臺灣博碩士論文加值系統

中文摘要
在神經系統發育的過程中,神經系統是藉由神經幹細胞產生各式各樣的神經細胞和神經膠細胞。之前,我們實驗室使用減差雜合法(subtractive hybridization),將發育10.5天大鼠胚胎在神經管所表現的cDNA,減掉在出生後1天大鼠大腦中所表現的cDNA,希望找到在早期神經管發育特殊表現的基因。Neural stem cell marker 1 (NSC1)基因在此項的研究中被找到,而透過蛋白質序列的比對, NSC1所產生的蛋白質很有可能是一個表現在細胞膜上的接受器(receptor)。
我的論文研究的主題是NSC1基因在神經系統的表現,我要探討NSC1基因的表現型式(expression pattern)。首先,我利用原位雜合法(in situ hybridization)去探討NCS1基因在不同時期胚胎的表現型式。在9天的大鼠胚胎,NSC1基因表現在剛剛發育出來,由幾層神經幹細胞所構成的神經表皮層(neuroepithelium)中。發育到10天的大鼠胚胎,NSC1基因仍表現在神經表皮層,並且表現在這個時期從神經表皮層所形成的神經管(neural tube)中,並且在體節(somites)也有表現。而在10.5天的大鼠胚胎,NSC1基因仍然在神經管有很高量的表現,但是在體節,NSC1基因的表現量已下降。到了11.5天的大鼠胚胎,NSC1基因在神經管的表現量下降,並且已經看不見在體節有表現的現象。而到E12.5天的大鼠胚胎,NSC1基因在神經管的表現量已經相當低,幾乎在原位雜合法上偵測不到。
接下來,為了進一步看NSC1基因在蛋白質層次的表現,我就去製造NSC1蛋白質的單株抗體(monoclonal antibody)。在1376個單株抗體中,我利用西方墨點法和細胞免疫螢光染色來篩選可以檢視NSC1蛋白質的單株抗體。最後,我篩選到兩個單株抗體,並且加以純化。其中編號6B4的單株抗體可以辨識NSC1蛋白質在細胞膜外的部位,另一株編號1D5的單株抗體則可以辨識NSC1蛋白質在細胞膜內的部位。兩株抗體在西方末點法和細胞免疫螢光染色的實驗中被證明,它們的確可以很專一地認識NSC1基因所表現出來的蛋白質。接下來,在細胞免疫螢光的實驗中,我利用單株抗體去染大量表現NSC1蛋白質的HEK293T細胞株,然後在共軛焦顯微鏡(confocal microscopy)底下觀察發現,NSC1的確表現在細胞膜上,進一步在活染(live staining)的實驗中,也得到同樣的結果。我也利用單株抗體做不同時期大鼠胚胎的組織免疫染色,發現NSC1蛋白質主要表現在早期胚胎的神經表皮層和神經管,到了晚期,NSC1蛋白質的表現量就下降了,與原位雜合法實驗的結果一致。從原位雜合法和組織免疫染色的結果可知,NSC1基因會在早期胚胎發育時期表現,但隨著在胚胎發育的過程中,表現量逐漸下降。

Abstract
Neural stem cells are involved in neural development and are capable of generating a variety of neural cells. Previously, we used a subtractive hybridization assay, in which cDNA of embryonic day 10.5 (E10.5) rat neural tube was subtracted from cDNA of postnatal day 1 (P1) rat brain, to identify genes preferentially expressed in early neural tube. Neural stem cell marker 1 (NSC1) gene was identified in this assay and was hypothesized as a membrane receptor.
In this project, I intended to detect the expression pattern of NSC1. First, I used in situ hybridization in different stages of rat embryos to detect NSC1 expression pattern. In the E9 stage, NSC1 was mainly expressed in neuroepithelium. Later in the E10 embryo, NSC1 gene was expressed in neuroepithelium, developing neural tube and somites. In the E10.5 stage, expression of the NSC1 gene was also strong in neural tube. However, the expression level in somites was reduced. In the E11.5 stage, the expression level in neural tube was down-regulated and the signals in somites were hardly detected. In the E12.5 stage, NSC1 expression was barely detected in the neural tube. Second, I generated monoclonal antibodies (mAbs) against NSC1. I had tested hundreds of clones for their specificity by western blotting and immunocytochemistry. Two clones, 6B4 against NSC1 extra-cellular domain and 1D5 against NSC1 intra-cellular domain, were selected and purified. It was shown that 6B4 and 1D5 were specifically against NSC1 in western blotting and immunocytochemistry. Results from cell fluorescence staining of HEK293T cells overexpressing NSC1 showed that staining of NSC1 was on the cell membrane, as demonstrated in the confocal microscopy and the live staining. Immunohitochemistry staining of different stages of embryos showed that NSC1 was expressed in neuroepithelium and neural tube in the early stage embryo. Expression of NSC1 protein was reduced in the later stage embryo. From the data of in situ hybridization and immunohitochemistry, expression of NSC1 was in a developmentally regulated pattern.

Contents
中文摘要 1
Abstract 3
Introduction 5
Materials and Methods 8
Results 18
Discussion 22
References 26
Figures and Tables 30

Reference
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