臺灣博碩士論文加值系統

基因編輯技術在近年來被廣泛應用在免疫疾病治療以及基因治療的研究上，自從2012年美國加州大學柏克萊分校教授道納(Jennifer Doudna)和法國生物學家夏彭提耶(Emmanuelle Charpentier)在Science期刊發表他們發明的CRISPR/ Cas9基因編輯系統，在很短的時間內就讓許多實驗室爭相開始在不同研究中使用。疾病治療由gene knockdown晉升為gene knockout，而在癌症疾病治療過程中已知的臨床藥物SMCL-4常搭配癌症化學治療使用。
SMCL-4藥物為已上市之核可非癌症治療藥物，但有研究發現其相關機制會抑制癌細胞，藉由老藥新用來探討引導癌細胞走向細胞凋亡(apoptosis)的分子機制。
爰此，本實驗利用CRISPR技術，將含有sgRNA的 LentiArray Human Kinase CRISPR Library以Lentivirus packing system送入AGS-Cas9細胞中，CRISPR-Cas9有效且精準地knockout特定的標的基因，再將SMCL-4藥物作用於AGS-Cas9細胞，在kinase library中篩選出陽性反應的基因，其所篩選出來的基因可能與AGS細胞凋亡有關，再以西方點墨法驗證篩選出來的基因是否對於癌細胞凋亡有正相關。

Gene editing technology has been widely used in immunological disease treatment and gene therapy research in recent years. In 2012, Jennifer Doudna and Emmanuelle Charpentier were the first to propose that CRISPR/Cas9 (enzymes from bacteria that control microbial immunity) could be used for programmable editing of genomes, which is now considered one of the most significant discoveries in the history of biology. In a short period of time, many laboratories began to use it in different researches. The disease therapy was from gene knockdown to gene knockout. The SMCL-4, which is a known-drug and used in the cancer therapy. It is often used together with cancer drugs
The exact mechanism of action of SMCL-4 drug in cancer cell is unknown. They are believed to increase the extracellular level by limiting its reabsorption into cell, increasing the level in the available to bind to the receptor.
Therefore, this experiment uses CRISPR technology to deliver LentiArray Human Kinase CRISPR Library containing sgRNA into AGS-Cas9 cells in Lentivirus packing system. CRISPR-Cas9 efficiently and accurately knockout specific target genes in AGS-Cas9 cells, and then use SMCL-4 drugs to treatment them. In the AGS-Cas9 cells, the positive reaction genes were screened in the kinase library. The selected genes may be related to the apoptosis of AGS cells, and the western blotting method was used to verify whether the selected genes were positively correlated with cancer cell apoptosis.

第壹章 緒論 - 1 -
一、CRISPR/Cas基因編輯技術簡介 - 1 -
二、CRISPR/Cas基因編輯機制作用 - 2 -
三、CRISPR/Cas基因編輯技術疾病治療上的應用 - 4 -
四、CRSIPR與癌症 - 5 -
五、胃癌 - 6 -
六、SMCL-4藥物 - 8 -
七、細胞死亡 - 8 -
(一)、細胞凋亡之外在途徑： - 8 -
(二)、細胞凋亡之內在途徑： - 9 -
(三)、Caspase級聯反應和效應caspase： - 9 -
八、研究動機與目的 - 10 -
九、實驗流程 - 11 -
第貳章 材料與方法 - 12 -
一、材料與儀器 - 12 -
(一)、實驗材料： - 12 -
(二)、實驗儀器 - 13 -
二、實驗方法 - 13 -
(一)、LentiArray Human Kinase CRISPR Library - 13 -
(二)、AGS細胞株與細胞培養 - 13 -
(三)、Blasticidin S HCl最佳濃度測試 - 14 -
(四)、細胞存活數量估計 - 14 -
(五)、建立AGS-Cas9穩定細胞株 - 15 -
(六)、SMCL-4藥物濃度測試 - 15 -
(七)、Western Blotting分析 - 15 -
(八)、流式細胞儀檢測 - 16 -
第參章 結果與討論 - 17 -
一、CRISPR篩選結果分析 - 17 -
(一) LentiArray CRISPR Library篩選結果分析： - 17 -
1.建立AGS_Cas9穩定細胞株 - 17 -
(1)測試Blasticidin抗生素篩選的最佳濃度 - 18 -
(2)篩選同時具有Cas9和抗Blasticidin基因的AGS細胞株 - 21 -
2.以PCR擴增法確認Cas9基因存在於AGS-Cas9細胞中 - 22 -
3.SMCL-4藥物(編號754)對AGS-Cas9細胞株濃度測試： - 23 -
4. LentiArray Human Kinase CRISPR Library Screening - 25 -
(1) LentiArray Human Kinase CRISPR Library Screening流程 - 26 -
(2) 利用CRISPR knockout screening篩選出影響SMCL-4藥物作用的基因 - 27 -
(二) Western Blot蛋白轉漬分析驗證 - 28 -
(三) 流式細胞儀檢測 - 29 -
二、討論 - 30 -
第肆章 參考文獻 - 34 -

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