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研究生:高越美
研究生(外文):Yue-Mei Gao
論文名稱:非病毒基因載體於子宮頸癌治療之研究
論文名稱(外文):Study on the effect of non-viral gene carrier for cervical cancer therapy
指導教授:劉澤英
指導教授(外文):Tse-Ying Liu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物醫學工程學系
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:69
中文關鍵詞:子宮頸癌基因治療超音波治療
外文關鍵詞:cervical cancergene thrapyultrasound therapy
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子宮頸癌(Cervical cancer)為女性常見的癌症之一,目前的臨床治療方法主要有三種,分別是手術治療、輻射治療、化療。其中以輻射治療與化療為最常見的主要與輔助治療方法,手術治療則侷限於前期(ⅡA)患者。針對子宮頸癌治療的方法最大的困難在於病患術後的生活品質不佳。放射治療容易造成尚未停經的婦女的卵巢功能喪失、陰道的彈性下降或引發膀胱或尿道的損傷,甚至是剝奪女性的生育能力。因此在保護子宮的同時治療子宮頸癌以保留生育能力或是減少輻射治療帶來的傷害與副作用是必要的趨勢。
本研究目的在探討超聲波與奈米載體搭配功能性基因誘導的子宮頸癌細胞凋亡。功能性基因由適當的啟動子和功能性蛋白基因片段組成。首先用螢光試驗功能性基因在細胞的轉染效率,再利用細胞存活率初步驗證功能性基因、載體與超聲波治療的效果。期望這樣的治療方式可以達到保護正常組織的同時治療子宮頸癌的目的。
Advances in the treatment of cervical cancer over the last decade have predominantly involved surgery, radiotherapy and chemotherapy. Among them, concurrent chemoradiation therapy (CCRT) is the most common primary and adjuvant treatment, and surgery is limited to patients with early stage (ⅡA). Although radiation therapy has a good treatment effect, radiation will be accompanied by many side effects. For example, menopause, uterus and vaginal tissue fibrosis, sore skin and other organs damage. Therefore, reduce the damage to normal cells to retain fertility or to reduce the damage and side effects caused by radiation therapy become more important.
The purpose of this study was to investigate the synergistic effect of nanocarriers and ultrasound on the expression of the engineering genes proposed for treating cervical cancer. The engineering gene consists of a suitable promoter and a functional protein gene fragment. First, the transfection efficiency of the functional gene in the cell was tested by fluorescence, and the cell viability was used to confirm the therapeutic effect of the engineering gene, nanocarriers and the ultrasound. We expect that the proposed gene therapy can inhibit cervical cancer cell without damaging normal tissues.
致謝 I
縮寫表 III
摘要 V
Abstract VI
目錄 VII
圖目錄 X
表目錄XII
第一章:緒論 1
第二章:文獻回顧 3
2.1子宮頸癌 3
2.1.1子宮頸癌之現有治療方法 3
2.1.2子宮頸癌之新興治療方法 6
2.2基因治療系統 7
2.2.1 質體DNA表達機制 7
2.2.2 非病毒載體 9
2.2.3 多孔性奈米載體 10
2.2.4 逃脫內體與藥物釋放機制 12
2.3 超聲波治療 13
2.3.1 超聲波機制 13
2.3.2 聲致發光 14
2.3.3 低強度超聲波治療 15
2.4 選擇性啟動子 17
2.4.1 特殊啟動子 17
2.4.2 早期生長反應蛋白1 18
第三章:材料與方法 19
3.1實驗設計 19
3.2材料 20
3.2.1儀器 20
3.2.2 化學合成材料 21
3.2.3生物實驗材料 22
3.3材料合成方法 24
3.3.1 不同大小的多孔性二氧化矽奈米粒子(SNPs-NH2)合成 24
3.3.2 SNPs-NH2-pDNA(SNP-pDNA)合成 24
3.3.3 螢光載體SNPs-FITC合成 24
3.4材料性質測試 25
3.4.1 穿透式電子顯微鏡(TEM) 25
3.4.2 粒徑與表面電性測試(DLS & Zeta Potential)25
3.4.3 載體材料官能基測定(傅立葉轉換紅外線光譜儀, FTIR) 25
3.4.4 介面活性劑CTAB定量(高效液相層析, HPLC)25
3.4.5 質體DNA濃度檢測(超微量分光光度計, Nano Drop Spectrophotometer)26
3.4.6 DNA載體型態觀察 (原子力顯微鏡, AFM)26
3.4.7 超聲波實驗裝置及參數 26
3.4.8 胞外活性氧檢測(Intercellular ROS assay, Elisa Reader) 27
3.5分子生物實驗 27
3.5.1 細菌培養/細菌放大DNA 27
3.5.2 質體DNA萃取 27
3.5.3 DNA Retardation Assay(Gel electrophoresis)27
3.6體外細胞實驗 (in vitro)28
3.6.1 細胞培養(Cell culture)28
3.6.2 核酸植質體轉染試驗(Plasmid DNA Transfection)28
