptibility. However, it is unclear whether these loci originated inform the same genes. In order to detect autoimmune glomerulonephritis at an early stage and provide subsequent therapy, it is essential to identify the candidate genes and aggravating factors of this disease. In this study, we created an MRL/MpJ-type congenic mouse that carries the telomeric region of chromosome 1 and is susceptible to autoimmune disease. We confirmed the application of this congenic strain in autoimmune glomerulonephritis models, pathology analyses, and the search for candidate genes or aggravating factors of this disease. We created an MRL/MpJ-type congenic mouse (B6.MRLc1) which was homozygous in the telomeric region of chromosome 1 (82-100 cM) [B6.MRLc1(82-100)] by applying serial backcrosses between C57BL/6 and (C57BL/6 × MRL/MpJ)F1. The B6.MRLc1(82-100) mice showed mild glomerular damage from 6 months of age. Their glomerular basement membranes were hypertrophic and contained IgG depositions. Glomerular damage in the B6.MRLc1(82-100) mice was more severe in the females; their glomerulus deteriorated with age. Furthermore, splenomegaly, which occurs due to proliferation of CD3- or B220-positive cells, was observed in the spleen of the B6.MRLc1(82-100) mice. Serum levels of blood urea nitrogen (BUN) and the anti-double strand DNA (dsDNA) antibody in the B6.MRLc1(82-100) mice were found to have increased with age and were also significantly higher than those of C57BL/6 at 6 months of age. From these findings, it became evident that the B6.MRLc1(82-100) mice had developed autoimmune glomerulonephritis and the candidate gene derived from MRL/MpJ was localized on chromosome 1 (82-100 cM). We named the loci in the B6.MRLc1(82-100) strain associated with autoimmune glomerulonephritis susceptibility "Mag" (MRL autoimmune glomerulonephritis). In order to reduce the number of regions of these loci, we produced 3 new B6.MRLc1 strains, namely, B6.MRLc1(82-86), B6.MRLc1(92-100), and B6.MRLc1(100), and assessed their pathology. Of these 3 strains, only the B6.MRLc1(92-100) strain showed glomerular damage with immune complex deposition in addition to elevated spleen weights, serum BUN and anti-dsDNA antibody concentrations, and urinary albumin excretions. These results confirm the localization of autoimmune glomerulonephritis candidate genes on chromosome 1 (92-100 cM). For the selection of the candidate gene, relative mRNA expression levels of Crp (pentraxin-related C-reactive protein), Apcs (serum amyloid P-component), Fcgr2b (low-affinity Fc receptor for IgG IIB), and Fcgr3, which are major immune-associated genes present in chromosome 1 (92-100 cM), were analyzed in the major organs. A high expression of Fcgr3 and an elevated Fcgr3/Fcgr2b ratio were observed in the kidney, spleen, and thymus of the B6.MRLc1(92-100) strain. Both the inhibitory and active Fc gamma receptors coded by Fcgr2b and Fcgr3, namely, FcγRIIB and FcγRIII, respectively, were localized on glomerular mesangial regions and dendritic cells in the lymphoid organs. The FcγRIII expressions of the B6.MRLc1(92-100) strains in these regions were stronger than those of C57BL/6. From these findings, the inhibitory and active FcγR genes were considered to be candidate genes. It was strongly suggested that the imbalance between those genes had a great impact on the pathogenesis of autoimmune glomerulonephritis. Female mice showed a higher incidence of systemic lupus erythematosus (SLE), which is the most common autoimmune disease. Further, similar