肌动蛋白染色协助分析肠上皮增殖和分化的相对定量研究

Actin staining quantitation in studying the proliferation and differentiation state of intestinal epithelial cells

目的明确肌动蛋白染色的相对定量是否能协助区分肠上皮细胞的增殖和分化状态。方法获取C57BL/6小鼠肠道隐窝-绒毛完整结构和单细胞,通过DAPI、鬼笔环肽免疫荧光染色、蛋白免疫印迹、流式细胞检测和细胞周期分析等方法分析β-Actin在增殖和分化细胞中的表达差异,以及不同β-Actin表达水平细胞的细胞周期分布。结果免疫荧光染色结果显示,以分化细胞为主的绒毛区细胞β-Actin的平均荧光强度高于增殖细胞为主的隐窝区细胞;隐窝中非增殖细胞β-Actin的平均表达量高于增殖细胞;基于CD24表面标记染色的流式细胞检测结果显示,隐窝细胞中Paneth细胞β-Actin平均表达量最高,肠内分泌细胞次之,增殖性细胞最低;对不同β-Actin表达水平的隐窝细胞进行流式细胞周期分析,结果显示低表达β-Actin的隐窝细胞在S期和G2/M的细胞比例显著高于β-Actin高表达的隐窝细胞。结论β-Actin蛋白表达的定性和定量分析可以作为一种标记区分肠上皮细胞增殖和分化的状态,该蛋白作为内参分析小肠上皮不同细胞蛋白表达存在一定的局限性。

Objective To study whether actin staining could distinguish the proliferation and differentiation state of intestinal epithelial cells.Methods The intestinal crypt-villus units were isolated using chelating agent at low temperature.These units were stained with Phalloidin FITC and DAPI for fluorescence imaging,and protein extraction for Western blotting.Single cells were dissociated from crypt and villus unit respectively and were stained by Phalloidin FITC and DAPI for flow cytometry testing and cell cycle analysis.Results The results of immunofluorescence staining showed that the average fluorescence intensity ofβ-Actin in villus cells dominated by differentiated cells was higher than that in crypt cells dominated by proliferating cells;the average expression level ofβ-Actin in non-proliferating cells in crypts was higher than that in proliferating cells.The results of flow cytometry based on CD24 surface marker staining showed that,in crypt cells,the average expression ofβ-Actin in Paneth cells was the highest,followed by enteroendocrine cells,and proliferative cells were the lowest.The flow cytometry analysis of crypt cells showed that the proportion of crypt cells with low expression ofβ-Actin was significantly higher than those with high expression ofβ-Actin in S phase and G2/M.Conclusions Qualitative and quantitative analysis ofβ-Actin protein expression can be used to distinguish the state of intestinal epithelial cell proliferation and differentiation,and reflects the limitation ofβ-Actin protein as an internal reference to analyze the protein expression of different epithelial cells in small intestinal.