選殖表達LAMR1基因的人類胚胎腎細胞; Cloning the LAMR1-overexpressed HEK293 cells

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Title:	選殖表達LAMR1基因的人類胚胎腎細胞;Cloning the LAMR1-overexpressed HEK293 cells
Authors:	蕭巧馨;Chiao-hsin Hsiao
Contributors:	生命科學研究所
Keywords:	LAMR1基因;LAMR1
Date:	2010-01-22
Issue Date:	2010-06-11 16:08:33 (UTC+8)
Publisher:	國立中央大學圖書館
Abstract:	LAMR1 是一個 295 胺基酸的非粘著蛋白受器 (non-integrin receptor)，且對層粘連蛋白(laminin)有很高的親合力。此蛋白質對於細胞的生長與轉移有很大的關係。本篇研究以人類胚胎腎臟細胞株-HEK293 為平台，建構了數種單株穩定型細胞株，包含全長、1-100 aa長的LAMR1 轉染的細胞株。另外為了利於偵測，也同時建構了一組在不同片段 LAMR1 之 C 端或 N 端接有 flag 序列的轉染細胞株，另外也針對 LAMR1 的 N 端剔除了不同片段的 LAMR1，分別是 43-295 aa以及 89-295 aa 共九種細胞株。結果發現，在RNA的表現下，所送入之各個片段的LAMR1都有表現，而蛋白質表現下，我們以flag抗體偵測到全長LAMR1 C端或N端的細胞株都有表現；偵測細胞膜上與細胞質中的蛋白質表現，結果顯示用 flag 抗體偵測 flag 蛋白質表現時，全長LAMR1基因帶有C端flag基因的細胞株其細胞膜上的flag蛋白質表現比LAMR1基因帶有N端flag基因的細胞株多；從全長LAMR1及LAMR1(1-100 aa)的細胞株其生長情形與控制組相比沒有太大差異，而在從N端設計不同片段之單株穩定細胞株中，43-295 aa生長比控制組細胞株慢，但是在89-295 aa這個細胞株中，其生長情形比控制組細胞快。而在有flag細胞株的組別中，全長LAMR1之N端帶有flag基因的細胞株中其生長較控制組慢，至於在C端皆有flag基因的細胞株中，其細胞數較控制組多。在處理Laminin的情況下，flag-LAMR1(1-295 aa)細胞株與LAMR1(89-295 aa)細胞株其細胞數比未處理Laminin的情況下多。根據以上的結果，我們推測在HEK293 細胞中，LAMR1的N端扮演著很重要的角色，尤其是在LAMR1的胺基酸 43-89 aa 區域。 LAMR1 is a 295 amino acids non-integrin receptor that has a high affinity to laminin. It was found to enhance the growth and metastasis of cells. To examine whether any of the specific amino acid domain of LAMR1 protein is responsible for its regulating growth of HEK293 cells, this study was using calcium phosphate transfection to clone several monoclonal stable cell lines of HEK293 which expressed the exogenous full length, C-terminus-truncated forms of LAMR1(1-100 aa), N-terminus-truncated LAMR1 (43-295 aa and 89-295 aa). The full-length LAMR1 fused with C-terminal flag protein stable cells displayed more LAMR1 in the membrane than did the full-length LAMR1 fused N-terminal flag protein cells. The cell growth of the full-length LAMR1-expressing HEK293 cells and LAMR1(1-100 aa)-expressing HEK293 cells as compared with empty vector-transfected cells were no significant difference. However, LAMR1(89-295 aa)-expressing cells had more cell numbers than control cells, but the cell numbers of LAMR1(43-295 aa)-expressing cells and the full-length flag LAMR1-expressing cells were less than control cells. With the coating treatment of laminin, LAMR1(89-295 aa)-expressing HEK293 cells and flag-LAMR1(1-295 aa)-expressing HEK293 cells had more cell numbers than cells without treating laminin. These data suggested that the N-treminus of LAMR1 at position of 43-89 aa plays an important role on cell growth.
Appears in Collections:	[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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