sex-related differences were observed in the B6.MRLc1(82-100) strain; glomerulonephritis was more severe in the female mice than in the male mice. Since sex hormones were considered to be one of the many definitive factors responsible for these sex-related differences, we castrated sex organs of both sexes of the B6.MRLc1(82-100) strain and assessed the effects of gonadectomy in inducing autoimmune glomerulonephritis. In sham operated groups, the female mice showed significantly higher values than the male mice with regard to the glomerular damage scores, the percentage of IgG- and CD3-positive glomerulus, the spleen weights, and the serum levels of total IgG and anti-dsDNA antibodies. The effects of gonadectomy in the male mice were more apparent than those in the female mice. The male gonadectomy group showed significantly higher values than the male sham operated group with regard to the glomerular damage scores, the percentage of IgG- and CD3-positive glomerulus, the urinary albumin excretions, the spleen weights, and the CD3-positive areas in the spleen. Interestingly, CD3-positive cells were observed in both the thymic cortex and the thymic medulla, in all animals except the males in the sham operated groups. On analysis of the active Fcgr3/Fcgr2b ratio in the kidney, the male gonadectomy groups and the female sham operated groups were found to have significantly higher values of the active Fcgr3/Fcgr2b ratio than the male sham operated groups. These results suggested that the pathological sex-related differences in autoimmune glomerulonephritis were strongly affected by the inhibitory roles of male sex hormones. The elevated levels of expression of inflammatory cytokines, chemokines, and their receptors were recently clarified by the array analysis using humor or biopsy samples from patients. These factors are attracting researchers as the targets for disease controls. We attempted to identify the aggravating factors for autoimmune glomerulonephritis in the B6.MRLc1(82-100) strain by studying the expressions of inflammatory mediators by comprehensive PCR array analysis. In the kidney of the B6.MRLc1(82-100) strain, several genes of chemokine ligands and their receptors showed remarkably high expression levels. The expressions of the Cxcl5 gene [chemokine (C-X-C motif) ligand 5] in the B6.MRLc1(82-100) strain were 72-fold higher than those in C57BL/6; the expressions of this gene increased over time in the B6.MRLc1(82-100) strain. The CXCL5 protein coded by the Cxcl5 gene was mainly localized on glomerular podocytes. In contrast, the gene that showed the highest expression in the kidney of the B6.MRLc1(82-100) strain was Il1f6 (Interleukin 1 family member 6), which is a member of the IL-1 family; these expressions were 186-fold higher than those of C57BL/6. Furthermore, Il1f6 gene expressions increased over time in the kidneys of the B6.MRLc1(82-100) strains. The expressions of the Il1f6 gene and its protein were observed in the kidneys from the distal tubules to the collecting ducts. The dilated tubules in the kidneys of the B6.MRLc1(82-100) strains, in particular, showed a higher level of expression of this protein. Interestingly, the number of renal tubules containing the IL-1F6 protein was in proportion to the number of renal tubules containing protein casts as indices for proteinuria. These results suggest that the Cxcl5 and Il1f6 genes, which were highly expressed in the kidney of the B6.MRLc1(82-100) strain, play important roles as aggravating factors of glomerular