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  • 學位論文

影響圈套寡核苷酸於細胞抑制效果之因子探討

A study of the factors that affect the cell inhibition efficacy for decoy oligonucleotides

指導教授 : 陳林祈
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摘要


臨床上已開發使用的癌症治療藥物,以化療藥物使用最為普遍,但是也帶給人體很大的副作用,因此現行藥物開發都希望能夠達到藥物使用量減少、副作用低、製造成本低的目標。近年來,標靶藥物開發盛行,圈套寡核苷酸此具選擇性的藥物,藉由模仿目標基因的序列,干擾轉錄因子結合上目標基因,進而阻止下游訊息的調控,抑制癌細胞生長。而在臨床上為了增加藥物的作用效率以及藥物在組織中的存在時機,在施予藥物時會利用載體攜帶至癌細胞附近累積。另外,除了載體能增加藥物在組織或體循環中的存在時間,也會改變藥物本身的結構。而在本研究將結合載體以及改良藥物結構的方式,來提升抑制癌細胞的效果。首先,會進行圈套寡核苷酸與載體Lipofectamine® 2000的最適化探討。然後將圈套寡核苷酸進行修飾,探討其在結構上和未修飾之圈套寡核苷酸的差異,以及修飾前後圈套寡核苷酸對癌細胞抑制效果是否有差異。最後也會將修飾與未修飾之圈套寡核苷酸於正常肝、腎細胞的是否有副作用。在圈套寡核苷酸與載體的轉染效率最適化實驗時,得知圈套寡核苷酸於6小時的時候,圈套寡核苷酸於細胞底層的累積是最大量的,而利用流式細胞儀時也量測出轉染效率於6小時之時間點達到90%。承接著上述的轉染條件,將修飾與未修飾之圈套寡核苷酸轉染於PC3前列腺癌細胞,修飾之圈套寡核苷酸於最高濃度下其細胞存活率為20%左右,抑制癌細胞能力高於未修飾的組別。測得修飾與未修飾之圈套寡核苷酸之抑制癌細胞的存活率後,將兩者於肝、腎細胞上測試其對於正常細胞的毒殺能力,結果顯示,兩者於肝細胞AML12只有超過300nM的時候才會有毒殺能力,相較於PC3癌細胞,圈套寡核苷酸可以說是幾乎不影響AML12細胞。然而,在腎293細胞測試時,卻發現圈套寡核苷酸的濃度和細胞的凋亡情形成正比,經過和文獻的比對之後,推測293細胞可能帶有腫瘤之特性所導致。

並列摘要


Chemotherapy drugs are common in clinical use, but they also come with a number of unwanted side effects. Nowadays, the research and development of drugs hope to minimizing the dosage, the side effects and the product cost. Over the past two decades, the concept of targeted therapy has developed, and decoy oligonucleotides, one of the targeted therapy drugs mimics the target gene sequence and interferes the transcription factors to bind to the corresponding target genes, thus prevents the regulation of downstream signaling and inhibits the proliferation of cancer cells. In consideration of enhancing the drug efficiency and its retention time in tissue, this drug will be translocated and accumulate to cancer cell by carriers. Additionally, the drug retention time can also prolong by adjusting the structure and the chemical characteristics of drug. In this study, we will combine the carrier and structure modification to improve the drug potency. First, we will discuss the maximal optimization of decoy oligonucleotide with Lipofectamine® 2000 carrier. Next, we take post-modified decoy oligonucleotide to compare with non-modified group on structure difference and their effect on cancer cell. Finally, to ensure if non-modified or post-modified decoy oligonucleotide have the side effects on human body, drugs will test on normal liver and kidney cell. During the optimization experiment, the transfection efficiency reach the maximal level at 6 hours after transfection. And next, modified and non-modified group were transfected in PC3 cancr cell line, the cell viability under maximal concentration of modified group was 20%, and it has better inhibition ability than non-modified group. Then, the side effects of these two groups on normal cells were test. The result shows that their toxicity on AML12 will only appear when the concentration is higher than 300nM, but the result was difference on 293 cell. The toxicity of decoy oligonucleotides on 293 had dose-dependent effect.

參考文獻


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