Wnt信号通路在大鼠气管干细胞增殖分化过程中的时空表达  

Temporal-spatial changes of Wnt signaling pathway during the proliferation and differentiation of rat tracheal stem cells

作　　者：吕威力[1] 邢雪松[2] 

机构地区：[1]沈阳医学院病理学教研室,辽宁省沈阳市110034 [2]沈阳医学院解剖学教研室,辽宁省沈阳市110034

出　　处：《中国组织工程研究与临床康复》2008年第21期4074-4078,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金(30170407)~~

摘　　要：背景:实验前期项目已协同有关人员创立了体外观察气管干细胞的模型,并解决了气管干细胞的定位、分离及性状分析。但在气管干细胞的增殖分化过程中,究竟有哪些基因参与及其各自作用、气管干细胞可产生功能细胞的分化程序至今仍不清楚。目的:利用氟尿嘧啶诱发离体气管上皮损伤,检测气管干细胞增殖分化损伤重建过程中Wnt信号途径成员的时空表达变化。设计、时间及地点:单一样本观察,于2006-06/2007-06在沈阳医学院中心实验室完成。材料:清洁级2周龄Wistar大鼠18只,用于制备离体气管环。氟尿嘧啶由天津人民制药厂生产。方法:将镜下观察纤毛摆动情况良好的气管环分为两组:损伤组将气管环置于含12.5mg/L氟尿嘧啶的HamsF-12液中,作用12h后去除氟尿嘧啶,换成新鲜的HamsF-12液继续培养,分别于3,6,12,24,48h取部分等量组织块。对照组用等体积HamsF-12液代替氟尿嘧啶,其余培养条件及取材时间同损伤组。主要观察指标:RT-PCR法检测气管上皮中Wnt-1、β-连环蛋白、T细胞因子、c-myc基因表达的变化。结果:氟尿嘧啶作用12h后,光镜下可见少数间隔分布、近于裸核的细胞,呈钉状位于基底膜上,前期实验已证明其为气管干细胞。RT-PCR结果显示,对照组气管上皮无Wnt-1和T细胞因子mRNA的表达,β-连环蛋白与c-myc mRNA轻度表达。损伤组经氟尿嘧啶作用后,可检测到Wnt-1 mRNA的表达,β-连环蛋白mRNA一时性轻度下降;Wnt-1、β-连环蛋白、T细胞因子、c-myc mRNA分别于去除氟尿嘧啶后3,6,12h达到高峰;24h后三者mRNA表达水平均下调;至48hWnt-1 mRNA无表达,后三者mRNA少量表达。结论:在整个气管上皮重建过程中,Wnt1、β-连环蛋白、T细胞因子和c-myc mRNA的表达变化基本遵循相似的规律,提示Wnt信号通路作为一个整体参与气管干细胞增殖分化过程的调控。BACKGROUND： In coordination with the relevant staff, tracheal stem cells have been modeled in vitro previously, in which the localization, isolation and characteristics of tracheal stem cells are also analyzed. However, it is still unclear that the effect of various genes involved in the proliferation and differentiation of tracheal stem cells, together with the procedure of differentiating into functioning cell. OBJECTIVE： To develop an extracorporeal injury model of rat tracheal epithelium induced by fluorouracil （5-FU）, and investigate the changes of spatio-temporal expression of Wnt signaling pathway members during the proliferation and differentiation of tracheal epithelial stem cells in the tracheal regeneration. DESIGN, TIME AND SETTING： A single sample observation was carried out in the Central Laboratory of Shenyang Medical College （Shenyang, Liaoning, China） between June 2006 and June 2007. MATERIALS： Eighteen Wistar rats, of clean grade and aged 2 weeks, were adopted to dissociate tracheal rings. 5-FU was purchased from Tianjin People＇s Pharmaceutical Factory （China）. METHODS： Rat tracheal rings representing good cilia beat frequency were divided into two groups： the injury group were put into Hams F-12 culture medium with the concentration of 12.5 mg/L 5-FU. Twelve hours later, the culture dishes were exchanged to the fresh Hams F-12 culture medium without 5-FU, and then some of equivalent tissues were extracted respectively at hours 0, 3, 6, 9, 12, 24 and 48. We applied the same volume of Hams F-12 to take place of 5-FU as the control group. The other conditions were the same as the injury group. MAIN OUTCOME MEASURES： Levels of Wnt-1, β -catenin, T cell factor-4 （Tcf-4） and c-myc mRNAs in the control and injury groups were detected by reverse transcriptase polymerase chain reaction. RESULTS： After treatment with 5-FU for 12 hours, the tracheal epithelium shed and cells with naked nuclei were seen sparsely on the basement membrane under the light microscope,

关 键 词：气管干细胞 WNT-1 Β-连环蛋白 T细胞因子 c—myc 增殖分化 

分 类 号：R394.2[医药卫生—医学遗传学]