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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、62、4 h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。
Objective To investigate the staining of Rhodamine 123 in bronchial epithelial cells of human bronchial epithelial cells before and after 5-fluorouracil (5-FU) injury and repair. Methods The human bronchial epithelial cells were treated with 5-FU and bronchial epithelial cells were harvested at 3,62 and 4 h, respectively. The cell suspension was obtained by digestion with protease and analyzed by flow cytometry. 1. PI staining was used to determine the number of cells in the cell cycle Accounting for percentage; 2. Rhodamine 123 staining, calculate Rhodamine 123 negative cells accounted for the proportion of living cells. Results After 1.5-FU treatment, the number of apoptotic cells in bronchial epithelial cells increased and the number of cells in proliferative phase (S + G2 / M phase) decreased significantly. Most of the remaining cells were in the stationary phase of cell cycle (G0 / G1) The number of Rhodamine 123-negative cells in the bronchial epithelium increased more than that in the normal group after 2.5-FU treatment; the proportion of Rhodamine 123-negative viable cells decreased gradually with the repair of injury Close to normal. Conclusions The role of 5-FU is to induce apoptosis and shedding of cells during the proliferative phase. Residual quiescent cells, which contain stem cells capable of excreting Rhodamine 123, are involved in the repair of the injury. Rhodamine 123 staining, flow cytometry sorting 5-FU residual cells, can be used for enrichment of purified bronchial stem cells.