小鼠胃 Cajal 间质细胞分离与培养实验方法探讨

目的:尝试优化体外培养Balb/c小鼠胃Cajal间质细胞(interstitial cells of Cajal,ICC)的实验方法,为深入探索该细胞的生理病理作用机制提供基础。方法:无菌条件下取出小鼠胃组织,使用酶解法消化分离细胞,将细胞悬液接种于含有SCF(干细胞因子)的M199培养基中培养,并进行传代。倒置显微镜下观察不同时间段细胞生长状态,采用ICC特异性标志物c-Kit(酪氨酸激酶受体)进行免疫荧光鉴定。结果:细胞培养24 h后基本已贴壁,呈梭形或三角形,有短突起;72 h后细胞胞体变大,突起伸长;5 d后,细胞之间通过突起彼此相互连接,开始形成网状结构;传代后细胞依然保持其固有特征。免疫荧光鉴定可见细胞c-Kit抗体荧光染色阳性。结论:使用酶解法成功分离细胞,细胞数量较多但不增殖,传代后可见细胞纯度较好,稳定培养3周以上后细胞形态逐渐发生变化并开始凋亡。

Experimental Study on Isolation and Culture of Interstitial Cells of Cajal in Mice Stomach

ABSTRACT Objective: To optimize the experimental method of culturing Interstitial Cells of Cajal in Balb/c mouse stomach in vitro, and to provide a basis for further exploring its physiological and pathological mechanism. Methods: The gastrictissues of mice were removed under the sterile conditions, and the cells were digested and separated by enzymatic digestion. The cell suspension was inoculated in M199 medium containing Stem Cell Factor and passaged. The cell growth state of different time points was observed under the inverted microscope, and the ICC specific marker c-Kit (tyrosine kinase receptor) was used for immunofluorescence. Results: After cultured for 24 hours, the cells were almost adherent, fusiform or triangular with short protuberances. After 72 hours, the cell bodies became larger and the protuberances were elongated. After 5 days, between the cells connected to each other through projection, began to form a network structure. And after passage cells still maintain its inherent characteristics. Immunofluorescence showed that cells stained positive for c-Kit antibody. Conclusion: The ICC was successfully separated by enzymolysis. The number of cells was larger but not proliferating. After passage, the cell purity was better. After more than 3 weeks of stable culture, the cell morphology gradually changed and began to apoptosis.