Sonic Hedgehog信号通路调控小鼠肺发育机制的研究

发布时间：2018-04-21 23:32

本文选题：Shh信号通路 + 肺发育　；参考：《南方医科大学》2017年硕士论文

【摘要】：背景胚胎的发育受到不同信号通路的调控,以及上皮-间质相互作用的影响,不同部位基因表达的异常激活或缺失,将会导致一系列的后果,包括引起肺发育异常的疾病,甚至死亡。正确认识肺发育过程中不同信号通路之间的关系,了解其作用机制,对理解导致人类肺先天发育异常相关疾病有所帮助。尽管小鼠与人类的肺的发育有所不同,但肺发育早期的分子信号通路是相对保守的。Hedgehog(Hh)信号通路在胚胎时期的细胞分化、组织与器官发育中扮演着重要的角色,主要由三种分泌型糖蛋白配体Sonic Hedgehog(Shh)、Indian Hedgehog(Ihh)和Desert Hedgehog(Dhh),两个跨膜蛋白受体Patchedl(Ptchl)、Smoothened(Smo),三个核转录因子Glil、Gli2、Gli3及下游的靶基因等组成。其中,Shh通路在小鼠胚胎肺的发育中发挥着重要作用。在Shh存在的情况下,Shh与Ptchl结合,解除了Ptchl对Smo的抑制,Smo则进入细胞内,释放微管活化的Glil和Gli2,并使其入核激活下游靶基因的表达。目的本课题以Shh信号通路为研究对象,利用免疫组化及免疫荧光技术来探索小鼠胚胎肺发育过程中经典Shh信号通路对上皮、间质转化的必要作用。首先利用免疫组化检测Shh通路中关键分子Smo以及血小板源性生长因子受体-α(platelet-derived growth factor receptor-α;Pdgfr-α)的时空表达特点,然后构建基因敲除小鼠模型,最后利用Pdgfr-αCre敲除Smo转基因小鼠模型,探索Shh信号通路对小鼠肺发育的作用及调控机制。方法及结果首先,我们利用免疫组化检测Shh信号通路受体Smo及Pdgfr-α在小鼠胎肺中的表达情况。结果显示,在整个假腺期,Smo在气道上皮和肺间质中均有表达;而在微管期,Smo的表达量迅速下降。假腺期Pdgfr-α在近端气道中表达,E12.5天Pdgfr-α在远端肺上皮细胞以及部分包绕着气管、支气管、细支气管周围的间质细胞表达;E14.5天,Pdgfr-α的表达严格限制在大支气管周围,与支气管平滑肌细胞毗邻的间质细胞表达Pdgfr-α,但在远端肺中未被发现有Pdgfr-α的表达。微管期,Pdgfr-α出现在更远端的肺间质。然后,利用Pdgfr-α Cre敲除Smo基因,构建基因敲除小鼠模型(由美国南加州大学/洛杉矶县儿童医院馈赠)。最后,利用免疫荧光检测Shh信号通路受体Smo基因用Pdgfr-α Cre敲除后对小鼠肺发育的影响。结果发现,与对照组肺相比,E12.5-E15.5天基因敲除组小鼠的肺体积缩小,支气管分支形成减少,TTF-1表达水平的下调,而E16.5天支气管分支及TTF-1水平未见明显差异。E12.5-E15.5天支气管间质产生软骨基质硫酸软骨素的量下调,气管环形成滞后,气管狭窄,而E16.5未见明显差异。E12.5-E13.5天,实验组近端及远端α-Sma的表达较对照组上调;E14.5实验组近端α-Sma的表达上调,远端α-Sma的表达未见明显变化;E15.5-E16.5天,实验组与对照组近端及远端α-Sma的表达未见明显差异。E12.5-E15.5天近端上皮标志物P63表达下调,而在E16.5天,P63表达无明显改变。在E12.5-E14.5天,均未见β4-tublin的表达,E15.5天远端上皮指标β4-tublin的表达量下降,E16.5天β4-tublin表达无明显改变。结论综上所述,本研究发现Shh信号通路准确的时空特异表达对小鼠胚胎支气管分支的形态发生,肺支气管软骨基质、软骨环的发育,近端及远端平滑肌以及上皮的发育起着重要的调控作用。Shh通路除了影响间质软骨、平滑肌的发育之外,对于上皮、肺泡的发育也起到重要作用,参与上皮-间质转化的过程。Shh通路可能参与支气管肺泡发育不良的发病过程,为阐述相关肺先天发育缺陷疾病带来一定的分子基础,为未来的诊疗提供参考靶分子,具有一定的临床意义。
[Abstract]:The development of background embryos is regulated by different signaling pathways and the effect of epithelial mesenchymal interaction. Abnormal activation or deletion of gene expression in different parts will lead to a series of consequences, including diseases that cause abnormal lung development, or even death. The mechanism of action helps to understand the disease related to human lung congenital abnormalities. Although the development of lung in mice is different from that of human lung, the molecular signaling pathway in the early stage of lung development is a relatively conservative.Hedgehog (Hh) signaling pathway, which plays an important role in the development of tissue and organs. Three secretory glycoprotein ligands, Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh), two trans membrane protein receptors Patchedl (Ptchl), Smoothened, three nuclear transcription factors and downstream target genes, are important in the development of mouse embryo lung. In the case of the combination of Shh and Ptchl, it relieves the inhibition of Ptchl to Smo, Smo enters the cells, releases the Glil and Gli2 activated by microtubules, and activates the expression of the downstream target gene. The purpose of this subject is to explore the classical Shh in the process of mouse embryo lung development by using the Shh signaling pathway as the research object. The necessary role of signal transduction on the transformation of epithelium and interstitial tissue. First, the spatio-temporal expression of the key molecules in the Shh pathway, Smo and the platelet-derived growth factor receptor- alpha (Pdgfr- a), was detected by immunohistochemistry, and then the gene knockout mouse model was constructed, and Pdgfr- alpha Cre knocked off the Smo transfer base. The effect and regulation