大鼠气管干细胞增殖分化过程中Wnt／β-Catenin信号分子的表达

Expression of Wnt/β-Catenin signaling molecule during proliferation and differentiation of rat tracheal stem cells

背景:干细胞和细胞外基质的互相作用需要Wnt信号途径的参与。在气管损伤后的修复过程中,不同的分子信号级联控制干细胞的迁移、增生与分化,Wnt信号途径是否参与其中?目的:检测并验证Wnt/β-Catenin信号途径在气管干细胞增殖分化中的作用。设计、时间及地点:单一样本观察实验,于2004-05/2006-07在沈阳医学院完成。材料:2周龄Wistar大鼠,雌雄不拘,作为离体气管来源。方法:实验组以含氟尿嘧啶的HamsF12培养液作用离体大鼠气管,作用12h后换成单纯Hams F12液,对照组只用Hams F12液培养。主要观察指标:去除氟尿嘧啶后于0,6,12,24,48h采用免疫组织化学SP染色及蛋白印迹法检测气管上皮中Wnt-1和β-Catenin的表达。结果:氟尿嘧啶作用12h后,大鼠气管上皮细胞绝大部分脱落,暴露出基底膜,仅剩余少数较分散的间隔分布的近于裸核的细胞,呈钉状位于基底膜上。氟尿嘧啶去除6h后,气管基底膜上可见少量扁平样细胞。24h后基底膜上细胞数目增加,并逐渐分化呈立方状,连接成片。48h后气管上皮中出现假复层柱状上皮,并可见纤毛细胞。免疫组织化学及蛋白定量结果显示:正常及损伤后0h气管上皮极少量表达Wnt-1及β-Catenin,损伤后6hWnt-1及β-Catenin表达最强,其后随时间延长,表达逐渐减少。结论:Wnt-1时间依赖性表达与气管上皮损伤修复进程相吻合,胞浆中游离状态的β-Catenin表达可能产生了促进气管干细胞的增殖并抑制其分化的作用。

BACKGROUND： Interaction of stem ceils and extracellular matrix requires the participation of Wnt signal. During reparation after trachea injury, whether Wnt signal participate in signal cascade of different molecules, controlling migration, hyperplasy and differentiation of stem ceils. OBJECTIVE： To determine the expression of Wnt/β -Catenin Signaling Pathway in the proliferation and differentiation of the tracheal stem ceils. DESIGN, TIME AND SETTING： The single sample experiment was performed at the Shenyang Medical College from May 2004 to July 2006. MATERIALS： Two-weeks old Wistar rats of both genders were selected as a source of ex vivo trachea. METHODS： Ceils in the experimental group were put into Hams F-12 culture medium with 5-fluorouracil （5-FU）. After 12 hours simple Hams F-12 culture medium was added. Ceils in the control group were incubated with Hams F-12 medium. MAIN OUTCOME MEASURES： Wnt-1 and β-Catenin expression in tracheal epithelium was detected by immunohistochemistry （SP method） and Western blotting 0, 6, 12, 24 and 48 hours after removing 5-FU. RESULTS： After treatment with 5-FU for 12 hours, the tracheal epithelium shed, basal membrane exposed and some of the retained ceils, nearly naked nucleus, were scattered, showing nail-like on the basal membrane. Six hours after the removal of 5-FU the tracheal rings were covered with flattened epithelial ceils. At 24 hours after the removal of 5-FU, most of the epithelial ceils were increased in number, showing cuboidal and merged into pieces. At 48 hours, only few positive ceils could be seen with some of the pseudostratified mucociliary epithelium, with ciliated ceils. Immunohistochemistry and Western blotting analysis showed that Wnt-1 and β - Catenin expression was minimally found in normal ceils and immediately after injury, expressed in a peak 6 hours after injury. Subsequently, with the prolongation of time, the expression decreased gradually. CONCLUSION： The expression of Wnt-1 corresponds with the wound and healing process of tracheal epithelium, suggesting that the increase in β -Catenin expression in kytoplasm may promote the proliferation of the tracheal stem ceils, while inhibit its differentiation.