damage and tubulointerstitial lesions. Thus, by research on disease model mice, it was concluded that the quantitative changes in candidate genes derived form mouse genome, sex hormones, and local aggravating factors in mouse kidney were closely related to the pathogenesis of autoimmune glomerulonephritis. These findings will provide fundamental knowledge that will enable early diagnosis and subsequent therapy of autoimmune glomerulonephritis in the fields of medicine and veterinary science. / Hokkaido University (北海道大学) / 博士 / 獣医学

1997 年香港で発生して以来、H5N1 ウイルスによる高病原性鳥インフルエンザの発生が続いている。筆者は2004 年、日本で発生した高病原性鳥インフルエンザの病原ウイルスであるA/chicken/Yamaguchi/7/2004 (H5N1) (山口株) および、2005 年モンゴルのErkhel 湖で発見されたオオハクチョウの斃死体から分離された高病原性鳥インフルエンザウイルス、A/whooper swan/Mongolia/3/2005(H5N1) (モンゴル株) の鳥類および哺乳動物に対する病原性を実験室内で確認した。山口株は調べた4 つの鳥類に対して高い病原性を示し、全身感染を起こすことが分かった。しかし、マウスに対する病原性は低く、ミニブタには感染しなかった。モンゴル株は、山口株に対する感受性が低かった幼ガモおよびマウスに高い病原性を示し、さらにミニブタには感染することが確認された。これらの結果から、山口株は鳥類に対して非常に高い病原性を示すが、哺乳類には病原性が低いものと考えられる。またモンゴル株は山口株よりも多くの種類の動物に対して病原性を示し、ミニブタに感染することから、公衆衛生上非常に重要であることが示唆された。次にＨ5 ウイルスによる高病原性鳥インフルエンザに対して有効なワクチンの開発およびその評価を行った。高病原性鳥インフルエンザの防疫の基本は摘発淘汰であるが、防圧困難な非常時に備え、高力価のワクチンを開発および備蓄することが必要である。そこで筆者はH5N2 およびH7N1 亜型の2 株の非病原性ウイルスからH5N1 亜型の遺伝子再集合ウイルスを実験室内で作出し、それをワクチン株とした。ワクチン株を鶏胚尿膜腔内に接種して得た尿液のウイルスを不活化し、256.512HA/0.1ml 相当の油中水型ワクチンを試製した。ワクチン0.5ml をニワトリ4 週齢のニワトリの下脚部筋肉内に1 回注射し、免疫3 週後にワクチン株と抗原性が類似する山口株または抗原性が異なるモンゴル株で攻撃したところ、いずれの場合もニワトリは臨床症状を示すことなく14 日間耐過した。さらに、免疫後の日数が異なるニワトリに、HPAI ウイルス株にて攻撃したところ、ワクチン接種後6 日以内のニワトリは攻撃ウイルスにより全て死亡したが、ワクチン接種8 日目のニワトリはHI 抗体が検出されないにも関わらず、HPAI ウイルスの攻撃に対して耐過した。これらの結果から、本ワクチンはアジアで近年流行している高病原性鳥インフルエンザの病原ウイルスに有効であり、発症防御効果も接種8 日目から確認されたことから、緊急用ワクチンとして有用であることが判った。 / Hokkaido University (北海道大学) / 博士 / 獣医学

The rat is an important model for understanding human physiology and disease. The rat contributes to the biomedical researches of human cardiovascular disease, diabetes, arthritis and many behavioral disorders. In particular, the rat has frequently been used in the fields of pharmacology and toxicology. Thus, pharmaceutical companies use rats for a large proportion of their mandatory toxicity testing. However, inter- and intrastrain differences in drug response are often observed among rat strains, and are an important issue to be overcome. Still, the fundamental mechanism of inter- and intrastrain differences regarding drug metabolizing enzymes have been overlooked. In this thesis, I focused on the CYP2D subfamily that is a key enzyme involved in individual differences in drug metabolism. CYP2D2 enzyme is known to metabolize the majority of typical substrates of the human CYP2D6 enzyme. Despite its impact on drug metabolism in rats, the transcriptional regulation of CYP2D2 remained to be elucidated. In chapter 1, I clarified the molecular mechanism of CYP2D2 gene expression. The CYP2D2 gene was positively regulated by the poly(C)-binding protein hnRNP K through a transcriptional regulatory element located in the 5'-flanking region from -94 to -113. Acting as a docking platform, the hnRNP K protein is known to be implicated in the regulation of transcription as an activator or a repressor, as a translational repressor, and as a participant in a variety of signaling systems. Although it has only been reported that hnRNP A1 protein, one of the hnRNPs, involves in the stabilization of the CYP2A5 and CYP2A6 mRNA, nothing is known about the potential role of hnRNP K in P450 gene regulation. Thus, this is the first report that hnRNP K protein is involved in CYP2D2 gene regulation. Furthermore, I