在体分离小鼠小肠隐窝、绒毛上皮细胞的生化完整性研究

Analysis of the Biochemical Integrity of the Isolated Mouse Small Intestinal Crypt and Villus Cells

目的:检测低温条件下用螯合剂沉淀法分离的小鼠小肠上皮隐窝和绒毛细胞是否具有生化完整性。方法:使用螯合剂在低温(冰浴)条件下分离和富集小肠上皮绒毛和隐窝细胞;抽提DNA、RNA和总蛋白,用电泳的方法检测完整性;用Real-time PCR检测溶菌酶Lysozyme的表达以判断隐窝、绒毛细胞富集程度。结果:低温条件下分离的肠上皮隐窝、绒毛细胞形态完整;基因组DNA完整,未出现明显的DNA ladder现象;富集细胞的RNA完整;富集隐窝、绒毛细胞的蛋白未降解,两组总蛋白具有表达谱差异性;隐窝细胞富集物溶菌酶mRNA表达水平较绒毛细胞富集物高30倍以上。结论:小肠隐窝绒毛的生物学性状可在低温螯合剂沉底法分离过程中得到保存,提示此方法可以用来分析生理和创伤病理条件下小肠上皮基因和蛋白表达改变。

Objective： To test the biochemical integrity of mouse intestinal crypt and villus cells isolated and enriched at low temperature using chelating agents. Methods： The crypt and villus units of small intestinal epithelium were isolated and enriched at low temperature （ice cold） using chelating agents. The genome DNA, total RNA and total protein were extracted and electrophoresed. The mRNA expression level of lysozyme was detected by real-time PCR to evaluate the purity of enriched crypt and villus units. Results： The enriched crypt and villus units remained morphological integrity. The integrity ofgenome DNA, total RNA and protein were preserved during isolation procedure. The expression level oflysozyme in the enriched crypt units is more than 30 times higher than that in villus units. Conclusion： The structural and biochemical integrity of crypt and villus cells can be preserved using the improved low-temperature method. In addition, this method may be useful for in vivo analysis of protein and mRNA expression in normal and stressed small intestinal epithelial cells.