Research Projects -
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Evaluate seed compounds for cancer suppressor SPRED2
2024.04 - 2025.03
AMED 橋渡し研究戦略的推進プログラム
Authorship：Principal investigator
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Development of DDS film and sheet materials that can be absorbed by the body
2024.04 - 2025.02
AMED 岡山県特別電源所在県科学技術振興事業
Authorship：Principal investigator
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Analysis of the mechanisms by which FPRs play a role in the immune regulation against cancer and organ transplantation and its application to treatments
Grant number：23K08070 2023.04 - 2026.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
吉村 禎造, 松川 昭博
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
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AI病理診断に向けた、組織切片の厚さの簡易計測法の開発と標準化技術の確立
Grant number：23K11895 2023.04 - 2026.03
日本学術振興会 科学研究費助成事業 基盤研究(C)
藤澤 真義, 大原 利章, 松川 昭博
Grant amount：\4160000 （ Direct expense: \3200000 、 Indirect expense：\960000 ）
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Mechanisms of cancer metastasis by message Capsule Exosomes
Grant number：22K19562 2022.06 - 2025.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory) Grant-in-Aid for Challenging Research (Exploratory)
松川 昭博
Grant amount：\6500000 （ Direct expense: \5000000 、 Indirect expense：\1500000 ）
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Development of Visualized Submucosal Injectables Using Polysaccharide Derivatives to Address Unmet Needs
2022.04 - 2023.02
AMED 特別電源設置県科学技術振興事業研究委託事業
Authorship：Principal investigator
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Role of Spred2 in the pathogenesis of cancer and its application for cancer thepapy
Grant number：21H02988 2021.04 - 2024.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
松川 昭博, 伊藤 嘉浩, 吉村 禎造, 宮武 秀行, 阪口 政清
Grant amount：\17420000 （ Direct expense: \13400000 、 Indirect expense：\4020000 ）
１）がんの増殖・転移・幹細胞性獲得におけるSpred2の役割解明
肝がん細胞株（HepG2, HepG3, HLE）のSpred2をノックダウンすると細胞増殖能はいずれも増加した。最もSpred2発現の多いHepG2細胞からCRISPR-Cas9法でSpred2を消去した細胞を作製し、がん細胞特性を確認した。その結果、がん細胞増殖能・浸潤能は増加し、細胞形態は紡錘形に変化、上皮間葉移行と幹細胞性は増強した。これらの変化は、Spred2の過剰発現で逆転した。Spred2発現は非がん部に比較してがん部で低く（データベース上、および自験例の検討）、Spred2高発現肝がん患者では低発現患者に比べ有意に予後は高かった（データベース解析）。以上より、Spred2は、ERK経路を抑制し、がん細胞のEMTや幹細胞性を負に制御することを明らかにした（現在、論文投稿中）。
２）ヒトがん組織におけるSpred2の発現
275の尿路上皮腫瘍の検討結果から、Spred2 mRNA発現は高異型度非浸潤性乳頭状尿路上皮癌 (HGPUC)で最も高く、上皮内癌 (CIS)と浸潤性尿路上皮癌 (IUC)で低下し、Spred2タンパク発現はHGPUCで高く、CISとIUCではHGPUCに比べて減少していることを見いだした。HGPUCでSpred2膜発現を示すものはSpred2膜陰性のものと比べ、ERK活性化レベルとKi67 indexが有意に低かった。以上より、Spred2は非浸潤性膀胱癌の進展を調節する鍵であるものと考えられた（PLoS One. 2021 Nov 24;16(11):e0254289）。
３）Spred2のがん治療への応用
SPR-domainのみのSpred2に加え、さらなる短鎖Spred2プラスミドを６種類作製した。これらを用いて、全長Spred2との活性比較を行っている。 -
Multimodal cancer therapy by combination of immune therapy and targeting nanostructured carrier containing anti-cancer drug
Grant number：21H04965 2021.04 - 2024.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A) Grant-in-Aid for Scientific Research (A)
伊藤 嘉浩, 上田 一樹, 鵜澤 尊規, 秋元 淳, 松川 昭博
Grant amount：\42380000 （ Direct expense: \32600000 、 Indirect expense：\9780000 ）
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アンメットニーズに応える多糖誘導体を用いた可視化粘膜下注入材の開発
2021.04 - 2022.03
岡山県特別電源所在県科学技術振興事業 岡山県特別電源所在県科学技術振興事業
Authorship：Principal investigator Grant type：Competitive
Grant amount：\2000000
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Development of a new concept of embolization material for colonic diverticulosis
2020.04 - 2021.03
AMED 橋渡し研究戦略的推進プログラム
Authorship：Principal investigator
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大腸憩室症に対する新発想の塞栓材開発
2020.04 - 2021.03
橋渡し研究戦略的推進プログラム 橋渡し研究戦略的推進プログラム シーズA
Authorship：Principal investigator Grant type：Competitive
Grant amount：\2000000
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リン酸化プルランの大量・精密製造技術の確立と短期骨再生を可能とするペースト状人工骨の開発
2019.04 - 2022.03
経済産業省 戦略的基盤技術高度化支援事業補助金
Authorship：Coinvestigator(s) Grant type：Competitive
Grant amount：\130000000
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Development of transplanted lung function-preserving method using a mouse model focusing on the anti-inflammatory related molecule Spred2
Grant number：19K09306 2019.04 - 2022.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Yamane Masaomi
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
A lung injury experiment using a thoracotomy and clamp model was also performed using Spred2 knockout mice and transgenic mice that are Spred2 gene overexpressing mice. Histological images by RT-PCR and H-E staining and arterial gas analysis were performed and evaluated as preliminary experiments, but no difference was observed. Furthermore, the expression level was confirmed by Western blotting using an anti-mouse SPRED2 antibody, but no difference was observed in the preliminary experiment.
