| 研究課題/領域番号 |
19K08428
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| 研究機関 | 大阪市立大学 |
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研究代表者 |
LE THITHANHTHUY 大阪市立大学, 大学院医学研究科, 特任講師 (10572175)
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| 研究期間 (年度) |
2019-04-01 – 2022-03-31
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| キーワード | Cytoglobin / Hepatic stellate cells / Anti-fibrosis / Liver cancer |
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| 研究実績の概要 |
We have examined therapeutic effects of 6-His tagged recombinant human Cytoglobin (rhCYGB) protein against mouse liver injuries, fibrosis and human HSC activation. In cultured HSCs, extracellular His-CYGB was endocytosed and accumulated in endosomes via clathrin-mediated pathway. His-CYGB significantly impeded ROS formation spontaneously or in the presence of ROS inducers in HSCs, thus leading to the attenuation of Col1a1 production and HSC activation. Intravenously injected His-CYGB markedly suppressed liver inflammation, fibrosis and oxidative cell damage in TAA- or DDC-administered mice. The injected His-CYGB predominantly localised to HSCs, suggesting specific targeting effects. His-CYGB exhibited no toxicity in humanised liver chimeric PXB mice.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
RNA-Seq analysis of His-CYGB-treated HHSteC samples in comparison with controls revealed the down-regulation of the fibrosis-related genes. Besides that, we also found IFN-stimulated genes such as the genes encoding IFN-inducible proteins IFI27, IFI6, and IFI44L, ISG15, IRF7 and IRF9, and OAS2 were upregulated, suggesting the involvement of IFNb signaling during His-CYGB treatment. His-CYGB treated HHSteC increased the levels of P-TBK1, and secreted IFN-β protein. Simultaneous with IFN-β secretion, STAT1 phosphorylation was observed, secreted IFNb 8 h after His-CYGB challenge. In opposite, the JAK1-specific inhibitor momelotinib attenuated the reduction in COL1A1 production in both His-CYGB- and rhIFN-β treated HHSteCs.
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| 今後の研究の推進方策 |
We have pointed that IFN- β is one of down-stream targets of Cygb in protecting liver injuries. Beside that, we overexpression of Cygb in human HSCs and cancer cells. Immunoprecipitation experiments using Pull-Down Poly-His Protein:Protein Interaction Kit, Sulfo-SBED Biotin Label Transfer Kit, and Dynabeads His-Tag Isolation and pulldown (Invitrogen) to triple confirm the binding targets. We have found some potential CYGB targets including Flotilin-1, Annexin A2, ATPb5. We are now explore the role of each target. For rhCYGB protein production and application: we will perform the shortening full-length of rhCYGB protein to be shorten peptides and explore the important portion of CYGB for its effect.
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| 次年度使用額が生じた理由 |
We need to shorten full-length of rhCYGB protein and explore the CYGB targets. We need to: design primers; PCR amplification of DNA fragments; Double digestion vectors; Ligation reaction; Transformation into DH5α, propagation the plasmids; Verify sequencing; Choose the corrected vectors and transformation into Ecoli BL21-AI; Produce rhCYGB short-peptides; Purify the peptides; In vitro application. Silencing or overexpression of each potential CYGB targets: Flotilin-1, Annexin A2, ATPb5. Examine the interaction between these targets with rhCYGB in vitro and in vivo.
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