elucidated the genetic basis of the extremely low expression of CYP2D2 mRNA in DA rats. Due to its relative low abundance, DA rats have been frequently used for the study of CYP2D substrate metabolism as the animal model of PM phenotype for CYP2D6 in comparison to SD rats as an EM phenotype. To take full advantage of physiological variation among rat strains, it is essential to further unravel the mechanism of low expression of CYP2D2 mRNA. I found a single substitution within the transcriptional regulatory element of the CYP2D2 gene in DA rats. The mutation was detected in the polypyrimidine sequence that is the preferred binding site for hnRNP K protein. Indeed, the mutation within the transcriptional regulatory element abolished the binding of hnRNP K protein. Thus, I conclude that decreased recruitment of hnRNP K protein to the mutated sequence causes the low expression of CYP2D2 mRNA in DA rats. Inter- and intrastrain differences in drug response in rats are a difficult problem for basic research as well as drug discovery as described above. Currently, information on genetic variation in laboratory rat strains is rapidly accumulating as a set of microsatellite markers, simple sequence length polymorphism markers and a variety of single nucleotide polymorphism in coding regions. Nevertheless, little is known about the differences in their drug metabolism characteristics that are ascribed to genetic variation. In chapter 2, I clarified the mechanism underlying inter- and intrastrain differences in diazepam p-hydroxylation among rat strains. The recent studies demonstrated that the pharmacokinetics of diazepam, which is one of the benzodiazepines, were quite different among rat strains because of its metabolic polymorphisms in diazepam p-hydroxylation. Although diazepam p-hydroxylation is a major metabolic pathway, SD and BN rats had 300-fold higher diazepam p-hydroxylation activity than DA rats at low concentration of diazepam. And Wistar rats showed 200-fold intrastrain differences in diazepam p-hydroxylation activity (EM-W > PM-W). In addition, it was suggested that CYP2D subfamily was involved in this activity. Based on these observations, I separated the specific protein expressed in liver microsomes of SD, BN and EM-W, and identified the specific protein to be CYP2D3. Then, I confirmed that only CYP2D3 but no other CYP2D isoforms had a diazepam p-hydroxylation activity. To date, there is little information about the catalytic specificity of CYP2D3. Thus, I demonstrated that CYP2D3 was involved in diazepam p-hydroxylation. Moreover, I analyzed the genetic polymorphism of the CYP2D3 gene among rat strains. I found a single insertion in exon 8 in DA and PM-W rats. A premature termination codon created by this frameshift consequently deleted the heme-binding region that is essential to maintain proper heme binding and active P450 enzymes. Therefore, I conclude that the deficiency of a functional CYP2D3 protein must be the cause of the significantly low diazepam p-hydroxylation in DA and PM-W rats. Additionally, the genotype frequency of CYP2D3 polymorphism is in good agreement with the phenotype frequency among rat strains. In this thesis, I clarified the genetic bases of inter- and intrastrain differences in CYP2D-dependent drug metabolism in rats. Clarification of inter- and intrastrain differences in rats will be further useful for predicting variability in human pharmacokinetics. Consequently, it is worthwhile to fully characterize animals used in pharmacokinetics studies from the point of view of the genetic expression of metabolic enzymes. Therefore, my work will strongly support the strain consideration in selection of a rat strain for drug metabolisms. / シトクロムP450（CYP, P450）は、生理活性物質の合成や代謝だけでなく、医薬品や環境汚染物質などの外来異物の代謝にも関与する酵素群である。