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

1.小鼠小肠肌组织分离培养方法的建立取0- 14日龄小鼠小肠肌层，用胶原酶消化后，用火焰钝头玻璃吸管研磨。将所得的小细胞簇置于胶原包被的盖玻片上，并与含有10%胎牛血清的DMEM在37 ℃下孵育2-3天。细胞团含有Cajal间质细胞（ICC），自发收缩，提示起搏功能存在.记录ICCC细胞团中的细胞内Ca 2+（[Ca 2 +]i）振荡用10 fEM Flu 4负载1-2 h，并通过CCD相机系统记录细胞内Ca 2+。当在孵育室中加入1 fEM硝苯地平（L-型Ca 2+通道阻断剂）时，平滑肌细胞中[Ca 2 +]i的收缩和振荡被抑制。经c-Kit免疫组化证实，ICC内[Ca ~（2+）]i的振荡与ICC内[Ca ~（2+）]i的振荡相一致。[Ca2 ICC +]i振荡与慢波同步。提示ICC内[Ca ~（2+）]i振荡与起搏功能密切相关。我们还证明了ICC内[Ca ~（2+）]i的振荡需要钙内流，这可能是由瞬时受体电位样通道4（TRP 4）介导的.小鼠胚胎干细胞肠样结构的器官发生胚胎干细胞是一种多能性细胞，可发育成多种细胞类型和组织，起源于三个胚层。采用悬滴法成功地形成了肠状结构。肠样结构由三层组成，即，上皮、固有层和肌肉组织。其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.