Overexpression of Spred2 at protein and mRNA levels could not be confirmed in Spred2 transgenic mice and conditional knockout mice specifically deficient in bone marrow-derived cells, compared with wild-type in experiments using a hilar clamp model. No significant difference was found in ischemia-reperfusion injury. -
多糖誘導体を用いた高操作性可視化粘膜下注入材の開発
2019.04 - 2020.03
岡山県特別電源所在県科学技術振興事業 岡山県特別電源所在県科学技術振興事業
Authorship：Principal investigator Grant type：Competitive
Grant amount：\2000000
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Inhibition of formylpeptide receptors for treatment of breast cancer
Grant number：18K08571 2018.04 - 2021.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Yoshimura Teizo
Grant amount：\2600000 （ Direct expense: \2000000 、 Indirect expense：\600000 ）
Tumor infiltrating leukocytes play an important role in the progression of cancer. In this study, we examined the role of the classical chemoattractant receptor Fpr expressed by non-tumor cells in the progression of breast cancer using a transplantable 4T1 mouse breast cancer model. Tumors grew at a similar rate in both strains at the injected sites; however, lung metastases were significantly reduced in Fpr2-deficient mice independently of the chemokine MCP-1 because serum MCP-1 levels were similar between tumor-bearing WT and Fpr2-deficient mice. In tumors of Fpr2-deficient mice, the development of blood vessels was weaker and necrosis of tumor tissue was more apparent. Ly6G+ neutrophils were detected mainly in the peripheral area and did not infiltrate inside tumors. Thus, Fpr2+ neutrophils appear to play a role in the progression of 4T1 breast cancer. Studies focusing on Fprs may lead to the identification of new targets and a potential prevention of breast cancer metastasis.
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Grant number：17K10930 2017.04 - 2020.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
TANAKA Masato
Grant amount：\4680000 （ Direct expense: \3600000 、 Indirect expense：\1080000 ）
The phosphorylated pullulan developed by the applicants is a functional polysaccharide derivative that firmly adheres to apatite, which is an inorganic component of living hard tissue. We aimed to develop a film-shaped phosphorylated pullulan that can be expected as a bioadhesive and a carrier for drug delivery, and to combine it with BMP to develop a safe and low-cost bone fusion promoter. The bone formation ability of the rat posterior lateral fixation model was examined, and good bone formation was observed in the BMP-2-added phosphorylated pullulan film group. In this study, phosphorylated pullulan was considered to have extremely low cytotoxicity in bone formation, and could be safely applied clinically.
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Grant number：16H05310 2016.04 - 2020.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
Ito Toshihiro
Grant amount：\16770000 （ Direct expense: \12900000 、 Indirect expense：\3870000 ）
Patients who have a chronic respiratory disease have a very high risk of respiratory infection due to viruses and bacteria, and it is necessary to elucidate the pathological condition and molecular mechanism and identify the factors that affect each other's diseases. In this study, we focused on SET domain, bifurcated 2 (SETDB2), which is one of the histone H3K9 (9th lysine) methylase, and the expression of SETDB2 showed a significant increase in the chronic respiratory disease model. The importance of SETDB2 in respiratory diseases was clarified by increasing the expression in chronic respiratory diseases and respiratory infections and by indicating the role of SETDB2 in pathological conditions.