特にヒトCYP2D6は、医薬品の約30％を代謝するにも関わらず多くの遺伝多型が存在する事から、薬効の個人差や副作用の発現に深く関与している。このため、CYP2D分子種は、毒性学上、非常に重要な分子種として位置付けられている。そこで本研究では、CYP2D分子種依存の薬物代謝反応におけるラットの系統差及び個体差、そしてそれらを引き起こすメカニズムについて、明らかにする事を目的とした。第１章では、Dark Agouti(DA)ラットにおけるCYP2D2mRNA低発現機構を解明した。DAラットは、ヒトCYP2D6の典型的基質を代謝する能力が著しく低いため、ヒトCYP2D6の代謝欠損者の動物モデルとして用いられている。ヒトCYP2D6の典型的基質の多くは、ラットCYP2D2によって代謝され、特にDAラットでは、Sprague-Dawley（SD）ラットやWistar系ラットに比べ、CYP2D2 mRNA及び蛋白質発現量が低い事が、CYP2D分子種依存の代謝活性が低い原因であると報告されている。しかし、DAラットにおいて、CYP2D2 mRNA発現量が低下している原因については、未だ明らかにされていない。そこで、CYP2D2遺伝子の5’上流域を、4kbにわたりシークエンス解析を行ったところ、DAラットにおいてTATAボックスの近傍にシトシンからチミンへの一塩基置換がある事を見出した。一方、SDラットでは同領域に変異は認められなかった。次にElectrophoresis mobility shift assayから、DAラットにおいて検出された一塩基置換は、核蛋白質(転写因子)/DNA複合体の形成を低下させる事が確認された。また、CYP2D2遺伝子のプロモーター領域の各種欠失変異体を用いたレポーターアッセイの結果、この一塩基置換により転写活性は約1/4に低下した。さらに、Matrix-assisted laser desorption/ionization time-of-flight mass spectrometryを用いて、CYP2D2遺伝子発現に関与する転写因子の同定を試みた。その結果、転写装置の足場蛋白として働く多機能な核蛋白質として知られる、ヘテロ核内リボ核蛋白質 K(heterogeneous nuclear ribonucleoprotein K: hnRNP K)が、コアプロモーター領域に結合する事を明らかにした。よって、DAラットにおけるCYP2D2mRNAの低発現は、CYP2D2遺伝子のプロモーター領域内の一塩基置換により、hnRNP K蛋白質のDNA結合が低下する事が原因である事を初めて明らかにした。第２章では、ジアゼパム代謝における系統差及び個体差発現機構を解明した。抗不安薬として世界で広く使用されているジアゼパムには、3位水酸化、N脱メチル化、p位水酸化の3つの代謝経路が存在する。これまでの研究から、低基質濃度ではジアゼパムp位水酸化が、ラットにおける主要な代謝経路であり、かつ著しい系統差(約300倍)及び個体差（約200倍）を示す事が報告されている。さらに、抗体を用いた阻害実験から、CYP2D分子種がこの代謝反応に関与する事が示唆されたが、CYP2D2の発現量の差では説明できない事が報告されており、未だその代謝酵素の同定には至っていない。そこで、ウエスタンブロット法を用いて、高活性個体のみに特異的に発現する蛋白質を単離した。アミノ酸シークエンスによってN末端配列を決定したところ、今まで主な触媒反応がわかっていない、CYP2D3のアミノ酸配列と完全に一致した。酵母に発現させたCYP2D3は、ジアゼパムp位水酸化活性を示したが、CYP2D2にはその活性がない事も再構成系実験を用いて確認した。さらに、定量リアルタイムPCR法により、CYP2D3 mRNA量を測定した。ところが、系統間及び個体間で、p位水酸化活性と相関のある発現量の差は認められなかった。そこで、CYP2D3のcDNAシークエンスを調べた。低活性個体では、一塩基挿入によるフレームシフトが認められ、P450の活性中心であるヘム結合領域の上流に終止コドンが形成されていた。このため、p位水酸化の低活性個体では、代謝機能を失った蛋白質が合成される事が予想された。したがって、CYP2D3の翻訳領域内の一塩基挿入により、機能的なCYP2D3をコードするmRNAの発現が低下する事で、ジアゼパムp位水酸化の系統差及び個体差が引き起こされる事を初めて明らかにした。以上の結果から、CYP2D分子種依存の薬物代謝反応における、ラットの系統差及び個体差発現の分子機構を明らかにした。ラットは、特に薬物代謝研究において多用されているにも関わらず、薬物代謝酵素に関する系統間及び個体間での遺伝的背景については、これまで明らかにされていなかった。また、CYP2D分子種は医薬品に対する感受性を決定する、最も重要な分子種の一つである事から、本研究成果は、薬物代謝研究におけるラットの系統を選択する際に、非常に有用な指標を与えるものと考えられる。 / Hokkaido University (北海道大学) / 博士 / 獣医学

For chronic kidney disease (CKD) patients with renal anemia, recombinant human erythropoietin (rhEPO) is a very effective drug; however, the treatment regime is troublesome, requiring multiple administrations each week. In veterinary field, rhEPO treatment has been also introduced; however, multiple administrations each week is necessary like human case. To resolve this problem, the method of sustained release of biologically active rhEPO over a period of two weeks or more should be necessary. Hydroxyapatite (HAp) is a biocompatible ceramic, widely used in the biomaterial field. HAp particles have been examined for application in the sustained release of various therapeutic agents, such as antibiotics, anticancer drugs and proteins. HAp has the ability to absorb therapeutic agents without deactivation and shows regulated release by biodegradation. The biodegradation speed of HAp can be regulated by calcination temperature. Spray-drying has been shown to be a good fabrication method for spherical porous HAp powder with a large surface area. In the present study, the efficiency of HAp as a drug