2018-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/705080摘要：在胚胎發育時期，肺臟神經會伴隨著呼吸上皮管路的發育發展出複雜的肺臟神經網路。這個肺臟 神經網路包括兩群神經元細胞：一群的細胞核是位於肺臟外如nodose神經節或延腦即所謂的外在神 經網路，他們會將神經突觸由肺臟外往肺臟内延伸並和呼吸道平滑肌以及肺臟的神經内分泌細胞體 (neuroendocrine body)相連結；另一群則是神經元位置就在肺臟内，即所謂的内在神經網路。利用 細胞追蹤技術已經知道這些内在神經網路其實是來自神經脊細胞（neural crest cell)。這些内在神 經網路也會和呼吸道平滑肌相連，被認為可能和肺臟發育與成熟相關，不過，它真正的功能其實不是 很清楚。除此之外，改變神經結構或神經傳導物質已經被證實會伴隨肺臟疾病的發生，例如自體氧氣 偵測相關疾病，肺臟血管的收縮或肺高壓，氣喘，慢性組塞性肺疾病等。最近一些證據顯示，慢性咳 嗽其實也是一種神經病變。Notch訊息對分臟的發育很重要，尤其是呼吸道上皮的分化。另外，若小鼠的Notch訊息被剔除， 肺臟呼吸道上皮會變鱗狀扁平化-這點和人類的慢性阻塞性肺疾病的呼吸道上皮病變特徵很像，此外 呼吸道上的神經内分泌細胞及神經内分泌體攝物目也會明顯增加。但是，到底這些上皮和肺臟神經是 如何互相影響則不是很清楚。另一方面，Notch訊息也會直接影響神經脊細胞的發育，進而影響腸道 神經系統的發育。但是，Notch訊息會不會影響以及如何影響同樣來自神經脊細胞的肺臟神經系統的 發育，目前仍是一無所知的。因為這麼複雜的肺臟神經網路連結到呼吸道平滑肌與肺臟的神經内分泌細胞體，傳統的二維組織 切片已經無法提供完整並全面的相關資訊。最近，有些研究試著要去建構全肺的神經網路，但是受限 於組織透明化技巧，他們無法建構完全擴張的全肺影像，因為脫水的步驟會導致組織萎縮變形。因此 在我們這個研究計劃，我們會和清華大學合作，利用江安世教授自行發展出的組織透明技巧及試劑， 及其實驗室先進的共軛影像設備，將他們研究果蠅腦神經並發表在Science及Cell雜誌的實驗技 巧應用在我們小鼠肺臟神經體的研究。同時，也會和清華大學團隊另一位成功將此研究果蠅腦神經技 巧轉移到研究小鼠腸道及胰臟神經的湯學成教授合作，如此將大幅提高此研究的成功率。最後，我們 期望能利用此有用的技巧建構出三維的肺臟神經網路體，以利後來研究某些神經群或各別神經元的功 能，及其在正常時期與肺臟疾病所扮演的角色。藉由和清大江安世教授與湯學成教授合作的優勢，我們將有很大的機會可以完成這些研究目標。 此研究計劃的目的如下：1. 建立小鼠全肺組織透明化的標準流程及三維共軛影像條件2.利用基因轉職標示細胞技巧來建構不同時期胎鼠的全肺神經網路體3.利用神經脊專一性的基因剔除技術來抑制神經脊Notch訊息以釐清Notch訊息如何影響肺臟 神經的發育以及肺臟神經對肺臟發育的影響4. 研究類似人類慢性阻塞性肺疾病的動物模式中肺神經是如何改變的（ShhCre;Pofut1肺臟上 皮專一性Notch基因剔除鼠）<br> Abstract: During embryogenesis, the complex pulmonary neural network form along with respiratory tubules. This network includes extrinsic and intrinsic neurons. Extrinsic neurons have cell bodies located in the jugular and nodose ganglia, and medulla oblongata, while their exons extend into the lungs and innervate the airway smooth muscle (ASM) and pulmonary neuroendocrine bodies (NEB) in the lung epithelium. In contrast, the intrinsic neurons are clustered in respiratory tracts. Using lineage labeling in mouse embryos has shown that these intrinsic nerve ganglia are derived from the neural crest cells. The intrinsic neurons in ganglia function to innervate airway smooth muscle, which was thought to be associated with lung development and maturation, but its role is still poor understood. The changes in neural structure and innervation have been shown to be associated with lung diseases such as oxygen sensing disorders, asthma, chronic cough or chronic obstructive pulmonary disease (COPD).Because the complex of pulmonary nerve and its associated ENBs and ASM, the standard 2-D histology cannot provide the global and spatial information of lung innervation. Recently, some reports try to demonstrate the whole lung innervation, however, due to the limitation of tissue clearing technique they cannot analyze the fully inflated lungs and the dehydrated step causes mild tissue shrinkage. Here, we will use the linage labeling technique to label each neuron and cooperate with Prof. Ann-Shyn Chaing and Prof. Shiue-Cheng Tang (bioimaging core at the Brain Research Center in Tsing Hua University) to study the lung connectome in whole mouse lungs. By using their optical-clearing solution to facilitate photon penetration for 3-dimensiontal (3-D) confocal microscopy and their high-resolution 3D imaging system, we will build the 3-D lung innervation map. Finally, we would like to provide a useful tool for 3-D imaging of lung neural tissues to better characterize their roles in health and lung diseases.Taking the advantage of cooperation with Prof. Ann-Shyn Chiang and Prof. Shiue-Cheng Tang, we will have a good chance to finish the following specific aims. The specific aims for this study are:Aim 1: Setup the optical-clearing condition and 3D image system for the whole embryonic lungs.Aim 2: Build up the 3D map of different developmental stage of whole lung innervation by using the transgenic lineage labeling technique.Aim 3: Study the role of Notch signaling in lung innervation by using neural crest specific deleter mouse and its effect on lung development.Aim 4: Study the changes of lung innervation in human chronic lung disease mouse model (ShhCre;Pofut1 conditional knockout mouse)