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Challenge to novel antiviral strategy by suppressing signal transduction pathway
Grant number：16K15258 2016.04 - 2019.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research Grant-in-Aid for Challenging Exploratory Research
MATSUKAWA AKIHIRO, Yang Xu, Sun Cuiming, Gao Tong, Miyatake Hideyuki, Kondo Masayuki
Grant amount：\3640000 （ Direct expense: \2800000 、 Indirect expense：\840000 ）
In this study, we clarified the mechanism of viral infection from the viewpoint of Ras-Raf-ERK / MAPK and its endogenous suppressor Spred2, and we challenged the antiviral strategy via ERK suppression by supplementation of Spred2. Activation of ERK-MAPK and viral infection were suppressed by Spred2 gene transfer. There was no difference in virus infection in Spred2 overexpressing mice, suggesting that the endogenous Spred2 is sufficient to inhibit the infection. Cell-penetrating Spred2 created in this study failed to inhibit ERK activation, however, supplementation of functional Spred2 protein may become a new antiviral drug.
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Grant number：16K10823 2016.04 - 2019.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
Arataki Shinya
Grant amount：\4680000 （ Direct expense: \3600000 、 Indirect expense：\1080000 ）
We analyzed the usefulness of new intervertebral disc regeneration treatment using intervertebral disc degeneration model of rats in combination with cultured disc annulus fibrosus cells and novel, visible light-induced, rapidly cross-linkable gelatin scaffold. Transplanted cells were found to engraft on the transplanted site, but regeneration of the annulus fibrosus was not identified. As a growth factor used for regenerative therapy, we studied BMP-7 that has been reported in the past, but this study suggested the possibility of contribution to degeneration in the annulus fibrosus.
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Grant number：16K10822 2016.04 - 2019.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
Sugimoto Yoshihisa
Grant amount：\4810000 （ Direct expense: \3700000 、 Indirect expense：\1110000 ）
We analyzed the usefulness of novel spinal cord injury (SCI) treatment using engineered hepatocyte growth factor with a collagen biding domain (CBD-HGF) and visible light-induced, rapidly cross-linkable gelatin in spinal cord injury model of mice. In motor recovery and immunohistochemical analyses, therapeutic effects were identified. CBD-HGF combined with the hydrogel scaffold may be promising for the treatment of serious SCI. About neural stem cells (NCS), we established stem cell culture technology using mouse embryos, however we could not reach transplantation into spinal cord injury model. We plan to continue the study by combining these methods.
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Development of fragility fracture treatment using molecular evolution engineering
Grant number：15K10445 2015.04 - 2018.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
Watanabe Noriyuki
Grant amount：\4810000 （ Direct expense: \3700000 、 Indirect expense：\1110000 ）
We investigated the availability of a titanium binding bone morphologic protein(TB-BMP), which is a new biological material. TB-BMP coating titanium wires and conventional titanium wires were implanted in the femur of rats. TB-BMP coating titanium wires significantly enhanced bone formation on the surface of titanium compared to conventional titanium wires. Pull-out strength was also significantly increased in TB-BMP group. These results suggest that TB-BMP is a useful material for the treatment for the osteoporotic patients.
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Grant number：15K21184 2015.04 - 2017.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B) Grant-in-Aid for Young Scientists (B)
Nosaka Nobuyuki, MORISHIMA Tsuneo, NISHIBORI Masahiro, TSUKAHARA Hirokazu, MATSUKAWA Akihiro, LIU Keyue, YASHIRO Masato, YAMADA Mutsuko, HATAYAMA Kazuki
Grant amount：\2600000 （ Direct expense: \2000000 、 Indirect expense：\600000 ）
Provision for an influenza pandemic is an urgent issue. We aimed to evaluate the therapeutic effects of anti-high mobility group box-1 (HMGB1) monoclonal antibody (mAb) treatment on severe pneumonia induced by influenza A virus in mice. Anti-HMGB1 mAb significantly improved the survival rate of mice with severe influenza pneumonia with attenuated histological changes in the lungs. In addition, anti-HMGB1 mAb also improved the survival rate of mice with influenza pneumonia with 50% lethality even with anti-influenza drug administration. There were no adverse effects found by anti-HMGB1 mAb administration.
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感染防御機能を有する革新的骨スクリューの開発
2014.04 - 2017.03
経済産業省 医工連携事業化推進事業
Authorship：Principal investigator Grant type：Competitive
Grant amount：\200000000
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New Implant Coating Technology with Phosphopullulan Bioadhesive Materials
Grant number：26462298 2014.04 - 2017.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
Kagawa Yohei
Grant amount：\4940000 （ Direct expense: \3800000 、 Indirect expense：\1140000 ）
In the orthopaedic surgery, the early fixation due to the new bone formations around the implant is important. Hydroxyapatite (HA) is often coated on the cementless implant surface. A variety of coating methods have been used, but the best coating procedure still remains controversial. A purpose of this study is to get promotion of the new bone formations by coating HA on implants with phosphopullulan (PP) as matrix. In the mechanical examination, in the quantitative elemental analysis, and in the histological examination, there was evidence of bone neoformation on the surface of the implants in PP+HA groups than in any other groups.
