| 研究実績の概要 |
[Purpose of research] Our previous observations found that chemokine receptor 5 (CCR5) had unique role for osteoclastic functions. To further investigate this research results, we will take combinatorial approaches of morphometericla, cytomorphhometrical and transcriptome analyses. The putative results would patio-temporally dissociate chemokine-mediated signals during osteoclast ontogeny and possibly contribute to therapeutic development for osteoporosis. In 1st year of this project, we conducted half of project as planned described above.
[Archievements] 1) Histomorphometrial analysis was conducted on femoral bone sections obtained from 8-10 week-old Ccr5 wild type and Ccr5 knock out mice; Ccr5-deficient mice had significantly increased number and size of osteoclasts, although they did not show significant difference in BMD compared to their wild-type littermates. In keeping with osteoclst dysfunction, Ccr5-deficient mice were less susceptible to RANKL-induced bone loss. 2) Transcriptome analysis was confirmed from chemokine knockout bone marrow cell culture, and Data of RNA-seq were analyzed using specific software; Maldi-Tof and RNA-sequencing demonstrated that CCR5-mediated signaling, cooperating with RANKL-mediated signaling, regulated cellular architecture and motility of differentiated osteoclast through activation of small-GTPases.
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| 今後の研究の推進方策 |
1) Cytomorphometrical analysis of Ccr5+/+ and Ccr5-/- osteoclasts by fluorescence bio-imaging; To evaluate three-dimensional architecture of cultured osteoclasts from Ccr5+/+ and Ccr5-/-, we will take 3D fluorescence images by confocal based super resolution microscopy.
2) Validation of candidate factor and signaling pathway from profiling; we will construct RNA interference (RANi) plasmid to block expression of candidate molecules using basic vector. These cells are analyzed by osteoclastic marker expression by qRT-PCR, osteoclast function assay and cytomorphometrical analyses.
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