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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17513
標題: | 高通量細胞表現型篩選平台 High-throughput cell-based assays |
作者: | Po-Cheng Chen 陳柏丞 |
指導教授: | 黃義侑 |
關鍵字: | 細胞表現型篩選平台,冷電漿,細胞微陣列晶片,微機電系統,微孔洞,微管柱, cell-based assay,plasma,cell microarray,MEMS,microwell,microcolumn, |
出版年 : | 2013 |
學位: | 博士 |
摘要: | 儘管在高通量藥物篩選以及進行和疾病相關的基因功能研究時,細胞表現型篩選平台已經越來越被普遍利用,但是受限於它複雜的操作步驟與昂貴的花費,大部分的實驗室仍無法進行此類型實驗;因此,我們希望開發出簡單且以晶片型式進行的細胞表現型篩選平台。首先,我們利用冷電漿(plasma)表面改質的技術開發出可進行反轉殖(reverse transfection)之菌落形式(colonial)的細胞微陣列晶片,以點放在聚丙烯(polypropylene) 薄膜上的刀豆素A(concanavalin A)讓S2 (Drosophila Schneider 2) 細胞可以附著在聚丙烯薄膜上面生長並進行質體(plasmid)DNA或小干擾RNA(Small interfering RNA;siRNA)之反轉殖實驗。 為了發展更高密度的細胞微陣列晶片,我們使用微機電系統(micro-electro-mechanical systems)技術,將光阻塗附在玻璃上製造微孔洞(microwell)晶片,並進行細胞基因表現(gene expression)與基因減弱(gene knockdown)的實驗。接著,藉由微機電系統技術,我們研發出與微孔洞晶片互補的微管柱(microcolumn)晶片,透過微管柱結構可以對生長在微孔洞中的細胞釋放藥物。我們利用塗附不同的上皮細胞生長因子接收器(epidermal growth factor receptor)之活性抑制劑在微管柱上,對微孔洞中的細胞進行藥物釋放測試,結果顯示可有效抑制上皮細胞生長因子接收器活性,證明此結構能提供高效率高通量的藥物篩選功能。因此,以上新開發的細胞表現型篩選平台,可以提供簡便有效率的基因功能分析與藥物篩選研究。 Although cell-based assay is becoming more popular for high-throughput drug screening and the functional characterization of disease-associated genes, most researchers in these areas do not use it because it is a complex and expensive process. We wanted to create a simple method of performing an on-chip cell-based assay. To do this, we developed a colonial cell microarray for reverse transfection assays with surface modification by plasma treatment. A colonial cell microarray of Drosophila Schneider 2 (S2) cells on a polypropylene (PP) thin film was demonstrated. The concanavalin A (ConA) micropatterns enabled the adhesion of S2 cells on the surface, resulting in the formation of a colonial cell microarray. Subsequently, the colonial S2 cells were transfected with plasmid DNA or siRNA by reverse transfection. For more high-density cell microarrays, we used micro-electro-mechanical systems (MEMS) to fabricate a microwell array chip comprised of a glass substrate covered with a photoresist film patterned to form multiple microwells and tested it in two reverse transfection experiments, an exogenous gene expression study and an endogenous gene knockdown study. It was used effectively in both. Then, using the same MEMS technology, we fabricated a complementary microcolumn array to be used as a drug carrier device to topically apply drugs to cells cultured in the microwell array. We tested the effectiveness of microwell-microcolumn on-chip cell-based assay by using it in experiments to identify epidermal growth factor receptor (EGFR) activity inhibitors, for which it was found to provide effective high throughput and high content functional screening. In conclusion, these new methods of cell-based screening proved to be a simple and efficient method of characterizing gene function and discovering drug leads. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17513 |
全文授權: | 未授權 |
顯示於系所單位: | 醫學工程學研究所 |
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