- 學位論文
單細胞分析阿黴素進入癌細胞研究 : 分佈及定量研究
Single Cell Analysis of Doxorubicin into Cancer Cell : Distribution , Kinetics , and Quantification
摘要
阿黴素(Doxorubicin)是一種廣範使用的抗癌藥物，其主要的作用機制是會和細胞的去氧核糖核酸(DNA)和拓樸異構酶(topoisomerase)結合並引發程序化細胞死亡來殺死癌細胞。一般評估藥物對細胞的作用主要是藉由測量IC50來得知，所謂的IC50就是指抑制細胞生長使其只有最大生長速率的一半所需要的藥物濃度。舉個例子，在我們的實驗中我們用了一種叫alamarBlue的染劑，當細胞功能正常時會將其轉成會放螢光的物質，若細胞功能受損則轉換效率變差，因此當我們測量到在某一藥物濃度處理過的細胞其alamarBlue螢光強度只有未用藥物處理細胞其alamarBlue螢光強度的一半時，這時的藥物濃度就是IC50 . 這種方法雖然能很快速的評估多少濃度的藥物對細胞會有影響，但並沒有告訴我們藥物的作用細節，例如 : 藥物在細胞內的分佈，實際進入細胞的藥物數目，個別細胞對藥物反應的差別性…等等，因此在這邊我們利用了阿黴素有螢光的特性並使用共軛焦雷射掃瞄顯微鏡(Confocal Laser Scanning Microscope)來幫助我們研究這些問題。用共軛焦雷射掃瞄顯微鏡的好處是它具有光學切片(optical section)的效果，因此我們可以看到每一層細胞的影像並且能排除非焦平面來的雜光干擾，這是傳統的螢光顯微鏡做不到的事。另一方面我們可以做單顆細胞的觀察，比較每一顆細胞吸收藥物的速率、吸收量的差異性，這些是一次做一群細胞實驗中無法得知的訊息。 最初我們預測阿黴素進入細胞的量應該會是隨時間增加，之後到達飽和，但實際觀察後我們觀察到並非每顆細胞皆如此 : 確實有些細胞吸收阿黴素達到飽和後然後死亡，但在許多細胞觀察到的現象是阿黴素進入的曲線到達飽和後一陣子又開始上升 ; 另外則有些細胞根本沒看到曲線到達飽和而是一直向上升，在這裡我們觀察到細胞間吸收藥物的差異性，也發現它們有別於群體行為的表現。另外在細胞吸收阿黴素達到飽和量方面我們發現細胞細胞間也有差異，細胞間可能差異蠻大，大部分的細胞在細胞核內累積阿黴素的濃度大約是其在培養液中濃度的100倍，但部分細胞會累積到更高的濃度，有些甚至可累積將近1000倍(見4uM Dox處理細胞的數據)。 在細胞死亡的觀察上我們利用了阿黴素本身會累積在核的特性直接將其當作是核染劑，用來判斷在特定時刻細胞核是否發生萎縮或是DNA產生碎裂。這樣做的好處是我們可以不用外加染劑，既可避免細胞毒性又可做及時的細胞死亡觀察。比較了用4uM及1uM阿黴素下處理的細胞我們發現用4uM阿黴素處理的細胞其吸收藥物的平均量比在1uM阿黴素處理的細胞平均量高，且死亡率也比較高，因此可以得知藥物在細胞的累積量確實和細胞死亡速率有關。
並列摘要
Doxorubicin is a widely used anti-cancer drug that kills cancer cells by binding to DNA and topoisomerase to induce programmed cell death. Scientists usually evaluate the effectiveness of drugs to cells through the IC50 test which is denoted by the half-maxima inhibitory concentration. For example, in this thesis we used the alamarBlue dye to test our system. In healthy cells alamarBlue can be metabolized to its reduced form to emit fluorescence. However, in cells where cell growth is inhibited, an oxidized environment is maintained in which alamarBlue will remain non-fluorescent. Therefore when the fluorescence intensity of drug-treated cells is only half of the untreated cells, the concentration of the drug is the IC50. Although we can find out the effective concentration of drugs to the cells through the IC50 test, it does not tell us the details of drugs such as the distribution of drugs in the cell, the number of drug molecules in the cell, the different responses between cells and et cetera. Therefore, to study these problems we used confocal laser scanning microscope by monitoring the fluorescence of Dox. The advantage of confocal laser scanning microscopes lies in their optical sectioning effect, which can exclude out-of-focus light and provides better image quality than conventional fluorescent microscopes. On the other hand, we can do single cell observation and compare the difference of rate and quantity of drug uptake between cells that cannot be obtained from the population experiment. At first we expected that the number of Dox molecules in the cell should increase with time until it reaches saturation. However, the results didn’t meet the expectation . In our experiment, we observed that in some cells the amount of Dox being absorbed into the cells rises at first and then gradually slows down as it reaches saturation. However, in some cases the amount of Dox continues to rise shortly after saturation while in others the amount of Dox never reached saturation but instead continues to increase. From this we observed the difference in the quantity and behavior of drug uptake between cells. Most of the concentration of Dox in the nuclei is higher than the concentration of Dox in the cell medium by about 100-fold; but some can even reach to 1000-fold or higher . We also used Dox itself as a nuclear marker to observe whether cells undergo nuclear condensation or DNA fragmentation. The advantage of using Dox itself as a nuclear marker is that we don’t have to add another nuclear marker which may cause cytotoxicity. After comparing cells subjected to the 4uM and 1uM Dox treatment we found that cells under the 4uM Dox treatment have higher death rates and higher Dox concentration in the nuclei than the cells under the 1 uM treatment. Therefore, from this we know that the higher the number of Dox molecules there are in the cells, the higher their death rates are and vice versa