評估藥物對細胞遷移影響篩選方法之改進

Improvement of a screening assay for evaluation of the effects of drugs on cell migration.

王秋月(Chiou-Yueh Wang)

指導教授 ： 徐祖安

國立清華大學/工學院/化學工程學系/碩士(2006年)

摘要

血管新生與癌症之轉移和組織修復息息相關，而細胞的遷移亦與血管新生密不可分。本研究主要有兩個目的即在於 – 找出細胞遷移試驗的最佳化條件以及利用此方法來篩選出具有促進或抑制血管新生的藥物。有很多方法能拿來評估細胞遷移的能力；本實驗主要用兩種方法 – wound healing 試驗以及modified Boyden chamber試驗，因為這兩種方法操作簡單且快速。Wound healing 試驗是在長滿細胞的培養皿上刮出一條痕跡，藉由觀察細胞修復此刮痕的現象進而評估不同化合物對細胞遷移能力的影響。Boyden chamber原本是被設計拿來研究細胞受到化學趨向物(chemotaxis)的影響而移動的裝置。 此裝置有上下兩層chambers，其操作方法為：在下層chamber中加入細胞培養液以及待測的分子，而在上下兩層chambers之間夾一片半透膜，此半透膜的孔徑大小比細胞直徑來的小，所以在正常情況下，細胞不會穿透半透膜到下面的chamber裡，然後在上層chamber中加入含細胞的細胞培養液。最後收集並分析受到化學趨向物而遷移到下層chamber的細胞數，此參數可以作為評估待測的分子對細胞遷移影響的參數。除此之外，MTS assay在本實驗中被使用於測試藥物對細胞的毒性，進而拿來評估藥物之後發展的可行性。利用本實驗我們已經發現有促進和抑制細胞遷移潛力的藥物。這些具有促進血管新生的藥物可以應用在組織工程裡；而能抑制血管新生的藥物則可以利用在抑制癌細胞的生長或擴散。

關鍵字

細胞遷移 ； 臍帶靜脈內皮細胞 ； Boyden chamber試驗 ； wound healing試驗 ； MTS assay ； 篩選方法

並列摘要

Cell migration is associated with angiogenesis, which is the phenomenon of new blood vessel formation. Angiogenesis plays an important role in cancer invasion or metastasis as well as tissue engineering. There are two specific aims in this thesis. One is to optimize cell migration assay and the other one is to discover compounds with pro- or anti-angiogenesis activity. Several published methods have been utilized to study cell migration, and we employed two methods, i.e., wound healing assay and modified Boyden chamber assay in this study because they were simple and fast. Wound healing assay was performed by scraping the monolayer of the cells with a pipette tip, and then observing the recovery ability of the cells that were treated with various compounds. The Boyden chamber was originally designed to study chemotaxis. It consists of two chambers and a chemo attractant is placed in the lower chamber, while cells are seeded on the upper chamber. There is a porous membrane between these two chambers, and only cells that migrate actively can go to the lower chamber. The degree of chemotaxis response can be determined by microscopy by counting the transmigrated cells. In addition to cell migration ability, cytotoxicities of the cells were also estimated. In this study, we found some small molecule compounds showed pro- and anti-angiogenesis potentiality. Drugs with pro-angiogenesis activity have potential application in tissue engineering, while drugs with anti-angiogenesis ability may be utilized for cancer treatment.

並列關鍵字

cell migration ； HUVEC ； Boyden chamber assay ； wound healing assay ； MTS assay ； screening assay

參考文獻

1.Klemke, R. L. et al. CAS/Crk coupling serves as a "molecular switch" for induction of cell migration. J Cell Biol 140, 961-72 (1998).

2.Pardanaud, L., Yassine, F. & Dieterlen-Lievre, F. Relationship between vasculogenesis, angiogenesis and haemopoiesis during avian ontogeny. Development 105, 473-85 (1989).

3.Poole, T. J. & Coffin, J. D. Vasculogenesis and angiogenesis: two distinct morphogenetic mechanisms establish embryonic vascular pattern. J Exp Zool 251, 224-31 (1989).

4.Holmgren, L., Glaser, A., Pfeifer-Ohlsson, S. & Ohlsson, R. Angiogenesis during human extraembryonic development involves the spatiotemporal control of PDGF ligand and receptor gene expression. Development 113, 749-54 (1991).

6.Arnold, F. & West, D. C. Angiogenesis in wound healing. Pharmacol Ther 52, 407-22 (1991).