成年C57BL／6小鼠空肠Cajal间质细胞的分离、培养及鉴定

目的尝试优化体外培养成年小鼠小肠Cajal间质细胞的操作方法，为深入研究该细胞的生理功能提供一定细胞模型。方法6～8周龄C57BL／6小鼠禁食24h，无菌条件下截取3～5cm中段空肠，分离平滑肌肌条并剪至小块后使用Ⅱ型胶原酶消化20min，离心后进行组织块原代培养，观察不同时期细胞形态。使用c—Kit免疫荧光染色鉴定细胞表型是否为Cajal间质细胞。Fluo-3AM染色细胞内钙，观察细胞能否自发产生钙离子振荡。结果分离所得组织不含空肠上皮和固有层组织，仅为小肠肌层。联合使用胶原酶消化和组织块原代培养能够较方便地培养出原代细胞，该细胞呈梭形，且具有细胞突起，彼此交织成网状。该细胞免疫荧光染色呈c—Kit阳性，平滑肌标志物SMA阴性。钙成像分析显示其能够产生钙离子振荡。结论联合使用酶消化和组织块培养能够较方便地培养出成年小鼠的空肠Cajal间质细胞，该方法为后续探讨Cajal间质细胞的生理功能及机制提供了一定的细胞模型。

Isolation, culture and identification of interstitial cells of Cajal from jejunum of adult C57BL/6 mouse

Objective To optimize the cultivation method of interstitial cells of Cajal(ICC) from jejunum of adult mouse,and provide a cellular model in vitro to study the biological function of ICC.Methods C57BL/6 mice(6～8 weeks old) were starved for 24 hours,and a jejunum fragment of 3～5 cm was isolated under sterile condition.The muscularis was dissected under anatomic microscope,digested using collagenase Ⅱ for 20 minutes and then collected for primary tissue culture.The morphology of ICC was observed.C-Kit staining was conducted to confirm the phenotype of ICC.Cytoplasmic calcium was stained using Fluo-3 AM to show the calcium oscillation.Results The dissected tissue did not contain epithelium and lamina propria,and only included muscularis.Collagenase digestion combined with tissue culture could get the primary cells conveniently.Cultured cells appeared to be spindle shaped with cell processes and formed network.They were positive for c-Kit,but negative for SMA.Fluo-3 AM staining showed they could generate calcium oscillation.Conclusion ICC can be isolated from jejunum of adult mouse using combination of collagenase digestion and tissue culture,which provides us a certain cellular model to further study the biology of ICC in vitro.