臺灣博碩士論文加值系統

對於癌症之治療，反譯寡核苷酸是十分引人注目的治療物質，是由短鏈的核苷酸組成，並設計成與特定的靶標mRNA序列互補，當反譯寡核苷酸特異地與與之互補的mRNA結合後，會干擾基因的表現，導至蛋白質的合成受抑制。
細胞凋亡是一種自然的生理機制，來選擇性的淘汰細胞，在細胞凋亡的過程中是藉由許多蛋白質之間的平衡所調控的，例如：Bcl-2。Bcl-2是一種對於抑制細胞凋亡很重要的蛋白質，許多癌細胞的異常增生是因為Bcl-2的過度表現。因此，為了降低癌細胞的Bcl-2的表現，給予標的細胞能夠抑制Bcl-2反譯寡核苷酸 (G3139) 為可能的辦法。
然而，反譯寡核苷酸在體內易被酵素、核酸酶破壞，且為帶負電的高分子量物質，使其不易穿透細胞膜。因此，本研究為改善反譯寡核苷酸的傳遞，而使用帶正電之脂質的系統，將G3139包覆至脂質載體中。此微脂粒由DC-Chol/egg-PC/PEG-DSPE以22.5:76:1.5 mol%組成，其粒徑大小平均為190.94±11.13 nm，表面電荷為4.07±3.18mV其包覆ODN的能力高達70%以上，粒子之膠體安定性可達十一週。為了增加標靶運輸的效果，可將運鐵蛋白當作配位體經後嵌入法修飾微脂粒。
在細胞攝入的實驗中，將帶有螢光的ODN/FITC包覆製不同處方之微脂粒，並給予細胞，利用流式細胞儀來定量攝入細胞的ODN含量。實驗結果可以發現進入細胞的ODN含量取決於給予細胞ODN的濃度；在競爭性實驗中，先給予有運鐵蛋白受體表現的K562細胞100μM的holo-transferrin，則有運鐵蛋白修飾的微脂粒的攝入受到了抑制。同時，利用西方墨點法來測定Bcl-2的蛋白質表現量，細胞之Bcl-2蛋白表現量確實因G3139的作用而受到抑制。

Antisense oligonucleotides (ODN) are attractively therapeutic agents for cancer therapy. They are short fragments of nucleic acids and designed to target the complementary mRNA sequence to specifically interfere with gene expression and inhibit encoded protein production.
Apoptosis is a physiological mechanism for selective elimination of cells, and regulated by the balance between many proteins during this process, such as Bcl-2, an essential inhibitory protein to apoptosis. Many cancer cells proliferate abnormally due to overexpression of Bcl-2. Therefore, to down-regulate the Bcl-2 expression, it is a potential strategy that delivered a phosphorothioate ODN (G3139) to Bcl-2 in targeting cells.
However, ODN are easily destroyed by enzymes and nucleases in vivo and negtive charged molecules with high molecular weight make them the ability across cell membranes is poor. In this study, to improve antisense delivery, we developed a cationic lipid system, encapsulating ODN in lipid vesicles. The liposomes made up of DC-Chol/egg-PC/ PEG-DSPE (22.5:76:1.5 mol%) containing G3139 with a mean diameter of 190.94±11.13nm, a zata potential of 4.07±3.18mV and showed colloidal stability can maintain up to 11 weeks. Encapsulation efficiency of ODN in the liposomes was up to 70%. In order to enhance the targeting effect, transferrin (Tf)-PEG-DSPE was inserted into the lipid bilayer by post-insertion.
Cell uptake of different formulations of liposomal ODN/FITC were observed by flow cytometry. The amount of ODN/FITC entering cells corresponds with the initial amount of ODN/FITC. In competition study, uptake of Tf-conjugated liposomes could be inhibited, when pre-treating free holo-transferrin 100μM to K562 cells, that were TfR positive . In the meantime, Bcl-2 protein level was evaluated by western blot to confirm G3139 work in selective cancer cells. The Bcl-2 protein level was down-regulation by G3139.

第一章 緒論 1
第一節 文獻探討 1
一、 癌症的基因治療 1
二、 反譯寡核苷酸 3
三、 基因傳遞系統 4
四、 標靶運輸系統 7
五、 細胞凋亡 9
六、 Bcl-2蛋白家族 11
第二節 研究動機 12
第二章 實驗材料與方法 13
第一節 實驗細胞及材料 13
一、 實驗細胞 13
二、 實驗藥品 14
三、 實驗儀器 17
第二節 實驗方法 18
一、 細胞培養 18
二、 細胞蛋白質之萃取及定量 20
三、 蛋白質SDS膠體電泳 22
四、 西方墨點法 23
五、 製備微脂粒 24
六、 氫硫基之定量分析 26
七、 微脂粒之包覆能力試驗 27
八、 細胞攝入試驗 28
九、 基因轉染試驗 30
十、 統計分析方法 31
第三章 實驗結果 32
第一部分 微脂粒之物理化學性質 32
一、 製備微脂粒之相關數據 32
二、 DC-Chol對微脂粒之粒徑大小及表面電荷影響 36
三、 注射針頭直徑對微脂粒粒徑大小之影響…………. 39
四、 乙醇濃度對微脂粒包覆能力及粒徑大小之影響 40
五、 ODN與脂質比例對微脂粒包覆能力 44
及粒徑大小之影響 44
六、 微脂粒之膠體安定性試驗 47
第二部分 微脂粒的體外試驗 52
一、 細胞的特質 52
二、 細胞攝入微脂粒之效力 54
三、 螢光顯微鏡之觀測 58
四、 基因轉染試驗 63
第四章 討論 73
第五章 結論 78
第六章 參考文獻 79

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