臺灣博碩士論文加值系統

多形性膠質母細胞瘤(Glioblastoma multiforme, GBM）是種即使傳統化療還是難以治療且高復發率的惡性腦瘤，目前超音波標靶微氣泡擊破(Ultrasound-targeted microbubbles destruction, UTMD)應用於血腦障蔽已被證實，並可以遞送治療基因至腦部，此外UTMD也可以應用於遞送殺腫瘤基因於癌症治療。GBM中的腫瘤血管的表皮細胞富含許多VEGF-A，可以與VEGFR2做結合，這也廣泛應用在標靶治療中。在本研究中，我們合成乘載基因並結合anti-VEGFR2抗體的正電微氣(Cationic microbubbles with anti-VEGFR2 antibody, VCMBs)進行血腦障蔽開啟和腦瘤治療，並使用VCMBs增加微氣泡表面的基因乘載量和癌細胞表面的吸附效率。
我們使用冷光基因(pLUC, 6.5 kb)進行基因轉染模擬並超音波參數最佳化，每隻大鼠(Male Sprague-Dawley rats)在左腦被種入5x105 C6大鼠膠質瘤癌細胞，種腫瘤後第7天，每隻大鼠頸靜脈注射2x109乘載基因的VCMBs後進行超音波照射，根據不同時間使用非侵入式活體分子影像系統(IVIS)偵測，使用有標靶VCMBs的腦瘤組別冷光表達量([5.5±1.5] to [11.6±4.4] x 103 photons/sec/cm2/sr,)高於使用無標靶的CMBs組別冷光表達量([4.4±1.4] to [7.3±1.1] x 103 photons/sec/cm2/sr, p*<0.5)
接著，我們使用自殺基因(Herpes simplex virus type 1 thymidine kinase, pHsv-TK, 7.2 kb)結合Ganciclovir藥物(GCV)已經被證實為最有高度療效於腦瘤治療。細胞實驗中，在細胞超音波轉染pHsv-TK基因24小時後，細胞培養於不同濃度的GCV藥物，細胞轉染後第4天，隨著GCV藥物濃度增加(0, 0.1, 1 and 50 μg/ml)而細胞存活率降低(100.0±10.5%, 90.2±21.4%, 63.6±8.9% and 57.6±4.9%)，而無轉染pHsv-TK基因的細胞，無論GCV濃度多寡皆不會影響細胞存活率(p*<0.5)。動物實驗中，大鼠在種腫瘤後第6天使用VCMBs結合超音波轉染pHsv-TK基因，接著連續8天腹腔注射0.2 ml (100 mg/kg/day) GCV藥物，種腫瘤後第18天MRI偵測結果顯示，有使用超音波結合VCMBs轉染pHsv-TK基因的組別的腦瘤體積(3.8 mm3)明顯小於無治療的組別的腦瘤體積(19.75 ± 8.3 mm3)，而且有治療的組別生存天數較長。總而言之，本研究建立一個承載基因的VCMBs結合超音波的方法應用於血腦障蔽開啟，並成為一個非病毒、非侵入式和標靶基因遞送的工具於腦瘤治療。

Glioblastoma multiforme (GBM) is a malignant brain tumor with poor prognosis and high recurrence rate despite traditional chemotherapy. Ultrasound-targeted microbubbles destruction (UTMD) has been approved to achieve local blood-brain barrier disruption (BBBD), enhancing therapeutic agents into the brain. Besides, UTMD has been employed to deliver tumor-killing gene for cancer therapy. Tumor vessels in GBM are highly rich in VEGF-A, which could bind VEGFR2 on endothelial cells, is widely used in targeted therapy. In this study, we fabricated DNA-loaded cationic microbubbles with anti-VEGFR2 antibody (VCMBs) for transient BBBD and targeted therapy in brain tumors. We used VCMBs for improving gene delivery by loading DNA on MBs shell and actively attaching on cancer cell.
Expression of reporter gene, luciferase (pLUC, 6.5 kb), was used for monitoring gene transfer and optimization ultrasound parameters. Male Sprague-Dawley rats were injected 5x105 C6 glioma cells in left hemispheres of the brain in vivo. At 7 days post injection, 2x109 DNA-loaded VCMBs were injected via jugular vein, then ultrasound- mediated gene delivery was actuated by insonation of the brain tumor xenografts. Comparisons of treatment conditions across all time points revealed that the use of VCMBs in brain tumors resulted in significantly higher luciferase expression measured by IVIS ([5.5±1.5] to [11.6±4.4] x 103 photons/sec/cm2/sr,) relative to the use of CMBs in brain tumors ([4.4±1.4] to [7.3±1.1] x 103 photons/sec/cm2/sr, p*<0.5).
Herpes simplex virus type 1 thymidine kinase (pHsv-TK, 7.2 kb) in combination with ganciclovir (GCV) has been shown as one of the most promising suicide gene systems for brain tumors treatment. For in vitro studies, 24 hours post transfection under FUS, the pHsv-TK transfected C6 glioma cells were incubated in the presence of 0-10 μg/ml GCV in medium. At day 4, cell viability of pHsv-TK transfected C6 glioma cells were decrease (100.0±10.5%, 90.2±21.4%, 63.6±8.9% and 57.6±4.9%) when GCV concentrations increase (0, 0.1, 1 and 50 μg/ml), respectively, showing significant cell death compared with cell viability of C6 glioma cells without treatment at different GCV concentrations (p*<0.5). For in vivo studies, rats were transfected with pHsv-TK with VCMBs under FUS after 6 days of tumor growth. Each rat was intraperitoneally injected with 0.2 ml (100 mg/kg/day) GCV every 24 h lasting for 8 days. The tumor volume measured by MRI on Day 18 was significantly smaller in the rats treated with pHSV-TK/GCV system with VCMBs under FUS (3.8 mm3) than in the rats without treatment (19.75 ± 8.3 mm3). Additionally, rats with treatment were significantly prolonged survival time compared to the rats without treatment. Overall, this study aimed to develop the DNA-loaded VCMBs, using UTMD for achieving local BBBD, as a non-viral, noninvasive and targeted gene delivery approach in brain tumors.

