2019-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/711307摘要：最初Wilms’ tumour 1 (Wt1) 基因被發現是和小孩腎臟腫瘤 (Wilms’ tumor)問題相關的一個基因。在胚胎發育時期，Wt1主要表現在間質細胞的組織,例如腎臟、性腺、脾臟以及肺臟。因此Wt1和這些相關的器官例如:泌尿系統、心臟、腎臟與肺臟的發育息息相關。 在發育中的肺臟，這些表現W T1的間質細胞會參與日後肺臟支氣管以及肺血管的平滑肌細胞的發育。但是W T1不只僅僅表現在肋膜的的間質細胞上，同時在肺芽(lung buds)剛開始發生時期，Wt1也會表現在前腸組織(foregut)內胚層的腹側上皮細胞上(日後發育成肺臟上皮)，而且他們發現這些表現Wt1的內胚層上皮細胞只會出現在日後發育中肺臟的氣管跟主要的支氣管上皮，而在不會出現在發育中肺臟的肺葉細胞中。但是他們並沒有仔細去看日後成鼠的肺臟。而且這種內胚層表現Wt1的上皮細胞對日後呼吸道上皮細胞的發育與分化的功能仍是不清楚的。 另外，先前的研究顯示，Wt1基因剔除的老鼠會表現間質組織(如肋膜與心包膜)發育的異常，以及過小的肺臟與先天性橫隔膜缺陷，就如同新生兒的先天性橫膈膜疝氣疾病一樣。但是他們認為這個過小的肺臟很可能是因為橫膈膜的缺陷，導致腹腔器官跑到胸腔，讓肺臟成長空間過小所導致。不過，Wt1對肺臟發育所造成的直接影響目前是不知道的。 在我們的初步結果顯示，我們有能力從新生鼠以及成鼠的肺臟分離出會表現Wt1的間質細胞，所以我們有機會可以利用體外的細胞實驗中去探討這些細胞的生物功能。另外，我們也將會利用基因轉殖鼠及螢光鼠來做細胞追蹤(lineage tracing)去探討這些表現Wt的肺臟上皮細胞在活體肺臟的生物功能。因此，在這個計劃裡面我們的主要目的如下:一、想利用體外細胞培養來瞭解這些從肺臟分離出來的Wt1陽性的間質細胞它的生物功能。二、利用Wt1-GFP-Cre 和 Wt1CreERT2老鼠去追蹤這些Wt1陽性的的肺臟上皮細胞在肺臟發育及日後肺臟恆定的角色。三、利用條件式基因剔除鼠(conditional knockout)的技術將肺臟上皮細胞的Wt1基因剃除去探討Wt1對肺臟發育的影響。<br> Abstract: The Wilms’ tumour 1 (Wt1) gene was first identified as a strong candidate predisposition gene for pediatric renal cancer-Wilms’ tumor. During embryogenesis, Wt1 is mainly expressed in mesodermally derived tissues, such as kidneys, gonads, spleen, and the mesothelial lining of thoracic and abdominal organs. Therefore, Wt1 is essential for mesothelial tissue, and its associated organs, such as urogenital, cardiac, and lung development. In developing lung, Wt1 positive mesothelial cells contributed to subpopulation of bronchial and vascular smooth muscle cells development. However, Wt1 not exclusively expressed in mesothelial cells (pleural membrance) during lung development, it also expressed in the endodermal epithelium of ventral foregut at E9.5 when lung buds just started to form. In the developing lung, Que et al. found these Wt1 positive endodermal epithelial cells were only seen within the trachea and main stem bronchi and never within the lobes of the developing lung. However, they did not look at it in detail in adult lung. In addition, the role of endodermal Wt1 in airway epithelial cells development and differentiation is still unclear. Previously, Wt1 mutants showed abnormal mesothelium development, such as pleural and pericardium defect), small lung, and diaphragm defect. They claimed that the small lung may secondary due to the defect of diaphragm (congenital diaphragmatic hernia) and pleural tissues. However, the role of Wt1 in lung development directly is still unknown. Our preliminary data showed that we can isolate mesothelial cells from neonatal and adult lungs by sorting the CD45-CD31-Epcam-Pdpn+Cxadr+ population, then we will have chance to analyze its functions in vitro. In addition, we will use transgenic and reporter mice to do lineage tracing to look at its biological function in lung development in vivo. Therefore, the specific aims of this project are:Aim 1: Explore the biologic function of isolated Wt1 positive mesothelial cells from lungAim 2: Lineage tracing of Wt1 positive lung epithelial cells in developing lung by using Wt1-GFP-Cre and Wt1CreERT2 miceAim 3: Study the function of epithelial Wt1 in developing lung by conditional knockout technology using ShhCre and Wt1 flox/flox mouse linesWt1間質細胞肺臟發育Wt1mesothelial celllung development