mechanism of Shh signaling pathway on lung development in mice was explored. Methods and results first, we detected the expression of Shh signaling pathway receptor Smo and Pdgfr- alpha in mouse fetal lungs by immunohistochemistry. The results showed that Smo was expressed in the airway epithelium and interstitial lung in the whole pseudo glandular stage, while in microtubule phase, Smo The expression of Pdgfr- alpha in the pseudo glandular stage was expressed in the proximal airway, and E12.5 days Pdgfr- alpha was expressed in the distal lung epithelial cells and some of the interstitial cells around the trachea, bronchus and bronchioles. On E14.5 days, the expression of Pdgfr- a was strictly restricted around the bronchioles and expressed in the stromal cells adjacent to the bronchial smooth muscle cells. Pdgfr- alpha, but the expression of Pdgfr- alpha was not found in the distal lung. In microtubule phase, Pdgfr- alpha appeared in the more distal pulmonary interstitium. Then, Pdgfr- alpha Cre was used to knock off the Smo gene and to construct a gene knockout mouse model (presented by the University of Southern California / Losangeles county children's Hospital). Finally, the Shh signaling receptor Smo gene was detected by immunofluorescence. The effect of Pdgfr- alpha Cre knockout on lung development in mice. The results showed that compared with the control group lung, the lung volume of the E12.5-E15.5 days gene knockout mice decreased, the branching of bronchial branches decreased, and the expression level of TTF-1 was down, while the bronchial branches and TTF-1 levels in E16.5 days did not show significant differences in the.E12.5-E15.5 days of the cartilaginous base of the bronchial interstitium in.E12.5-E15.5. The quantity of chondroitin sulfate decreased, tracheal ring lag, tracheal stenosis, and E16.5 no significant difference.E12.5-E13.5 days, the expression of proximal and distal alpha -Sma in the experimental group was up up compared with the control group; the expression of the proximal alpha -Sma in the experimental group was up and the expression of the distal alpha -Sma was not obviously changed; E15.5-E16.5 days, the experimental group and the control group were in the proximal and distal end of the experimental group. The expression of alpha -Sma was not significantly different from.E12.5-E15.5 day proximal epithelial marker P63 expression, but on E16.5 days, there was no obvious change in P63 expression. In E12.5-E14.5 days, there was no expression of beta 4-tublin, the expression of beta 4-tublin in E15.5 day distal epithelial index decreased, and E16.5 day beta 4-tublin expression did not change significantly. The exact temporal and spatial expression of the present Shh signaling pathway plays an important role in the morphogenesis of the branched branching of the mouse embryo, the cartilage matrix of the lung, the development of the cartilage ring, the development of the proximal and distal smooth muscle and the development of the epithelium. In addition to the development of the interstitial cartilage and the smooth muscle development, the development of the epithelium and the alveoli is also the same. The.Shh pathway involved in the process of epithelial mesenchymal transition may be involved in the pathogenesis of bronchioloalveolar dysplasia, which provides a molecular basis for the elaboration of related pulmonary congenital defects and provides a reference target for future diagnosis and treatment, and has a certain clinical significance.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563

【参考文献】