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

1. 小鼠小肠肌肉组织的分离与培养0 ~ 14日龄小鼠小肠肌肉层分离，切成小块，胶原酶处理，用火钝化玻璃移液管培养。将得到的小细胞团置于胶原包被的盖子上，用含有10%胎牛血清的DMEM在37℃下孵育2-3天。细胞团中含有Cajal间质细胞（ICC），自发收缩，表明心脏起搏器功能完整。利用CCD摄像系统记录细胞内Ca2+ ([Ca2+]i)在icccl细胞团中1 ~ 2 h的振荡。在培养室中加入1fem硝苯地平（l型Ca2+通道阻滞剂），可抑制平滑肌细胞内[Ca2+]i的收缩和振荡。剩余的[Ca2+]i振荡与c-Kit免疫组化证实的ICC一致。[Ca2+]i振荡与慢波同步。这表明ICC中[Ca2+]i振荡与起搏器功能密切相关。我们还证明，ICC中的[Ca2+]i振荡需要钙内流，这可能是由瞬时受体电位样通道类型4 （TRP4）介导的。小鼠胚胎干细胞（ES）是一种多能细胞，在胚胎胚层发育成多种细胞类型和组织。我们成功地用吊滴法形成了肠状结构。肠样结构由上皮、固有层和肌肉组织三层组成。它们的发育过程与胚胎肠相似。因此，我们认为该系统是研究肠道器官发生的良好模型和工具。

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.