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系統識別號 U0026-0812200910412985
論文名稱(中文) 利用長時間縮時錄影系統對活體外傷痕癒合的分析
論文名稱(英文) In Vitro wound healing assay revisited: aided by a long-term, time-lapse recording system
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 91
學期 2
出版年 92
研究生(中文) 江旆萱
研究生(英文) Pei-Shuan Chiang
電子信箱 hsing0325@ms52.url.com.tw
學號 t1690402
學位類別 碩士
語文別 英文
論文頁數 50頁
口試委員 指導教授-蔡少正
召集委員-劉校生
口試委員-江美治
指導教授-何中良
中文關鍵字 細胞移動  長時間縮時錄影系統  傷痕癒合 
英文關鍵字 wound healing  time lapse recording  functional screening  cell migration 
學科別分類
中文摘要 活體外傷痕癒合分析是一種常用來研究細胞移動的分析法。將單層細胞劃一道傷痕後,在傷痕邊緣的細胞會分裂、移動,並將整個空缺的區域填滿。普遍認為計算整個區域面積的改變,或是傷痕寬度的改變即代表細胞移動的能力。但是傷痕寬度的改變是否可完全代表細胞的移動是令人懷疑的。利用測量傷痕寬度改變的方法,發現高密度的T24單層細胞其傷痕寬度改變的速率顯著的較低密度的為高。以傳統的方法解釋,會認為細胞在高密度時移動較快。為了說明這項觀察,利用我們實驗室中的一套長時間縮時錄影系統對活體外傷痕癒合的現象做更進一步的研究。這個錄影系統可使研究者對每一單顆細胞在傷痕癒合的過程中做最詳盡的觀察與分析。研究結果發現只有在傷痕旁的前幾排細胞參與整個傷痕癒合的過程。細胞在高密度時都具有較好的順著傷痕癒合方向移動的方向性,但是平均路徑總長在密度高或密度低的實驗中並沒有顯著的差別。此結果可解釋其傷痕癒合速率較快的現象。因此,計算細胞移動的路徑總長可能是計算細胞移動速率較好的方法。另外,也觀察了mitomycin C以及甘草次酸(β-glycyrrhetinic acid)對於傷痕癒合的過程中寬度的改變以及細胞移動速率的影響。先前的學者為了減少細胞分裂對傷痕癒合的影響,建議在做活體外傷痕癒合分析觀察細胞的移動能力是否有受影響時,應控制細胞的分裂,或是盡量簡短觀察的時間。在利用mitomycin C抑制細胞分裂後發現,此藥物在傷痕癒合的早期即會抑制細胞移動。另外,在加入甘草次酸抑制細胞間隙連結(gap junction)以後,結果發現順著傷痕癒合方向移動的細胞有明顯減少的現象。
在此篇論文的第二部份,我們將長時間縮時錄影系統應用於建立一套利用過渡性轉染(transient transfection)將未知基因送入活體細胞觀察基因對細胞功能影響的功能分析平台。在此初步建立的步驟中,將EMP2基因構築(construct)至可共同表現綠色螢光蛋白(green fluorescent protein, GFP)的質體中,送入NIH3T3細胞表現,可分析帶有EMP2基因的細胞其細胞形態、生長、分裂、或移動等等現象或功能是否有受到影響。此步驟可用於快速的分析基因的功能。
英文摘要 Wound healing is one of the most frequently used methods to study cell motility. A monolayer of cells is scratch-wounded and cells alongside the wound would proliferate and migrate to fill up the denuded area. The area change or the wound closure distance is considered to be a measurement of cell motility. However, the rate of wound closure may not be a true measurement of cell motility. By measuring the distance of wound closure, we found that high-density monolayers of T24 cells (a bladder cancer cell line) showed faster wound-closure rates than low-density ones, which, by conventional interpretation, implied that T24 cells at higher cell densities would have greater cell motilities. To clarify such an observation, we investigated the details of wound healing with our long-term, time-lapse recording system, which was able to record and depict the migration path of a single cell through the entire healing process. Only the first few rows of cells behind the wounded edge contributed to wound closure. These cells showed better moving directionality (toward the direction of wound closure) at higher cell-densities, explaining the greater wound-closure rate, whereas the average lengths of the migration paths are the same in high- and low-density experiments. The lengths of migration paths over a period of time are the better measurement of cell motility, whereas the wound-closure rate represents the combinational effect of cell motility and directionality. The effects of mitomycin C and β-Glycyrrhetinic acid on wound-closure rate and cell motility in wound healing were further investigated. It had been suggested that in order to minimize the effect of cell proliferation on wound healing, the proliferation activity should be inhibited or the assay time should be kept as short as possible. However, we found that inhibition of cell proliferation by mitomycin C treatment may affect cell motility in a short period of time. Gap junctional communication was thought to play a role in wound healing. By inhibition of gap junction with GCA, we found that the percentage of forward moving cells as well as the migration rate significantly decreased.
In the second part of the study, we applied the long-term time-lapse recording system to the functional analyses of genes through transient transfections. A preliminary procedure was established. Using a construct to co-express green fluorescent protein and EMP2 in NIH3T3 cells, we established a procedure to evaluate potential effects of EMP2 on cell morphology, viability, apoptosis, membrane ruffling and cell motility. The procedure could be utilized as a rapid screening test for gene functions.
論文目次 Abstract………………………………………………………………I
中文摘要..…………………………………………………………III
誌謝……………………………………………………………………V
Content………………………………………………………………VI
Figure content……………………………………………………VII
Introduction…………………………………………………………1
Materials and methods ……………………………………………6
Results………………………………………………………………13
Discussion …………………………………………………………21
References …………………………………………………………27
Figure legends ……………………………………………………31
Figures………………………………………………………………36
作者簡歷 ……………………………………………………………50
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