人支气管上皮损伤修复过程中Rhodamine 123 染色的动态观察

目的探讨离体人支气管上皮经5-氟尿嘧啶（5-FU）损伤前后及修复过程中支气管上皮细胞Rhodamine 123的染色情况。方法分别取5-FU作用前后及修复3、6、24h的人支气管上皮，蛋白酶消化法获取细胞悬液，进行流式细胞仪分析：1．应用PI染色，确定细胞周期中各期细胞所占百分数；2．Rhodamine 123染色，计算Rhodamine 123阴性细胞占活细胞的比例。结果1．5-Fu处理后支气管上皮细胞中凋亡细胞增多，增殖期（S＋G2/M期）细胞明显减少，剩余细胞绝大部分处于细胞周期的静止（G0/G1）期；随支气管上皮的恢复，S＋G2/M期细胞增多；2．5-FU作用后支气管上皮中Rhodamine 123阴性细胞占活细胞的比例较正常组增多；随着损伤的修复，Rhodamine 123阴性活细胞所占比例逐渐降至接近正常。结论5-Fu的作用使增殖期细胞凋亡、脱落，残留静止期细胞，其中包含具有Rhodamine 123排出能力的干细胞，由它们完成损伤的修复。应用Rhodamine 123染色，经流式细胞仪分选5-Fu作用后残留的细胞，可用于富集纯化支气管干细胞。

DYNAMIC ANALYSIS OF RHODAMINE 123 STAINING OF HUMAN BRONCHIAL EPITHELIUM DURING THE WOUND REPAIR PROCESS INDUCED BY FLUOROURACIL

Objective To analyze the condition of Rhodamine 123 staining of human bronchial epithelium in vitro during the wound-repair process induced by fluorouracil(5-FU). Methods The bronchial epithelial cells in normal and 5-FU injurious group were obtained by enzymatic digestion and analyzed by flow cytometry.1.PI staining to identify the percentage of cells in different cell phase;2.Rhodamine 123 staining in live cells was to contrast the percentage of negative cells in two groups. Results 1.Analysis of cell cycle:apoptotic cells in injurious group increased,cells in S+G-2/M phase almost disappeared,majority of the remain live cells of injurious group were in G-0/G-1 phase;with the recover of bronchial epithelium,cells in S+G-2/M phase increased;2.The percentage of Rhodamine 123 negative staining live cells in injurious group was higher than that of normal group,and with the recover of bronchial epithelium,this percentage lowered to nearly normal.Conclusion 5-FU can make the cycling cells apoptosis and reserve quiescent phase cells.Among them there were bronchial stem cells which had the capacity to efflux Rhodamine 123.Just the proliferation and differentiation of these stem cells regenerated bronchial epithelium.Rhodamine 123 staining to sort the remain cells after 5-FU treatment by FACS can be used to enrich and purify bronchial stem cells.