【作者】 曲杨；

【导师】 贾心善；

【作者基本信息】 中国医科大学 ， 病理学与病理生理学， 2006， 硕士

【摘要】 组蛋白去乙酰化酶1在人支气管干细胞增殖分化过程中的时空表达 前言 目前,在气管干细胞研究中,我们建立了氟脲嘧啶（Fluorouracil,5-FU）引发离体气管损伤修复模型,论证了气管干细胞位于具有5-FU抗性的G0期细胞中。为阐明气管干细胞增殖分化的调控机制,我们已检测了wnt信号中的wntl,β-catenin,cyclinD1等成员在气管干细胞增殖分化过程中的时空变化情况,证明了Wnt/β-catenin信号传导途径参与调控气管干细胞增殖分化全过程。近来有学者在研究斑马鱼视网膜发育中发现组蛋白去乙酰化酶1（Histone deacetylase 1,HDAC1）促进视网膜干细胞分化成神经元和胶质细胞,提出HDAC1是视网膜干细胞由增殖到分化的开关。那么HDAC1在气管干细胞的增殖分化过程中是否也具有这种开关作用?这在国际上尚无报道。本研究利用免疫组织化学和western blotting方法检测了HDAC1在气管干细胞增殖分化过程中的动态表达情况。 材料和方法 1.制备人支气管损伤模型:在因肺癌肺叶切除组织中取距肺癌组织2cm处支气管（该材料经患者家属及中国医科大学伦理委员会同意）放入无菌袋中在2h内用冰盒运回实验室,无菌PBS反复冲洗,洗净血液和粘液,置于DMEM/F12培养液中（含10%胎牛血清）,剪成约2-3mm的气管环,于倒置显微镜下观察纤毛摆动良好,于培养液中加入终浓度为120mg/ml的5-FU,37℃,5%CO₂孵育12h,弃去上述培养液,换成新鲜DMEM/F12液（含10%胎牛血清）继续培养,于换液后0、3、6、12、24、48h分别取出一组织块,10%中性福尔马林固定,石蜡包埋,制成4μm厚的组织切片。另于换液后0、3、6、24、48h取气管组织,解剖显微镜下机械剥离上皮,每个样品约更多还原

【Abstract】 Introductionwe have previously constructed a bronchial injury model induced by 5 - flu-orouracil (5 - FU) ex vivo to localize the bronchial stem cells and idendified bronchial stem cells exist in the GO cells. To explore the mechanism of involvement of the Wnt/ β - catenin pathway during regulated growth of bronchial epithelial stem cells in rats, we used this injury model to observe changes in levels of Wntl, β - catenin, and cyclinDl mRNAs, and localization of B - catenin protein during rat bronchial epithelial regeneration. Our findings indicate that the Wnt/ β - catenin pathway plays a role in proliferation and differentiation of bronchial epithelial stem cells. However,the mechanism that determines how the bronchial epithelial stem cells switch from proliferation to differentiation is largely unknown. Recently, it has been reported that Histone deacetylase 1( HDAC1) is required for the switch of retinal stem cells from proliferation to differentiation in the zebrafish retinal neurogenesis. To determine the function of HDACl during regulated growth of human bronchial epithelial stem cells, we want to observe initially its changes in space - time expression and explore the relationship between HDACl and bronchial stem cell proliferation and differentiation.Materials and Methods1. Preparation of bronchial Epithelium Regeneration Model Materials were provided by chest department of the first hospital of China Medical University. Human bronchial epithelium were dissociated from the unin-volved parts obtained from patients who underwent lung cancer operation ( under protocol approved by the IRB at the university of CMU). Bronchial samples were delivered to lab sterilly in 2 hours. Then they were cut into 2 —3mm wide rings and cultured in 1:1 mix of Dulbeccos modified Eagles medium and Hams F -12 medium (DMEM/F12) containing 120 mg/ml 5 -FU and 10% fetal bovine serum (FBS) for 12 h at 37 °t. The tissues were then refreshed by culturing in DMEM/F12 containing 10% FBS. Bronchial rings were taken out at 0,3,6, 12,24,48 h time points after removing 5 - FU. For western blotting, epithelium of some bronchial rings were stripped under anatomical microscope, snap - frozen and stored at -80°C until use. Some other bronchial rings were fixed in 4. 5 % formaldehyde, and made into paraffin - embedded tissue sections for hema-toxylin - eosin stain and immunohistochemistry observation. Normal bronchial rings were also analyzed as controls.2. hematoxylin - eosin stain was used to observe the morphological changes during bronchial epithelium regeneration.3. ImmunohistochemistryStreptavidin - Peroxidase ( SP) immunohistochemical technique was used to detect expression of HDACl in the dynamic process during bronchial epithelium regeneration. PBS replaced the first antibody as negative group. Result determination;positive cell was brown, negative cell was not coloured. HDACl is positive in nuclear.4. western blot analysis was used to identify changes in levels of HDACl protein during bronchial epithelium regeneration.Results1. Morphological Changes in Bronchial EpitheliumWe found that the bronchial epithelium desquamated after 5 - FU treatment. The residual were trifle nude - nucleus cells distributed intervally on the basement membrane (GO phase cells) . When removing 5 - FU, the bronchial epithelium began to recover. The bronchial rings were covered with flattened epithelial cells at 3 -6h after the removal of 5 - FU. At 12h, most of the epithelialcells were cuboidal cells and merged into pieces, the number of the cells increased. At 24h, pseudostratified mucociliary epithelium appeared in some region of bronchial epithelium, and the ciliated cells can be seen. At 48h after removal of 5 - FU, pseudostratified mucociliary epithelium was restored to its original mode similarly.2. Expression of HDACl in Bronchial EpitheliumWe found the Expression of HDACl was negative in normal bronchial epithelium and in the epithelium at oh after 5 - FU treatment, only few of the epithelial cells were HDACl positive at 3h - 6h after the removal of 5 - FU. HDACl positive cells increased obviously at 12h. and was in top expression at 24h ,then decreased slightly at 48h.3. Changes in HDACl protein Levels during Bronchial Epithelial RegenerationResult of western blotting indicated that there was no detectable level of HDACl protein in normal bronchial epithelium and in the epithelium at oh after 5 - FU treatment. The level of HDACl protein was elevated slightly at 3 -6 h after the removal of 5 - FU, reached a top at 24h and then decreased slightly at 48h.ConclusionsOur work showed the expression of HDACl was few during bronchial stem cell proliferation and increased obviously when differentiation occurs. With more differentiating cells appeared, more HDACl -positive cells were observed,suggesting that HDACl plays a significant role in the switch of bronchial epithelial stem cells fom proliferation to differentiation, we conclude that HDACl function as a molecular switch of bronchial epithelial stem cells fom proliferation to differentiation during regulated growth of human bronchial epithelial stem cells.更多还原