本文選題：Shh信號(hào)通路 + 肺發(fā)育　； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景胚胎的發(fā)育受到不同信號(hào)通路的調(diào)控,以及上皮-間質(zhì)相互作用的影響,不同部位基因表達(dá)的異常激活或缺失,將會(huì)導(dǎo)致一系列的后果,包括引起肺發(fā)育異常的疾病,甚至死亡。正確認(rèn)識(shí)肺發(fā)育過(guò)程中不同信號(hào)通路之間的關(guān)系,了解其作用機(jī)制,對(duì)理解導(dǎo)致人類肺先天發(fā)育異常相關(guān)疾病有所幫助。盡管小鼠與人類的肺的發(fā)育有所不同,但肺發(fā)育早期的分子信號(hào)通路是相對(duì)保守的。Hedgehog(Hh)信號(hào)通路在胚胎時(shí)期的細(xì)胞分化、組織與器官發(fā)育中扮演著重要的角色,主要由三種分泌型糖蛋白配體Sonic Hedgehog(Shh)、Indian Hedgehog(Ihh)和Desert Hedgehog(Dhh),兩個(gè)跨膜蛋白受體Patchedl(Ptchl)、Smoothened(Smo),三個(gè)核轉(zhuǎn)錄因子Glil、Gli2、Gli3及下游的靶基因等組成。其中,Shh通路在小鼠胚胎肺的發(fā)育中發(fā)揮著重要作用。在Shh存在的情況下,Shh與Ptchl結(jié)合,解除了Ptchl對(duì)Smo的抑制,Smo則進(jìn)入細(xì)胞內(nèi),釋放微管活化的Glil和Gli2,并使其入核激活下游靶基因的表達(dá)。目的本課題以Shh信號(hào)通路為研究對(duì)象,利用免疫組化及免疫熒光技術(shù)來(lái)探索小鼠胚胎肺發(fā)育過(guò)程中經(jīng)典Shh信號(hào)通路對(duì)上皮、間質(zhì)轉(zhuǎn)化的必要作用。首先利用免疫組化檢測(cè)Shh通路中關(guān)鍵分子Smo以及血小板源性生長(zhǎng)因子受體-α(platelet-derived growth factor receptor-α;Pdgfr-α)的時(shí)空表達(dá)特點(diǎn),然后構(gòu)建基因敲除小鼠模型,最后利用Pdgfr-αCre敲除Smo轉(zhuǎn)基因小鼠模型,探索Shh信號(hào)通路對(duì)小鼠肺發(fā)育的作用及調(diào)控機(jī)制。方法及結(jié)果首先,我們利用免疫組化檢測(cè)Shh信號(hào)通路受體Smo及Pdgfr-α在小鼠胎肺中的表達(dá)情況。結(jié)果顯示,在整個(gè)假腺期,Smo在氣道上皮和肺間質(zhì)中均有表達(dá);而在微管期,Smo的表達(dá)量迅速下降。假腺期Pdgfr-α在近端氣道中表達(dá),E12.5天Pdgfr-α在遠(yuǎn)端肺上皮細(xì)胞以及部分包繞著氣管、支氣管、細(xì)支氣管周圍的間質(zhì)細(xì)胞表達(dá);E14.5天,Pdgfr-α的表達(dá)嚴(yán)格限制在大支氣管周圍,與支氣管平滑肌細(xì)胞毗鄰的間質(zhì)細(xì)胞表達(dá)Pdgfr-α,但在遠(yuǎn)端肺中未被發(fā)現(xiàn)有Pdgfr-α的表達(dá)。微管期,Pdgfr-α出現(xiàn)在更遠(yuǎn)端的肺間質(zhì)。然后,利用Pdgfr-α Cre敲除Smo基因,構(gòu)建基因敲除小鼠模型(由美國(guó)南加州大學(xué)/洛杉磯縣兒童醫(yī)院饋贈(zèng))。最后,利用免疫熒光檢測(cè)Shh信號(hào)通路受體Smo基因用Pdgfr-α Cre敲除后對(duì)小鼠肺發(fā)育的影響。結(jié)果發(fā)現(xiàn),與對(duì)照組肺相比,E12.5-E15.5天基因敲除組小鼠的肺體積縮小,支氣管分支形成減少,TTF-1表達(dá)水平的下調(diào),而E16.5天支氣管分支及TTF-1水平未見(jiàn)明顯差異。E12.5-E15.5天支氣管間質(zhì)產(chǎn)生軟骨基質(zhì)硫酸軟骨素的量下調(diào),氣管環(huán)形成滯后,氣管狹窄,而E16.5未見(jiàn)明顯差異。E12.5-E13.5天,實(shí)驗(yàn)組近端及遠(yuǎn)端α-Sma的表達(dá)較對(duì)照組上調(diào);E14.5實(shí)驗(yàn)組近端α-Sma的表達(dá)上調(diào),遠(yuǎn)端α-Sma的表達(dá)未見(jiàn)明顯變化;E15.5-E16.5天,實(shí)驗(yàn)組與對(duì)照組近端及遠(yuǎn)端α-Sma的表達(dá)未見(jiàn)明顯差異。E12.5-E15.5天近端上皮標(biāo)志物P63表達(dá)下調(diào),而在E16.5天,P63表達(dá)無(wú)明顯改變。在E12.5-E14.5天,均未見(jiàn)β4-tublin的表達(dá),E15.5天遠(yuǎn)端上皮指標(biāo)β4-tublin的表達(dá)量下降,E16.5天β4-tublin表達(dá)無(wú)明顯改變。結(jié)論綜上所述,本研究發(fā)現(xiàn)Shh信號(hào)通路準(zhǔn)確的時(shí)空特異表達(dá)對(duì)小鼠胚胎支氣管分支的形態(tài)發(fā)生,肺支氣管軟骨基質(zhì)、軟骨環(huán)的發(fā)育,近端及遠(yuǎn)端平滑肌以及上皮的發(fā)育起著重要的調(diào)控作用。Shh通路除了影響間質(zhì)軟骨、平滑肌的發(fā)育之外,對(duì)于上皮、肺泡的發(fā)育也起到重要作用,參與上皮-間質(zhì)轉(zhuǎn)化的過(guò)程。Shh通路可能參與支氣管肺泡發(fā)育不良的發(fā)病過(guò)程,為闡述相關(guān)肺先天發(fā)育缺陷疾病帶來(lái)一定的分子基礎(chǔ),為未來(lái)的診療提供參考靶分子,具有一定的臨床意義。
[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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R563

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本文編號(hào)：1784625