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研究生：	陳睿妤 Chen, Jui-Yu
論文名稱：	篩選標靶FXYD2的適體應用在卵巢亮細胞癌的偵測 The Selection of FXYD2 Targeting Aptamers for Detection of Ovarian Clear Cell Carcinoma
指導教授：	陳玉玲 Chen, Yuh-Ling
學位類別：	碩士 Master
系所名稱：	醫學院 - 口腔醫學研究所 Institute of Oral Medicine
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	52
中文關鍵詞：	卵巢亮細胞癌、FXYD2 、適體
外文關鍵詞：	OCCCs, FXYD2, Aptamer
相關次數：	點閱：83 下載：2
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卵巢癌在婦科中是常見的惡性腫瘤之一，也是女性癌症死亡的主要原因之一。上皮卵巢癌有四種亞型，在卵巢癌中佔了約90%的發生率。卵巢亮細胞癌在台灣及日本在上皮卵巢癌中大約佔了25%的發生率。罹患卵巢亮細胞癌的病人通常有較高的復發率並且對化療藥物有較高的抗性。因此對卵巢亮細胞癌的早期診斷及開發新的藥物是急迫的。在我們之前的研究顯示，FXYD2是鈉鉀幫浦的亞基，並且會參與調控鈉鉀幫浦的活性，在卵巢亮細胞癌有大量表現的情形。透過強心苷Digoxin阻斷FXYD2可以有效的抑制卵巢量細胞的生長。然而，強心苷的可利用濃度間距太小，容易有生物毒性，造成許多副作用甚至是死亡，因此不太適用於臨床。適體是一可被化學合成的寡核苷酸，透過形成三級結構，對目標物的分子有專一性的辨識，可被應用於偵測癌細胞或是當做抗癌的治療方式。在本研究中，我們透過IP- SELEX篩選出可以靶向FXYD2的DNA適體，APF2，並且與控制組的細胞相比，APF2比較傾向抓在大量表現FXYD2的細胞上。利用即時定量聚合酶連鎖反應 (Real-time PCR) 偵測，發現候選的APF2對於大量表現FXYD2的細胞都有較高的親和力及專一性。接下來我們透過流式細胞儀觀察，發現APF2-38傾向辨認表現FXYD2的細胞，像是卵巢癌亮細胞。接著，在細胞生長實驗中，APF2-38 並沒有表現出毒殺卵巢癌亮細胞的能力，並且也沒有抑制卵巢癌亮細胞中FXYD2 的表現。另外，在捕獲試驗中，APF2-38 表現出對於卵巢癌亮細胞有好的捕獲能力。這些結果顯示 APF2-38 可以專一性地辨認並抓取FXYD2表現的細胞。這暗示著APF2-38 具有被開發為卵巢亮細胞癌檢測器的潛力。

Ovarian cancer is one of the most common gynecological malignancies and one of the leading causes of cancer mortality in women. Epithelia ovarian cancer (EOC) with four subtypes conclude 90% ovarian cancer and ovarian clear cell carcinoma (OCCC) accounts for approximately 25% of all EOC diagnoses in Taiwan and Japan. Patients with OCCC have a high recurrence rate and a higher resistance to platinum-based chemotherapy. Therefore, the early detection and developing new targeting drugs of OCCC are urgent. Our previous studies have shown that FXYD2, a γ subunit of Na,K-ATPase and it modulates the activity of Na,K-ATPase, highly expresses in OCCC. Blocking FXYD2 by cardiac glycosides such as Digoxin effectively inhibits growth of OCCCs. However, limit available concentration of Digoxin causes toxicity easily to patients, such as some side effects and even dead. Aptamers are oligonucleotides that can form three-dimensional structure to recognize specific molecular of their cognate targets and can be applied in cancer cells detection and anticancer therapies. In this study, we used immunoprecipitation and systematic evolution of ligands by exponential enrichment (IP-SELEX) to screen DNA aptamers targeted FXYD2. We identified APF2 that preferentially bound FXYD2-overexpressed cells compared with control cells. Quantitative real time polymerase chain reaction (Real-time PCR) detection showed APF2 candidates with high affinity and specificity for FXYD2-overexpressed cells. Next, we observed that APF2-38 preferentially bind to FXYD2-expressed cells, such as OCCC cells via flow cytometric assay. Following, in the cell growth assay, APF2-38 didn’t exhibit cytotoxic effect in OCCC cells. Either, it couldn’t suppress FXYD2 level in OCCC cells. Additionally, APF2-38 performed good capacity to catch OCCC cells in the capture assay. These results showed that APF2-38 could recognize and capture FXYD2-expressed cells specifically. It implied APF2-38 possessed the potentiality to be developed as OCCC detector.