The PP has little harm to the living body, and does not remain in the body as an alien substance to be absorbed in vivo. The PP coating had a beneficial effect on interfacial shear strength and peri-implant new bone formation in rabbit femurs during the early stages of bone healings. -
The effect of the phospho-pullulan cement for the treatment of metastatic bone tumor
Grant number：25462335 2013.04 - 2016.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
Shinohara Kensuke, OZAKI TOSHIFUMI, MATSUKAWA AKIHIRO
Grant amount：\4940000 （ Direct expense: \3800000 、 Indirect expense：\1140000 ）
We investigated the availability of phosphorylated pullulan using with the therapeutic agents of bone malignant tumor. Breast cancer cell line (MDA-MB231), which is high frequency as the primary focus of metastatic bone tumor, was injected into the bone hole that was created by using a 24G needle to the central condylar of the nude mouse femur.These are divided into four groups, non-treatment group, the anti-cancer agent alone group, the anti-cancer agent + phosphorylated pullulan group, and phosphorylated pullulan group. We evaluated the antitumor effect using IVIS successively. Anti-cancer agent was using docetaxel. The antitumor effect was observed in the anti-cancer agent alone group and the anti-cancer agent + phosphorylated pullulan group. This result showed that the phosphorylated pullulan was suggested to be useful as a containing cement of anticancer agent.
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Regulatory mechanism of inflammation and inflammation-related cancer by regulating Ras-Raf-ERK/MAPK
Grant number：25293095 2013.04 - 2016.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
MATSUKAWA AKIHIRO, ITO Toshihiro, TAKAHASHI Sakuma, FUSHIMI Soichiro, TAIRA Asako, ITAKURA Junya, WATANABE Haruyuki, SATO Miwa
Grant amount：\17160000 （ Direct expense: \13200000 、 Indirect expense：\3960000 ）
In this project, we have analyzed the role of Ras-Raf-ERK/MAPK and its endogenous inhibitor, Spred-2 in various animal models, using Spred-2 knock-out (KO) mice. We have demonstrated that Spred-2 plays a central role in the regulation of inflammation in various types of inflammation. Our results suggest that Spred-2 can be a new target for controlling inflammation. We also demonstrated that Spred-2 expressed in clinical cancer tissue may be involved carcinogenesis and cancer progression. Furthermore, our results indicate that Spred-2 may be essential regulator of cancer metastasis by modulating epithelial mesenchymal transition (EMT).
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リン酸化プルランを用いた世界初の多目的接着性人工骨の開発
2012.04 - 2015.03
研究成果最適展開支援プログラム A-STEP 起業挑戦タイプ
Authorship：Coinvestigator(s) Grant type：Competitive
Grant amount：\150000000
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インフェクションコントロール型チタン骨プレートの開発
2012.04 - 2014.03
研究成果最適展開支援プログラム Ａ-ＳＴＥＰ A-STEP 復興促進プログラム シーズ顕在化タイプ
Authorship：Principal investigator Grant type：Competitive
Grant amount：\7997000
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2012 - 2013
産学が連携した研究開発成果の展開 復興促進プログラム A-STEP シーズ顕在化タイプ
松川 昭博
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可視光硬化型ゼラチンを用いた新規歯周病治療用接着材の開発
2011.04 - 2013.03
研究成果最適展開支援プログラム A-STEP 起業検証タイプ
Authorship：Coinvestigator(s) Grant type：Competitive
Grant amount：\8000000
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可視光硬化型ゼラチンを用いた新規歯周病治療用接着材の開発
2011.04 - 2013.03
研究成果最適展開支援プログラム A-STEP 探索タイプ
Authorship：Coinvestigator(s) Grant type：Competitive
Grant amount：\1700000
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Grant number：23592822 2011 - 2013
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
NAKAMURA Mariko, YOSHIDA Yasuhiro, MATSUKAWA Akihiro, ITO Yoshihiro
Grant amount：\5200000 （ Direct expense: \4000000 、 Indirect expense：\1200000 ）
The purpose of this study is to develop a multi-purpose bio-adhesive system by the visible light-induced crosslinkable geratin and phosphorylated pullulan. The following things were confirmed from these findings. Phosphorylated pullulan-based composite is adhesive to dentin and possesses high biocompatibility. The visible light-induced crosslinkable gelatin is adhesive to dentin and low cellular toxicity. It was suggested that the visible light-induced crosslinkable gelatin and phosphorylated pullulan can be used as key materials of biodegradable adhesives for regeneration and reconstruction of bone and tooth.
