Notch signaling promotes the corneal epithelium wound healing
Notch signaling promotes the corneal epithelium wound healing
复制标题
DOI：
--
复制
发表时间：
2012-02
期刊：
影响因子：
2.2
通讯作者：
Huayi Lu;Qingxian Lu;Yajuan Zheng;Qiutang Li
中科院分区：
文献类型：
--
作者：
Huayi Lu;Qingxian Lu;Yajuan Zheng;Qiutang Li
目的Notch信号通路在多种组织和细胞类型中对细胞增殖、分化和细胞命运的决定起着重要的调节作用。本研究旨在利用在角膜上皮细胞中过表达激活的Notch1(NICD)的转基因小鼠，测试Notch活性增强对角膜上皮细胞动态平衡和伤口愈合的影响。方法在R26fN1-ICD转基因小鼠上进行研究，该转基因小鼠携带NICD基因，其表达被“Lox-Stop-Lox”盒阻止。当这只转基因小鼠被培育成携带由组织特异性角蛋白14(K14)启动子驱动的Cre重组酶表达盒的小鼠品系时，花朵状的“STOP”盒被切除，NICD在角膜上皮中表达。用定量聚合酶链式反应(QPCR)检测转基因角膜上皮中NICD及其下游靶基因Hes1和与YRPW基序1相关的Hes1基因和Hes1基因的表达水平。将转基因角膜上皮细胞的表型和形态与野生型(WT)对照组进行比较。用5-溴-2‘-脱氧尿苷(BrdU)掺入法检测角膜上皮细胞的增殖率，用K14、肿瘤蛋白p63(P63)、K12和闭锁带1(ZO-1)免疫反应检测角膜上皮细胞的分化状态。分别于伤后0、6、12、18、24 h用荧光染色和Richardson‘s染色观察角膜上皮细胞对创面愈合的反应，显微镜下观察角膜上皮细胞对创面愈合的反应。结果虽然NICD在角膜上皮细胞中的过度表达导致其下游靶基因Hes1和Hey1表达上调，但这并不改变角膜上皮细胞的增殖和分化。而伤口愈合诱导的Notch活性和NICD的过表达促进了角膜上皮损伤的愈合，这与NICD转基因小鼠比野生型对照小鼠更快的早期增殖反应是一致的。结论进一步证实了Notch信号在角膜上皮损伤修复反应中的作用。
Purpose The Notch signaling pathway plays crucial roles in regulation of cell proliferation, differentiation and cell fate decision in multiple tissues and cell types. This study was designed to test the effects of enhanced Notch activity on corneal epithelium homeostasis and wound healing using the transgenic mice that overexpressed an activated Notch1 (NICD) in cornea epithelium. Methods The studies were performed on R26fN1-ICD transgenic mice that carry a NICD cDNA (cDNA) whose expression is prevented by a “Lox-STOP-Lox” cassette. When this transgenic mouse is bred to a mouse strain carrying a Cre recombinase expression cassette driven by a tissue-specific keratin 14 (K14) promoter, the floxed “STOP” cassette is excised and NICD is expressed in the cornea epithelium. The expression level of NICD and its downstream target genes, hairy and enhancer of split 1 (Hes1) and hairy/enhancer-of-split related with YRPW motif 1 (Hey1), in the transgenic corneal epithelium was examined by quantitative PCR (qPCR). The phenotypes and morphology of the transgenic corneal epithelium were compared with that of wild type (WT) controls. The proliferation rate of the epithelial cells was assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the differentiation statues were examined by K14, tumor protein p63 (p63), K12, and zona occludens 1 (ZO-1) immunoreactivity at either normal developmental condition or after corneal epithelial debridement. The corneal epithelial response to wound healing was studied by fluorescent staining and Richardson’s staining macroscopically and by H&E staining at microscope level at 0, 6, 12, 18, and 24 h post injury. Results Although overexpression of NICD in cornea epithelium led to upregulation of its downstream targets, i.e., Hes1 and Hey1, this did not alter corneal epithelial cell proliferation and differentiation. However, wound healing induced Notch activity and overexpression of NICD promoted corneal epithelial wound healing, which was in agreement with more rapid early proliferation response in NICD transgenic mice than in the wild type control mice. Conclusions These findings further demonstrate the functional role of Notch signaling in corneal epithelium wound healing response.