Contents
1 Introduction 11
1.1 Overview of brain tumors 11
1.1.1 Introduction of brain tumors 11
1.1.2 The role of blood-brain barrier 12
1.1.3 Current status of brain tumors treatment 13
1.1.4 Gene therapy in brain tumors 14
1.1.5 Research of ultrasound-mediated gene delivery in brain tumors 17
1.2 Antiangiogenic-targeting MBs in gene therapy 18
1.2.1 Properties of DNA-loaded MBs 18
1.2.2 Properties of antiangiogenic-targeting MBs 19
1.3 Mechanisms of ultrasound-mediated gene delivery 20
1.3.1 Properties of MBs combined with FUS 20
1.3.2 FUS-induced blood-brain barrier opening 20
1.3.3 Ultrasound-mediated gene delivery in vitro 21
1.3.4 Ultrasound-mediated gene delivery in vivo 22
1.4 Overview of dissertation 23
2 Material and Methods 25
2.1 DNA-loaded VCMBs 25
2.1.1 Plasmid preparation 25
2.1.2 Preparation of DNA-loaded VCMBs 25
2.1.3 Characterization of DNA-loaded VCMBs 27
2.1.4 Measurement of avidin-biotin binding efficiency 27
2.1.5 Measurement of DNA loading efficiency 28
2.1.6 Nuclease protection assay 28
2.1.7 Microscope and cryo-TEM image 29
2.1.8 Measurement of C6 glioma cells targeting efficiency 30
2.2 Acoustic properties of DNA-loaded VCMBs 30
2.2.1 Stability Analysis 30
2.3 Ultrasound-mediated gene delivery in C6 glioma cells 31
2.3.1 Cell membrane permeability test 31
2.3.1.1 Experiment setup for cell membrane permeability test 31
2.3.1.2 Experimental design 32
2.3.1.3 Analysis of cell membrane permeability 32
2.3.2 pLUC transfection in vitro 33
2.3.2.1 Experiment setup for pLUC transfection in vitro 33
2.3.2.2 Experimental design 34
2.3.2.3 Analysis of pLUC expression and cell viability 34
2.3.3 pHsv-TK transfection in vitro 34
2.3.3.1 Experiment setup for pHsv-TK transfection in vitro 34
2.4 Ultrasound-mediated gene delivery in vivo 35
2.4.1 Monitoring of pLUC delivery in subcutaneous tumors 35
2.4.1.1 Experiment setup for pLUC delivery in subcutaneous tumors 35
2.4.2 pLUC delivery in brain tumors 36
2.4.2.1 Brain tumor model 36
2.4.2.2 Confirmation and quantification of FUS-BBB opening 37
2.4.2.3 Experiment setup for pLUC delivery in brain tumors 37
2.4.2.4 Analysis of pLUC expression 39
2.4.3 pHsv-TK delivery in brain tumors 39
2.4.3.1 Experiment setup for pHsv-TK delivery in brain tumors 39
2.4.3.2 Magnetic resonance imaging (MRI) 40
3. Results and Discussion 41
3.1 DNA-loaded VCMBs 41
3.1.1 Characterization of DNA-loaded VCMBs 41
3.1.2 Measurement of avidin-biotin binding efficiency 42
3.1.3 Measurement of DNA loading efficiency 43
3.1.4 Nuclease protection assay 44
3.1.5 Microscope and cryo-TEM Images of VCMBs 45
3.1.6 Measurement of C6 glioma cells targeting efficiency 46
3.2 Acoustic properties of DNA-loaded VCMBs 47
3.2.1 MBs stability analysis 47
3.2.2 Measurement of inertial cavitation dose and subharmonic intensity 48
3.3 Ultrasound-mediated gene delivery in C6 glioma cells 49
3.3.1 Cell membrane permeability test 49
3.3.2 Ultrasound-mediated gene delivery of pLUC in vitro 54
3.3.3 Ultrasound-mediated gene delivery of pHsv-TK in vitro 62
3.4 Ultrasound-mediated gene delivery of pLUC in subcutaneous tumors 63
3.4.1 Assessment of pLUC Expression 63
3.5 Ultrasound-mediated gene delivery of pLUC in brain tumors 65
3.5.1 In vivo FUS-induced BBB opening and EB release 65
3.5.2 Analysis of pLUC Expression 66
3.6 Ultrasound-mediated gene delivery of pHsv-TK in brain tumors 67
4. Discussion 69
4.1 Safety in ultrasound-mediated gene delivery 69
4.2 CMBs for increase gene loading efficiency 69
4.3 Antiangiogenic-targeting MBs applied in ultrasound-mediated gene delivery 70
4.4 Limitation of pHsv-TK/GCV system 71
4.5 Overall conclusion 72
5. Future Work 72
Reference 73

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