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進化分子工学と先端接着技術の応用による骨粗鬆症の新しい骨折予防法・治療法の開発
Grant number：23592187 2011
日本学術振興会 科学研究費助成事業 基盤研究(C) 基盤研究(C)
塩崎 泰之, 尾崎 敏文, 松川 昭博
Grant amount：\5200000 （ Direct expense: \4000000 、 Indirect expense：\1200000 ）
進化分子工学の手法を用いて創製する結合性改変成長因子Collagen Binding Domain (CBD)等の新規生体材料を効果的に用いることにより,骨粗鬆症患者の骨折予防や骨折治療に有効な新しい骨組織再生・再建技術を開発する。
CBDの遺伝子配列と,骨形成促進蛋白-4(BMP4)の遺伝子配列を連結した融合遺伝子をベクターにいれ,蚕の卵に導入することによりトランスジェニックカイコを作製する。この時,遺伝子は選択的に絹糸腺で働くようにしおく。絹タンパク質と同時に繭に吐き出されたCBD-BMPは,できた繭から精製することにより得られた。
ウエスタンブロット法を用いて、作製したタンパク質を測定すると55kDaであった。
続いて、このCBD-BMPと通常のrhBMPとの効果の比較をマウス大腿骨へ投与する事で検討した。まず、蛍光標識を行ったそれぞれの成長因子を投与後1,3,7日でマウスを屠殺後に非脱灰凍結切片を作成し蛍光顕微鏡で評価した。CBD-BMPに関しては7日後であってもマウス大腿骨内への残存が確認できた。
続いて、投与後4週後に屠殺し、両群をμCTで撮影し骨密度を比較した。これも2群間で有意にCBD-BMP群が優れていた。(P=0.0489)
また、これらの大腿骨骨髄からRNAを抽出しRT-PCRを用いて両者を比較した。(CBD-BMP,BMP,生理食塩水の3群)HPRTをハウスキーピング遺伝子としALP,Osteocalcin,Osterixを計測し、P=0.035,0.0599,0.0126とCBD群が優れていた。
これらの結果よりCBD-BMPは、成長因子の効果を発現するために必要な標的組織への定着が確認できた。また、その効果に関しても放射線学的、遺伝子学的にも確認できた。これらを用いる事で効果的な骨形成が得られ,骨粗鬆症患者の骨折予防や骨折治療に有効である。 -
Grant number：22220009 2010.04 - 2015.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (S) Grant-in-Aid for Scientific Research (S)
ITO Yoshihiro, TADA Seiichi, UZAWA Takanori, UEDA Motoki, MATSUKAWA Akihiro, YOSHIDA Yasuhiro, SAKURAGI Makoto, KITAJIMA Takashi
Grant amount：\217880000 （ Direct expense: \167600000 、 Indirect expense：\50280000 ）
The concept of growth factor immobilization from Japan was confirmed by not only chemical immobilization, but also design of binding growth factor and extended from biological tissue or organic materials to metal or inorganic materials. For the creation of binding growth factors not only conventional recombinant gene method but also bioorthogonal chemistry, molecular evolutionary engineering, or the fused method was developed. The prepared binding growth factors were applied for direct injection or with substrate for implantation into animals and the effects were confirmed for future regenerative medicine. The developed methodology is also universally applicable for creation of novel functional molecules.
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Role of T cells in the innate immune response during sepsis
Grant number：20390111 2008 - 2010
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
MATSUKAWA Akihiro, OKAZAKI Yasumasa, HIDA Akira, KUBO Masato
Grant amount：\11960000 （ Direct expense: \9200000 、 Indirect expense：\2760000 ）
T cells exist in the peritoneum under healthy conditions. Rag2Ko mice (T and B cell deficient) showed increased mortality rate during sepsis, which was recovered after adoptive transfer of T cells, but not B cells. Mice with overexpression of SOCS5 in T cells (SOCS5-cTg) demonstrated increased Th1 response in the peritoneum whereas SOCS3-cTg mice exhibited decreased systemic inflammatory response during septic peritonitis, leading to the increased survival rate during sepsis. Thus, T cells appear to be important in innate immunity, which can be controlled by SOCS3/5 in T cells. We also elucidated the crucial roles of T cells in different types of inflammation models, such as T cell-dependent hepatitis and arthritis.
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Grant number：20249053 2008 - 2010
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A) Grant-in-Aid for Scientific Research (A)
MORISHIMA Tsuneo, OKABE Nobuhiko, NAKAGOMI Osamu, NUNOI Hiroyuki, KONDOU Kazuhiro, HOSOYA Mitsuaki, MATSUKAWA Akihiro, KIMURA Hioroshi, YOSHIKAWA Tetsushi, ICHIYAMA Takashi, OKUMURA Akihisa
Grant amount：\49400000 （ Direct expense: \38000000 、 Indirect expense：\11400000 ）
We have studied on the clinical features and pathogenesis of acute encephalitis/encephalopathy caused by viruses among children. As an causative agent, Influenza virus was a most common virus, followed by HHV-6 and rotavirus, and overall mortality was 7.7%. In terms of the pathogenesis of CNS damage caused by HHV-6 primary infection (E. subitum), virus growth has not been found in the brain tissue, showing encephalopathy not encephalitis. For treatment of this illness, TRX could be an important target in the near future.
