罗丹明123及P-糖蛋白在人支气管损伤修复过程中的表达(英文)

Study of Rhodamine 123 staining and P-glycoprotein expression of human bronchial epithelium during wound-repair process induced by fluorouracil

目的研究离体人支气管上皮经氟尿嘧啶(5-FU)损伤前后及修复过程中罗丹明123(Rh123)的染色及P-糖蛋白的表达情况。方法取5-FU作用前后及修复3、6、24h的人支气管上皮,进行如下实验:采用酶消化法获取细胞悬液,Rh123染色,流式细胞仪分析;用免疫荧光及蛋白印迹法观察P-糖蛋白的表达,RT-PCR法检测P-糖蛋白mRNA的表达。结果5-FU作用后支气管上皮中Rh123阴性染色细胞占活细胞的比例较正常组增多;随着损伤的修复,Rh123阴性活细胞所占比例逐渐降至接近正常。免疫荧光法可见5-FU作用后的上皮细胞涂片上有P-糖蛋白的表达,在蛋白及mRNA水平,5-FU作用后支气管上皮的P-糖蛋白表达均较正常组增加。结论5-FU作用后,Rh123染色阴性、表达P-糖蛋白的支气管干细胞占残留活细胞的比例增多,由它们完成损伤的修复。

[Objective] To explore the condition of Rhodamine123 (Rh123) staining and the expression of P-glycoprotein(P-gp) of human bronchial epithelium in vitro during the wound-repair process induced by fluorouracil(5-FU). [Methods] The normal and 5-FU injurious bronchial epithelial cells were obtained by enzymatic digestion and stained with Rh123, then analyzed by flow cytometry. The expression of P-gp in normal and injurious groups were detected by immunofluorescence and western-blotting. Reverse transcriptase polymerase chain reaction (RT-PCR) was applied to detection of P-gp mRNA. [Results] The percentage of Rh123 negative staining live cells in injurious group was higher than normal group. With the wound-repair process, the percentage of Rh123 negative live cells gradually fall to nearly normal level. The expression of P-gp could be seen on the smear of bronchial epithelium in injurious group by immunofluorescence. The expression levels of P-gp and its mRNA were all higher after 5-FU treatment than the normal group. [Conclusions] There are bronchial stem cells which have the capacity to efflux Rh123 and express P-gp after 5-FU treatment. The proliferation and differentiation of these stem cells can regenerate bronchial epithelium.