臺灣博碩士論文加值系統

目的：聚焦式超音波搭配微氣泡，可強化奈米抗癌藥物微脂體在大鼠腦腫瘤組織的累積量及療效。本研究欲探討在不同成長階段的大鼠腦腫瘤，其在有無搭配聚焦式超音波搭配微氣泡所造成的藥物在腫瘤組織累積量的差異、藥物抑制腫瘤生長的效果差異，以及在微觀下的特殊組織染色分析腫瘤組織的差異。
方法與材料：本實驗的超音波參數頻率為0.5 MHz，壓力為0.5 MPa，微氣泡劑量為100 ul/kg；奈米抗癌藥物微脂體劑量為6 mg/kg。使用神經膠質瘤細胞C6 glioma cells (106 cells) 植入右半腦以及相同體積的生理食鹽水植入左半腦。奈米抗癌藥物微脂體在腫瘤細胞植入後第8天、第11天或第14天經尾靜脈注射，分別代表早期、中期和晚期階段腫瘤；另外設計一組分別在第8天和第11天都施藥；除此之外，分為有無施打聚焦式超音波搭配微氣泡。一半的老鼠在奈米抗癌藥物微脂體注射入體內後２４小時犧牲做酵素免疫分析法定量腫瘤組織內藥物，另外一半的老鼠會在第16天犧牲評估腫瘤治療效果，組織樣本會做特殊免疫染色：H＆E、CD31、CD133、VEGF，以及螢光染色。我們會在第15天注射Evans blue (EB, 100 mg/kg)，老鼠總共為136隻。
結果：奈米抗癌藥物微脂體的萃取量隨著腫瘤的生長其藥物在組織中的萃取濃度越高，而且經過施打聚焦式超音波搭配微氣泡有顯著的差異，在第8天的強化成效較第11天以及第14天顯著。除此之外，治療方面，腫瘤生長體積在第16天的結果，發現奈米抗癌藥物微脂體結合聚焦式超音波搭配微氣泡比只有施打奈米抗癌藥物有顯著的抑制效果；單次治療組別，在第8天施打奈米抗癌藥物微脂體結合聚焦式超音波搭配微氣泡其腫瘤生長抑制效果較在第11天做治療顯著；相較於其他治療組別，兩次治療分別在第8天和第11天施打奈米抗癌藥物微脂體結合聚焦式超音波搭配微氣泡有最顯著的抑制腫瘤生長效果。在組織染色的部份，隨著腫瘤生長時間增加，細胞分佈越濃密，特殊組織染色分析結果也越明顯；比較治療後第16天的腦腫瘤組織，其細胞核變異性，細胞數，特殊蛋白表現較控制組第16天的腦腫瘤組織細胞核單一性，細胞數少量，特殊組織染色分析結果降低，尤其是治療兩次施打奈米抗癌藥物微脂體結合聚焦式超音波搭配微氣泡的組別。
結論：就奈米化療藥物累積量的結果來看，得知晚期的腫瘤組織通透性較早期的腫瘤組織佳，原因或許可以從CD31、VEGF的特殊組織染色分析結果其表現量增加而推出晚期腫瘤組織具備較多的血管，也就是說在中期與晚期階段的腫瘤組織其血管通透性較好，在早期第8天其腫瘤組織的血管通透性較差，另外隨著腫瘤體積增加其血腦屏障的完整性可能被腫瘤組織破壞，因此聚焦式超音波搭配微氣泡強化奈米藥物累積，雖有其顯著強化效果，但尤其是對早期腫瘤組織特別顯著。就腫瘤治療結果來看，治療兩次施打奈米抗癌藥物微脂體結合聚焦式超音波搭配微氣泡腫瘤生長抑制效果最為顯著，歸因於其具有較高藥物濃度的累積，而其抑制效果除了腫瘤體積減少，腫瘤細胞數減少，其CD133的表現量也減少，也就是所殘存的腫瘤細胞其抗藥性和再分化能力降低。所以藥物注射入體內後利用聚焦式超音波搭配微氣泡以提昇腦腫瘤組織內累積量達到藥物毒殺細胞效率進而抑制腫瘤的生長的效果，特別是在早期階段性治療以及多次治療具備更好的抑制效果。