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Role of signal transduction pathway in host defense during sepsis
Grant number：17590352 2005 - 2006
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
MATSUKAWA Akihiro
Grant amount：\3600000 （ Direct expense: \3600000 ）
Cytokine signaling mediated by STAT pathway is negatively regulated by SOCS proteins. In this project, we have investigated the role of SOCS3/5 that inhibit STAT3/4/6 pathway, in innate immunity during sepsis. Mice with a cell-specific overexpression of SOCS5 in T cells (SOCS5Tg) were resistant to the lethality relative to the wild-type (WT) mice. This was due to the enhanced innate immunity in SOCS5Tg mice whereby CD4+T cells with overexpressed SOCS5 augment type-1 immune response of neutrophils and macrophages. Enhanced type-1 response during sepsis was also seen in mice lacking chemokine receptor 8 (CCR8), suggesting an important role of chemokine signaling in innate immunity. In inflammation, resident macrophages, but not other cell types, play a regulatory role through a Stat3 signaling pathway by mediating the anti-inflammatory role of IL-10. Macrophages lacking STAT3 showed decreased expression of Fcy receptor and compliment receptor 1 (CCR1), which was conversely augmented in macrophages lacking SOCS3. This might be responsible for the augmented phagocytic activities of macrophages lacking STAT3/SOCS3. Mice with a cell-specific deletion of STAT3 in phagocytes augmented Thl and Th2 type acquired immune response, possibly due to enhanced APC activities off the cells. On the other hand, mice with a cell-specific overexpression of SOCS3 in T cells (SOCS3Tg mice) were resistant to sepsis due to decreased type-1 response in tissue, resulting in alleviated organ damage. The SOCS3Tg mice were also deleterious in drug-induced hepatotoxicity by increasing STAT1 activation while decreasing STAT3 activation in hepatocytes.
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Mechanisms of neuroendocrine differentiation of lung carcinoma cells and their cell biological siginifcances : trials from the points of proteomics analyses
Grant number：16590318 2004 - 2005
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
ITO Takaaki, UDAKA Naoko, MATSUKAWA Akihiro, ARAKI Norie
Grant amount：\3600000 （ Direct expense: \3600000 ）
Aims of this research projects include following issues : 1. analyses alterations of mRNAs and proteins expressed in human lung cancer cell lines transfected with a lung neuroendocrine master gene, hASH1, 2. analyses cell biological effects including cell differentiation, proliferation and morphogenesis by gain or loss of the neuroendocrine genes, 3. focuses some key molecules among the molecules found by DNA miroarray and proteomics analyses ad study functions of these molecules.
We have established hASH1-transfected human lung adenocarcinoma cell lines and Notch1-transfected human small cell carcinoma cell lines. HASH1 transfection induced neuroendocrine phenotypes in adenocarcinoma cells and upregulation of neuroendocrine markers such as chromogranin A, but, the tumors grown in the subcutaneous tissue of nude mice showed usual adenocarcinoma morphology even though they have neuroendocrine differentiation. On the other hand, the Notch1-transfected human small cell carcinoma cell lines lose neuroendocrine phenotypes, showing nuclear translocation of HES1 and loss of nuclear hASH1. Moreover, Notch1 transfection made the cuture cells, which were floating in the medium before transfection, adhere to the plastic dishes, and up-regulated expression of E-cadherin, integrins, and CD44. Moreover, we studied cross-talks between the Notch1-Hes1-hASH1 system and other cell signalling systems. Cell proliferation signals via MAP kinase and ATT/PI3kinase downregulated hASH1 and alter cell differentiation toward non-neuroendocrine. We found that small cell carcinoma cells posesse large amount of STAT3, but STAT3 was underphosphorylated though non-small cell carcinoma cells have phosphorylated STAT3. Thus, neuroednodrine differentiation is affected by various signalling systems. We recently established Flag-, HA-, hASH1-transfected human adenocarcinoma cell lines to study proteins associated with hASH1 and its DNA binding sites. In the near future, we will clarify important molecules involved in neuroendocrine differentiation, and more precise molecular mechanisms of lung epithelial cell and lung carcinoma cell differentiation. -
Analysis of signal transduction pathway in innate immunity during sepsis
Grant number：15590349 2003 - 2004
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
MATSUKAWA Akihiro
Grant amount：\3600000 （ Direct expense: \3600000 ）
Stat3 is a transcription factor mediating anti-inflammatory properties of IL-10. To understand the role of Stat3 in inflammation, mice with targeted disruption of Stat3 in macrophages and neutrophils were succumbed to septic peritonitis. The mice displayed an excessive local and systemic inflammation relative to the control mice, an event that was accompanied by substantial increases in the level of multiple cytokines. Hepatic and renal injury was significantly exacerbated in mice with Stat3 deficiency. In addition, the mice exhibited an increased lethality after intraperitoneal inoculation of live bacteria recovered. In vitro, productions of inflammatory cytokines were significantly augmented when cells were stimulated with a synthetic lipopeptide, macrophage-activating lipopeptide-2(MALP-2) and LPS. Thus, macrophage/neutrophil-specific Stat3 is crucial in modulating multiple organ failure associated with systemic inflammation. Experiments were further carried out to understand the precise role of Stat3 in inflammation. For this purpose, mice lacking Stat3 in macrophages and neutrophils were intraperitoneally injected with thioglycollate and the subsequent inflammatory responses were investigated. We obtained evidence that Stat3 expressed in resident macrophages, but not other cell types, play a central role in the regulation of inflammatory response. Resident macrophages are important in triggering inflammation, but the cells in the same breath appear to control inflammation through Stat3 signaling pathway.
