Rasd1在由地塞米松引發於斑馬魚幼魚脈絡叢之壓力反應中所扮演的角色

Abstract

壓力是現代生活中常會碰到的問題，並會引發許多嚴重的病症，諸如肥胖、心血管疾病、心理問題等等。在壓力狀態下，腎上腺會分泌可體松(cortisol)一類的葡萄糖皮質素(glucocorticoid)，引發一系列的反應以對抗壓力。地塞米松(dexamethasone)是一種人工合成的葡萄糖皮質素，且與內生性的葡萄糖皮質素有非常相似的結構與功能，常被用來模擬身體在壓力下的狀態。在本實驗室先前的研究中，已證實地塞米松會在斑馬魚幼魚的脈絡叢(choroid plexus)中引起rasd1的表現。Rasd1被認為是一種位於膜上的小三磷酸鳥苷酶(small GTPase)，並且可能與一些訊息傳導路徑有關聯。而Rasd1在這由地塞米松所引發的脈絡叢壓力反應中究竟扮演著何種角色目前仍不得而知。 為了釐清Rasd1在脈絡叢的壓力反應中所扮演的角色，我們進行了核糖核酸定序(RNA sequencing)。由於當時仍無法準確地取脈絡叢的細胞，因此核糖核酸定序所使用的RNA是取自斑馬魚幼魚的全頭。我使用了反轉錄即時定量聚合酶連鎖反應(qRT-PCR)以及全固定原位雜合反應(whole-mount in situ hybridization)來驗證核酸定序的結果，並且發現與缺氧壓力相關的hif1al基因同樣也會在地塞米松的影響之下表現於脈絡叢。另外我也發現在進行地塞米松加藥處理後，rasd1表現量上升的時間較hif1al要早。 雖然全頭的核糖核酸定序分析已經幫助我得到一些初步的結果，但若要更進一步的探討脈絡叢的免疫反應，則針對脈絡叢的核糖核酸分析仍是不可或缺的。為了實現精準的定位脈絡叢細胞，我使用了由實驗室學長製造，在脈絡叢過量表現增強型綠螢光蛋白(eGFP)的基因轉殖斑馬魚(Wu, 2019)來進行此實驗。我使用極細的針頭將位於脈絡叢的綠螢光細胞切割出來，並且使用從這些細胞抽取的核糖核酸進行定序。而根據針對脈絡叢的核糖核酸定序結果，我發現在經過地塞米松加藥處理後，許多心臟相關的基因在脈絡叢的表現量會顯著上升，但此結果仍需進一步的驗證。另外，在根據此定序結果進行的全固定原位雜合反應實驗結果中，我也發現經地塞米松處理後，與物質運輸相關的slc26a2基因在脈絡叢的表現量會顯著上升。 為了便於針對定序的結果進行後續的篩選與驗證，我製造了一條在脈絡叢過量表現Rasd1的基因轉殖斑馬魚。利用這條轉殖魚，我發現rasd1的過量表現並不會引發hif1al的上升，綜合先前得到rasd1表現量較早上升的結果，我認為這兩個基因很可能是在不同的反應路徑上。未來我們將持續探討rasd1、hif1al與slc26a2等基因之間的關聯性，相信可以幫助我們對脈絡叢的壓力反應有更進一步的認識。

Stress is a common problem in modern society, which leads to many serious illnesses, including obesity, cardiovascular diseases and mental problems, etc. Under stress conditions, adrenal glands secret glucocorticoids such as cortisol, that leads to a series of responses to confront stress. Dexamethasone (DEX) is a synthetic glucocorticoid that has similar predicted functions as endogenous glucocorticoids, often used to mimic stress conditions. Previously, our laboratory proved that the expression of rasd1 is upregulated in choroid plexuses (CP) of 3 dpf zebrafish larvae after DEX treatment. Rasd1 is predicted to be a membrane-bound small GTPase, may be involved in signal transduction. However, the role Rasd1 plays in the DEX-induced stress response of CP still remains unknown. To clarify the role of Rasd1 in the stress response of CP, we did RNA-seq for comparing the gene expression of DEX-treated and control zebrafish larvae. Due to the difficulty in collecting CP cells from zebrafish larvae, RNA extracted from whole head of DEX-treated and control larvae was used for RNA-seq. I utilized qRT-PCR and whole-mount in situ hybridization (WMISH) to verify the RNA-seq result, and found that hif1al is also induced in CP after DEX treatment. With time-course WMISH, I found that the induction of rasd1 by DEX treatment is earlier than that of hif1al. Although I have gained some data with whole-head RNA-seq, CP-specific RNA-seq is still needed for further research. To specifically harvest CP cells, I used a transgenic zebrafish line generated by our lab member (Wu, unpublished, 2019), which expresses eGFP in CP cells. Utilizing syringe needles, eGFP expressing cells in CP were carved out, and RNA extracted from these cells was used for RNA-seq. With the CP-specific RNA-seq result, I found that many heart-related genes were significantly upregulated in CP after DEX treatment, while further verification of the result is still needed. Also, based on CP-specific RNA-seq result, I selected several genes to do WMISH, and found that slc26a2 was also induced in CP by DEX. To help me verify the RNA-seq result, I generated a transgenic zebrafish line that overexpresses rasd1 in CP. With this transgenic line, I found that hif1al was not induced in CP by rasd1 overexpression. Combining the time-course WMISH data, I believe the two genes are highly possible to be in separate pathways. We will keep investigating the relationship among rasd1, hif1al and slc26a2, and the results should let us have a deeper understand of the stress response in CP.