Purpose: Focused ultrasound (FUS) with microbubbles (MB) could enhance the delivery of nanodrug into brain tumors in rodent model. In this study, we investigated the difference of PEGylated liposomal doxorubicin (PLD) accumulation, tumor growth response after treatment and immunohistochemistry in different stages of brain tumor tissue-bearing with or without FUS/MBs.
Methods and Materials: The ultrasound frequency and peak pressure at the focal zone were 0.5 MHz and 0.5 MPa, respectively. The doses of MB and PLD through IV injection were 100 ul/kg, and 6 mg/kg, respectively. C6 glioma cells (106 cells) were injected into the right hemisphere and same volume of saline were into the left hemisphere (sham). PLD solution was injected on Day 8, Day 11, or Day 14 after tumor inoculation for the early-, medium-, or late-staged tumors, respectively, and a shot on both Day 8 and Day 11 was also studied for both with and without FUS/MBs. A half of the rats were sacrificed 24 hours after PLD injection to quantify the amount of PLD accumulated in tumor tissues by ELISA and the other half were sacrificed on Day 16 to evaluate the tumor growth response. Some tissue samples were analyzed by immunohistochemistry, CD31, VEGF and CD133, and immunofluorescence assay. We also injected Evan blue (EB, 100 mg/kg) on Day 15 and N was equal to 6 for each group.
Results: The result of PLD accumulation shows that there was higher concentration of doxorubicin in later staged tumor tissue with or without sonication, FUS/MBs sonication could significantly enhance the amount of PLD accumulated in tumor tissues on Day 8 but not so drastically enhanced by sonication on Day 11 or Day 14. In addition, the tumor size of the PLD+MBs+FUS group was significantly smaller than the PLD groups on Day 16; in the groups with one treatment, we found the tumors size of the PLD+MBs+FUS groups treated on Day 8 was significantly smaller than that of group treated on Day 11and Day 14; and an additional PLD+MBs+FUS treatment causes a significantly further inhibition. Furthermore, the immunochemistry analysis of brain tumor tissues on Day 16 showed that (1) the nuclei were more pleomorphic and the cell distribution was more condensed in the control groups, and the responses of CD31, VEGF, and CD133 in brain tumor tissues were stronger in different stages; (2) these above phenomena were decreased in the treated groups, especially in the group with two PLD+MBs+FUS treatment.
Conclusion: The result of nanodrug accumulation shows that there was a higher concentration of doxorubicin in a later staged tumor tissue, due to the compromised BBB with tumor growth, and the responses of CD31 and VEGF immunochemistry for a later-staged tumor tissue were stronger, indicating a high vascular permeability for medium- and late-staged tumors but a low permeability for the early-staged tumor. Therefore FUS/MBs is able to enhance the delivery of nanodrug into brain tumors, especially in early staged tumors. The result of tumor therapy shows an additional PLD+MBs+FUS strategy could successfully inhibit tumor growth due to a high concentration of nanodrug accumulation. The strategy decreases not only tumor volume and tumor cells but also CD133 immunochemistry response, displaying that abilities of drug resistance and differentiation of tumor cells would decrease. As a results, FUS/MBs can enhance the delivery of nanodrug and then achieve a greater cytotoxic ability of nanodrug to achieve an effective inhibition for tumor growth. A better hinder for early-staged tumors can be seen and multiple treatment of PLD+MBs+FUS can further damage the tumor.

口試委員會審定書 #
誌謝 i
中文摘要 ii
ABSTRACT iv
CONTENTS vii
LIST OF FIGURES ix
LIST OF TABLES x
Chapter 1 INTRODUCTION 1
1.1 Problems of brain tumor therapy 1
1.2 Blood vessel architecture and angiogenesis in glioma 1
1.3 Cancer stem cell markers 3
1.4 Blood-brain barrier (BBB) and blood brain-tumor barrier (BTB) 3
1.5 Biologic therapy for malignant glioma 4
1.6 Delivery strategies across the BBB 5
1.7 Disadvantages of passive targeting 5
1.8 BBB disruption via focused ultrasound combined with microbubbles 6
1.9 PEGylated Liposomal Doxorubicin (PLD) 7
1.10 Specific Aim 7
Chapter 2 MATERIALS AND METHODS 8
2.1 Cell culture 8
2.2 Animals 8
2.3 Tumor implantation 8
2.4 Study design 9
2.5 Ultrasound 10
2.6 Chemotherapy 11
2.7 Quantification 11
2.8 Immunofluorescence analysis 12
2.9 Immunohistochemistry 12
2.10 Statistical Analysis 13
Chapter 3 RESULTS 17
3.1 Tumor growth and blood brain-tumor barrier (BTB) 17
3.2 Effect of burst length of FUS sonication on nanodrug delivery and tissue damage 17
3.3 PLD accumulation in tumor tissues related to FUS sonication and tumor stages 18
3.4 Influence of FUS sonication times with MBs on tumor growth response for chemotherapy with anticancer nanodrug 19
3.5 Immunohistochemistry 19
Chapter 4 DISCUSSIONS 28
Chapter 5 CONCLUSIONS 32
REFERENCES 33

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