Cell biology of interstitial cells of Cajal as pacemakers in the gastrointestinal tract

作为胃肠道起搏器的卡哈尔间质细胞的细胞生物学

基本信息

批准号：
13670008
负责人：
TORIHASHI Shigeko
金额：
$ 2.3万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670008/
关键词：
interstitial cells of Cajal pacemaker gestrointestinal tract calcium smooth muscle culture cell biology 自動運動緩徐波 TRP

项目摘要

1. Establishment of isolation and culture of the musculature in mice small intestinesMuscle 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 ICCCell 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) cellsES 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.

1。在小鼠小鼠的小肠层中建立肌肉组织的隔离和培养物（0-14天老）小肠分离，切成小块并用胶原酶处理，并用大吹动的玻璃管移玻璃移液管。将所得的小细胞簇放在胶原蛋白涂层的盖玻片上，并与含有10％胎牛血清的DMEM在37 <c时孵育2-3天。细胞簇包含Cajal（ICC）的间质细胞，自发收缩，然后表明存在完整的起搏器功能。2。将ICCCELL簇中的细胞内Ca2+（[Ca2+] I）记录在ICCCELL簇中的振荡持续1-2 h，并通过CCD摄像头系统记录细胞内Ca2+。当将1个FEM硝苯地平（L型Ca2+通道阻滞剂）添加到孵育室时，在平滑肌细胞中[Ca2+] I的收缩和振荡被抑制。剩余的[Ca2+] I振荡与C-KIT免疫组织化学确认的ICC相对应。 [Ca2+] ICC中的I振荡与慢波同步。这表明ICC和起搏器功能中的[Ca2+] I振荡之间存在亲密关系。我们还证明了ICC中的[Ca2+] I振荡所需的钙涌入可能是由瞬态受体电位（如通道类型4（TRP4））介导的。3。小鼠胚胎干（ES）细胞的肠道结构的器官发生是多能细胞，并发展为许多细胞类型和组织，源自三个胚胎菌层。我们成功地使用悬挂式下降方法形成了类似肠道的结构。肠状结构由三层，即上皮，椎板和肌肉组织组成。它们的发育过程类似于胚胎肠的发展过程。然后，我们得出结论，该系统是研究肠道器官发生的好模型和工具。