C57BL/6 and BALB/c mice are prototypical Th1- and Th2-type mouse strains, respectively. We attempted to characterize the innate immune response of macrophages from these mouse strains. We demonstrated that macrophages from BALB/c mice showed impaired bactericidal activity relative to those from C57BL/6 mice, resulting from a lack of effector molecules for bacterial killing by the cells. Thus, innate immune response of macrophages is different between these mouse strains, which may affect the development of Th1 and Th2 adaptive immunity in these strains. In different experiments, we showed that C10, regarded as Th2-type chemokine in acquired immune response, is a potent monocyte chemoattractant in a sterile peritonitis model. -
Role of Th1/Th2 cytokines and their regulation in a murine model of septic peritonitis
Grant number：13670222 2001 - 2002
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
MATSUKAWA Akihiro
Grant amount：\3500000 （ Direct expense: \3500000 ）
Signal transducer and activator of transcription (Stat)4 and Stat6 are transcription factors that provide type-1 and type-2 response, respectively. Here, we explored the role of Stat4 and Stat6 in innate immunity during septic peritonitis. Stat4^<-/-> and Stat6^<-/-> mice were resistant to the lethality, as compared to wild-type (WT) mice. At the mechanistic level, bacterial levels in Stat6^<-/-> mice were much lower than WT mice, which was associated with increased peritoneal levels of IL-12, TNFα, macrophage derived chemokine (MDC) and C10, known to enhance bacterial clearance. In Stat4^<-/-> mice, hepatic inflammation and injury during sepsis were significantly ameliorated without affecting local responses. This event was associated with increased hepatic levels of IL-10 and IL-13, while decreasing those of MIP-2 and KC. Sepsis-induced renal injury was also abrogated in Stat4^<-/-> mice, which was accompanied by decreased renal levels of MIP-2 and KC without altering IL-10 and IL-13 levels. Thus, Stat6^<-/-> and Stat4^<-/-> mice appeared to be resistant to septic peritonitis by enhancing local bacterial clearance and modulating systemic organ damage, respectively, via balancing cytokine responses. These results clearly highlight an important role of local type-1 and systemic type-2 cytokine response in protective immunity during sepsis, which can be regulated by Stat proteins. We are currently investigating the involvement of Stat proteins in the production of MDC and C10. In addition, the contribution of Stat4 and Stat6 in thymic apoptosis during sepsis is under investigation.