delivery carrier for the sustained release of rhEPO was examined to reduce the frequency of administration. Spray-dried HAp microparticles, formed from zinc-containing HAp (Zn-HAp) or Zn-HAp calcined at 400℃, were used as carriers of EPO, and five Zn-HAp formulations incorporating rhEPO were prepared; no formulation, zinc (Zn) formulation, poly-L-lactic acid (PLA) formulation, Zn/PLA formulation, and calcined/Zn/PLA formulation. ICR mice were administered subcutaneously these formulations or rhEPO alone as a control from dorsal neck, and hematological and histopathological analyses, including enzyme-linked immunosorbent assay for plasma EPO concentration, were performed. The efficiency of the sustained release was the lowest in no formulation among the five formulations. For the other formulations, the peak hematopoiesis was delayed and higher hematological values remained until day 14. Further, plasma EPO levels were higher in these formulations than those in control on day 3. A reduction in the initial burst was observed in Zn/PLA formulation, and plasma EPO levels remained high until day 8 in Zn formulation and Zn/PLA formulation. This indicates that sustained EPO release could be achieved by using the Zn and/or PLA formulations on the Zn-HAp microparticles. The ICGN (ICR-derived glomerulonephritis) mouse is an inbred strain showing the hereditary nephrotic syndrome due to a mutation of the tensin2 gene. With the deterioration of renal function, ICGN mice developed a normochromic and normocytic anemia, which is consistent with clinical reports on patients with renal anemia. The expression of EPO mRNA in the kidneys was significantly reduced in ICGN mice. Thus, ICGN mice are an authentic model for human anemia with CKD. In the present study, Zn and Zn/PLA formulations were examined whether these formulations could ameliorate the severe anemia in ICGN mice more efficiently compared with EPO alone. As a result, the hematological parameters in anemic ICGN mice administered rhEPO alone were not different from that of day 0, indicating that anemic ICGN mice might be resistant to rhEPO treatment similar to human CKD patients. As a matter of fact, rhEPO should be administered to patients three times a week to maintain suitable levels of serum rhEPO. The hematological parameters in ICGN mice administered both formulations increased and the peak of hematopoiesis was observed on day 7 and slightly decreased after day 14; however; they were always higher than that of rhEPO alone until day 21. These data suggest that both formulations are useful for sustaining the release of rhEPO in vivo. Although macroscopic observation showed that both formulations still remained in the subcutaneous tissue on day 21, these formulations did not cause any significant inflammatory reactions. The biodegradability of HAp microparticles injected subcutaneously was known, however, more detailed examinations for degradation of both formulations remaining in the subcutaneous tissue are necessary as well as the adverse long-term effects and the excretion mechanism to be studied. In conclusion, HAp was recognized as a novel drug carrier to achieve sustained release of rhEPO. According to in vivo release test of rhEPO from HAp in ICGN mice, elevated hematopoiesis were maintained for 21 days. There was no adverse effect during and after the administration. Further optimization study is necessary to achieve longer sustained release of rhEPO to establish curative DDS for anemia. / Hokkaido University (北海道大学) / 博士 / 獣医学