3.6.3 冷光蛋白測試(Luciferase Assay) 28
3.6.4 西方墨點法(Western blot)29
3.6.5 細胞存活率測試(Presto Blue Cell Viability Assay) 29
3.6.6 胞內活性氧測試(Intracellular ROS Assay)29
3.6.7 細胞吞噬(Cell uptake) 30
3.7動物實驗(in vivo)30
3.7.1 載體毒性試驗(Safety evaluation test)30
第四章:結果與討論 31
4.1 材料特性分析 31
4.1.1 載體外觀及粒徑分析 31
4.1.2 FTIR鑑定 33
4.1.3 Zeta potential鑑定 34
4.1.4 DNA載體表面觀測 35
4.1.5 DNA Retardation assay 37
4.2 hEGR1 promoter細胞選擇性開啟效果驗證 38
4.3 hEGR1-caspase3重組基因表達差異驗證 40
4.4 超聲波加強hEGR1-caspase3重組基因表達差異驗證 42
4.5 SNPs載體ROS增效 44
4.6 SNPs載體之細胞毒性試驗 46
4.7 SNPs載體細胞吞噬 48
4.8 SNPs偕同超聲波誘導hEGR1啟動子開啟 49
4.9 SNPs體內毒性的組織病理學評估 50
第五章:結果與討論 51
第六章:參考資料 53

圖目錄
Figure 1 1 Global cancer statistics 2018. [1] 1
Figure 2 1 Staging of cervical cancer. [2] 3
Figure 2 2 The CTLA-4 and PD-1/PD-L1 pathways in cervical cancer. [5] 6
Figure 2 3 Transcription and translation process. [6] 7
Figure 2 4 Problems of intracellular nucleic acid delivery. [6] 8
Figure 2 5 Gene therapeutics and delivery strategies for gene therapy. [8] 9
Figure 2 6 Enhanced permeability and retention effect. [12] 10
Figure 2 7 Scheme for the main pathways of nanoparticle endocytosis. [14] 10
Figure 2 8 Multifunctional silica nanoparticle. [21] 11
Figure 2 9 Endosomal escape mechanisms. [25] 12
Figure 2 10 Possible mechanisms of SDT. [26] 13
Figure 2 11 Low intensity ultrasound in cancer treatment. [30] 15
Figure 2 12 Schematic of low‐intensity ultrasound‐induced molecular signaling cascades on activation of the apoptotic machinery. [33] 16
Figure 2 13 The principle of promoter-operating targeted expression in cancer gene therapy. [38] 17
Figure 2 14 Overview of signaling partners involved in oxidative stress mediated Egr-1 signaling. [40]. 18
Figure 3 1 Schematic of Ultrasonic probe in vivo test. 26
Figure 4 1 TEM images and particle-size distribution of Silica nanoparticles. 32
Figure 4 2 Material properties analysis. 33
Figure 4 3 Zeta potentials of the SNPs before and after the extraction and surface modification. 34
Figure 4 4 AFM images of phEGR1-case3, SNPs-NH2, SNPs-phEGR1-case3. 36
Figure 4 5 The effects of SNPs-NH2 on the condensation of phEGR1-case3. 37
Figure 4 6 Activity of hEGR1 promoter in Hela cells and L929 cells. 39
Figure 4 7 Schematic diagram of plasmid constructs of phEGR1-case3. 40
Figure 4 8 The protein expression of Caspase3 in Hela cells and L929 cells transfected with Effectene / hEGR1-Caspase3. 41
Figure 4 9 The protein expression of caspase9 and active caspase3 in Hela and L929 cells transfected with Effectene/hEGR1-Caspase3 and ultrasound. 43
Figure 4 10 Extracellular and intracellular ROS assay production of reactive oxygen species. 45
Figure 4 11 Cytotoxicity of SNPs. 47
Figure 4 12 SNPs-FITC cell uptake images of HeLa cells. 48
Figure 4 13 phEGR1-Lucia actived by ultrasound and SNPs in cervical cancer cell. 49
Figure 4 14 Safety evaluation of Silica nanoparticles in vivo. 50
Figure 5 1 TEM image of degradation of SNPs in MEM. 52

表目錄
Table 2 1 FGIO staging of cervical cancer. [3] 4
Table 2 2. Different stage of treatment for patients with cervical cancer. 5
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