| Project/Area Number |
13670008
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General anatomy (including Histology/Embryology)
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| Research Institution | Nagoya University |
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Principal Investigator |
TORIHASHI Shigeko Nagoya University, Graduate School of Medicine, Associate Professor, 大学院・医学系研究科, 助教授 (90112961)
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| Co-Investigator(Kenkyū-buntansha) |
NAKAYAMA Shinsuke Nagoya University, Graduate School of Medicine, Associate Professor, 大学院・医学系研究科, 助教授 (30192230)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2001: ¥3,000,000 (Direct Cost: ¥3,000,000)
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| Keywords | interstitial cells of Cajal / pacemaker / gestrointestinal tract / calcium / smooth muscle / culture / cell biology / 自動運動 / 緩徐波 / TRP |
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| Research Abstract |
1. Establishment of isolation and culture of the musculature in mice small intestines
Muscle layers of the mice (0-14days old) small intestines were separated, cut into small pieces and treated with collagenase and they were triturated with fire-blunted glass pipettes. The resultant small cell clusters were placed onto collagen-coated coverslips and incubated with DMEM containing 10% fetal bovine serum at 37 < C for 2-3 days. Cell clusters contained interstitial cells of Cajal (ICC), contracted spontaneously and then indicated existence of intact pacemaker function.
2. Recording of intracellular Ca2+ ([Ca2+]i) oscillation in ICC
Cell clusters were loaded with 10 fEM fluo4 for 1-2 h and intracellular Ca2+ was recorded by CCD camera system. When 1 fEM nifedipine (L-type Ca2+ channel blocker) was added to the incubation chamber, contractions and oscillations of [Ca2+]i in smooth muscle cells were suppressed. Remaining [Ca2+]i oscillation was corresponded to that of ICC confirmed by c-Kit immunohistochemistry. [Ca2+]i oscillation in ICC was synchronized with slow waves. That indicates intimate relationship between [Ca2+]i oscillation in ICC and pacemaker function. We also demonstrated that [Ca2+]i oscillation in ICC required calcium influx probably mediated by transient receptor potential like channel type4 (TRP4).
3. Organogenesis of gut like structure from mouse embryonic stem (ES) cells
ES cells are pluripotent cells and develop to many cell types and tissues originated from three embryonic germ layers. We succeeded to form gut-like structures using hanging drop method. Gut-like structures were composed of three layers, i.e., epithelia, lamina propria and musculature. Their developmental process was similar to that of embryonic gut. We then concluded this system was a good model and tool for study of the gut organogenesis.
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