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敗血症時の自然免疫におけるCCケモカインリセプター8とそのリガンドの機能解明
Grant number：13226103 2001
日本学術振興会 科学研究費助成事業 特定領域研究(C) 特定領域研究(C)
松川 昭博
マウス腹膜炎誘発敗血モデル(cecal ligation and puncture ; CLP)時の浸潤マクロファージにはCCR8の発現増強がみられた。そこで,本モデルにおけるCCR8の生物学的意義を知るために、CCR8-/-およびCCR8+/+にCLPを行い,敗血症に伴う致死率を比較すると、CCR8-/-マウスではCCR8+/+に較べその生存率は有意に高かった。このとき,腹腔内および血中の菌数はCCR8-/-マウスで有意に低く,in vitroにおける菌消化能はCCR8-/-マクロファージで増強していた。腹腔マクロファージのLPS刺激培養上清中の活性酸素、ライソゾーム酵素、一酸化窒素(NO),TNFαおよびCXCL10の産生量は,CCR8-/-マクロファージで有意に高かった。また、脾細胞をconAで刺激した培養上清では,CCR8-/-脾細胞はIFNγ,IL-12の高値,IL-4,IL-13の低値を示した。以上より、CCR8-/-マウスでの免疫反応は自然免疫に必須なI型免疫反応にシフトし、細菌排除に効率的であることが示唆された。
次に、本モデルでのCCR8のリガンドを検索した.すると,CLP後の腹腔内でのCCL17の一過性上昇が検出され,CLPマウスに抗CCL17抗体を投与し、腹腔内および抹消血中の細菌数を検討すると、抗体投与群ではコントロールに比べ有意な菌数の増加が観察された.このときの腹腔内TNFαおよびCXCL10の産生量はコントロールに較べ有意に増加していた。したがって、CCL17は敗血症時の自然免疫反応を抑制する作用を持つことが示され、CCR8-CCL17の作用は自然免疫時の生体防御に負に働くことが示唆された。 -
炎症局所での好中球浸潤支配機構とサイトカインカスケードの解明
Grant number：09770154 1997 - 1998
日本学術振興会 科学研究費助成事業 奨励研究(A) 奨励研究(A)
松川 昭博
Grant amount：\1800000 （ Direct expense: \1800000 ）
ウサギ急性炎症モデルでのGROの発現動態およびその役割を調べるためには,その遺伝子組み換え体および抗体の作製が必要となる.そこで,LPS刺激ウサギ脾細胞のcDNAライブラリーからGRO cDNAをクローニングし,これを発現ベクター(pQE-30:QIAGEN社)に組み込み,試大腸菌(E.coli M15[pREP4])に導入,ウサギGROの発現を拭みた.次に,大腸菌抽出液からGROタンパクを単離精製し,これをマウスおよびヤギにアジュバントとともに免疫し,モノクローナル抗体およびポリクローナル抗体を作製した.得られた抗体はウサギGROは認識するものの,他のウサギサイトカイン(TNFα,IL-1β,lL-8,MCP-1,IL-1 receptor antagonist)は認識せず,したがって炎症局所のGROの産生動態を知るうえで有用なツールと考えられた.そこで,ウサギGROをウサギ膝関節内に投与し,その時誘導される炎症反応を観察しようと計画した.しかしながら,上記GROは不溶性(8M Ureaには可溶)であったため,これをin Vivoに投与できなかった.そのためBaculovirus Transfer Vectorを用いる方法(Invitrogen社)で再度遺伝子組み換え体の作製を行い,その培養上清および細胞中から可溶性のGROを得た.現在このタンパクを精製中であり,精製後はin Vivoに投与し,その時の炎症反応を観察するとともに,ウサギ炎症モデルでのGROの産生動態を解明しその炎症反応への関与を明らかにしていく予定である.
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炎症局所の好中球による単核球浸潤の支配機構の解明
Grant number：08770161 1996
日本学術振興会 科学研究費助成事業 奨励研究(A) 奨励研究(A)
松川 昭博
Grant amount：\900000 （ Direct expense: \900000 ）
炎症反応の局面におけるMCP-1の発現動態、その産生細胞を決定し、好中球浸潤と単核球浸潤との相互関係をしるためにこのプロジェクトを計画し,以下の結果を得た.
1)Con A刺激ウサギ脾細胞のcDNAライブラリーからMCP-1 cDNAをクローニングし、これを発現ベクターに組み込んだ.2)発現ベクターを動物細胞(COS-7 cell)に組み込み、ウサギMCP-1を発現させ,その培養上清からウサギMCP-1を単離、精製した.3)ウサギMCP-1をウサギ膝関節内に投与し,経時的な炎症反応の推移を観察したが,好中球浸潤を伴わない単核球浸潤は誘導できなかった.4)遺伝子組み替えMCP-1を用いて,ポリクローナル抗体およびモノクローナル抗体を作製し,免疫染色およびELISA測定系を開発した.5)ウサギLPS関節炎におけるMCP-1の産生動態は,LPS投与後2-4時間で最高になり,以後その産生量は急速に減少した.これは,好中球浸潤のピーク(9時間)に先行するものであり,その産生細胞は免疫組織化学的に滑膜表層細胞と判明した.6)抗MCP-1抗体の投与による細胞浸潤の変化を観察すると,LPSによる好中球浸潤に影響はなかったものの,単核球浸潤は有意に抑制された.
以上の結果から,MCP-1は炎症の極めて早い時期から産生され,単核球浸潤を誘導していることが明らかとなった.しかし我々の結果から,先行して誘導される好中球から産生される単核球遊走因子が,つづいておこる単核球浸潤を誘導するという従来の仮説は否定的であると思われた.今後は,in vivoにおける他の炎症性サイトカインとのカスケード反応について検討する予定である.