摘要	I
Abstract	II
Acknowledgement	IV
Contents	VI
Introduction	1
Ovarian carcinoma	1
Ovarian clear cell carcinoma	2
Na/K-ATPase	3
FXYD2	3
Aptamer	4
Application of aptamer	5
Rationale and Specific Aims	7
Material and Methods	8
Cell Lines	8
Single-stranded DNA library selection	8
Single-stranded DNA (ssDNA) Library and Primer	8
Plasmid and FXYD2-flag Overexpression	8
IP-SELEX	9
Polymerase Chain Reaction (PCR)	10
Aptamers Purification	10
Western Blot	10
RNA Isolation	11
Reverse transcription (RT)-PCR	11
Quantitative real-time PCR (qPCR)	11
Buffer preparation	12
IP Lysis Buffer (pH7.5)	12
SELEX Buffer (pH7.5)	12
20X SSC Buffer (pH7.0)	12
Aptamer Properties Assays	12
Cell-based Affinity Assay	12
Flow Cytometric Analysis	12
Aptamer Functions Assays	13
Pull Down Assay	13
Cytotoxic Assay	13
Proliferation Assay	13
Immunofluorescence Staining	14
Capture Assay	14
Results	15
1.	FXYD2-specific aptamers were identified by IP-SELEX strategies.	15
2.	The selected aptamers had high affinity toward FXYD2 proteins.	15
3.	FXYD2-specific aptamers had no cytotoxicity for OVCA cells.	16
4.	APF2-38 preferentially targeted FXYD2-overexpressed cells.	16
5.	APF2-38 preferentially bound OCCC cells compared to non-OCCC cells.	17
6.	APF2-38 increased the proliferation but not influenced the expression levels of FXYD2 in OCCC cell lines.	18
7.	The capture ability of FXYD2-specific aptamers toward FXYD2.	18
8.	APF2-38 effectively captured FXYD2- expressed cells.	19
9.	The localization of APF2-38 binding to OCCC cells was examined by a fluorescent assay.	19
Discussions	20
Conclusions	24
Reference	25
Tables	29
Table 1. The procedure of aptamer refolding.	29
Table 2. The components for IP-SELEX.	29
Table 3. The processes of IP-SELEX.	30
Table 4. The procedures of reverse-transcription PCR.	31
Table 5. The progress of qPCR with TaqMan System.	32
Table 6. The progress of qPCR with SYBR Green System for cell-based affinity test.	32
Figures	33
Figure 1. The procedure of IP-SELEX.	33
Figure 2. The results of IP-SELEX.	34
Figure 3. The secondary structures of FXYD2-specific aptamers.	36
Figure 4. The FXYD2-specific aptamers had high affinity for FXYD2.	37
Figure 5. FXYD2-specific aptamers didn’t affect viability of ovarian cancer cells.	39
Figure 6. The FXYD2-specific aptamers preferentially bound to FXYD2 overexpressed cells.	42
Figure 7. APF2-38 preferentially bound to ovarian clear cancer cells.	43
Figure 8. APF2-38 promoted the proliferation in OCCC cells.	46
Figure 9. APF2-38 didn’t affect the expression level of FXYD2 in ovarian cancer cells.	47
Figure 10. FXYD2-specific aptamers could pull down FXYD2-flag proteins.	48
Figure 11. APF2-38 could identify FXYD2 in TOV21G by immunofluorescence assay.	50
Figure12. APF2-38 captured OCCC cells specifically compared with SC cells.	52


                                    

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