臺灣博碩士論文加值系統

背景與研究目的：癌症的免疫治療是近年來致力於發展的癌症治療方式，透過提升宿主的免疫能力，配合目前的主流療法來達到抑制腫瘤轉移以及復發的效果。過去研究顯示，超音波的熱效應和機械效應能協助釋放腫瘤相關抗原因子，提升宿主對抗腫瘤的能力；如配合提升宿主免疫反應的藥物，則有可能達到腫瘤治療的加乘效果。本研究主要目的是探討原位癌腫瘤在不同治療方法所產生的免疫反應，對轉移及再發腫瘤之影響。

材料與方法：腫瘤細胞為CT26和4T1；小鼠品系為BALB/cByJNarl。主要治療腫瘤方式為超音波及OK-432免疫刺激藥物。超音波雙頻物治探頭，發射頻率：1 MHz；發射強度：3 W/cm2；工作週期：50 %；治療持續時間：15 min。動物實驗分成三部分：第一部分分為控制組(n=10)；OK-432治療組(n=10)；脈衝超音波熱治療(pUSHT)組(n=10)以及OK+pUSHT治療組(n=10)。對原位腫瘤做兩次治療後手術切除，之後在對側背部植入相同數量的腫瘤細胞，觀察是否能夠長成實質腫瘤。也會記錄存活率來評估腫瘤轉移的情況。第二部分則增加治療次數(四次)是否能更有效提升對於CT26腫瘤的免疫反應，分組情形與第一部分動物實驗相同，但每組n=5。第三部分則是再發腫瘤測試，從皮下注射改為尾靜脈注射，以模擬轉移的情形，使用細胞株為4T1。

結果： 第一部份再發腫瘤測試不論CT26和4T1腫瘤細胞，控制組與治療組在再發腫瘤測試中沒有差異性，而在存活率方面OK-432治療組與OK+pUSHT治療組在統計上有顯著差異(p<0.05)，結果顯示在影響4T1轉移的部分，合併治療並沒有看到較好的效果。第二部分增加治療次數，結果顯示不論控制組或是治療組，對於CT26腫瘤的免疫反應皆有所提升，與第一部分實驗相比，再發腫瘤測試收到更好的效果。第三部分實驗在肺腫瘤結節數量上，各組間沒有統計顯著差異。

結論：結果顯示超音波結合免疫刺激劑OK-432不足以影響再發腫瘤的生成與原位腫瘤的轉移，不同癌細胞的再發腫瘤測試有不一樣的效果。

Background and Purpose: Recently, immunotherapy is being developed for treating different kinds of cancers. Researchers hope that tumor can be treated by boosting host immune response and combining with mainstream cancer therapeutics. Recent studies showed that the thermal and mechanical effects of therapeutic ultrasound facilitate the release of tumor related antigens. Boostung immune response by increasing antigen presentation may further inhibit tumor growth and its metastasis. The main purpose of this study is to investigate the effect of immune response induced by primary tumor treatment on the development of metastasis and rechallenge tumor.

Materials and Methods: Female BALB/cByJNarl strain mice and cancer cell lines CT26 and 4T1 were used in this study. The main treatment was the combination of pulsed-wave ultrasound hyperthermia (pUSHT) and immunostimulant OK-432. The parameters of ultrasound hyperthermia: frequency 1 MHz; intensity 3 W/cm2; duty cycle 50%; heating duration 15 min. There were three portions for animal studies. The first portion includes four groups: Control (n=10); OK-432 alone (n=10); pUSHT alone (n=10), and OK+pUSHT (n=10). The primary tumor was treated twice and then removed by surgery. The same number of cancer cells was implanted on the contralateral side of flanks as a rechallenge model. The study was investigated tumor metastasis by survival rate. The treatment for the second portion was four times instead of twice and five mice for each group. The rechallenge model for the third portion was by injecting cancer cells via the tail vein to mimic tumor metastasis. The evaluation was based on the amount of lung tumor nodules.

Results: There was no significant difference between control group and treatment groups in the first portion study for both CT26 and 4T1 cancer cell lines in the rechallenge tumor model. For the survival rate, there was significantly different between the OK+pUSHT group and the OK-432 group (p<0.05). In the second portion study, the immune response was increased in all groups but there was still no significant difference among the groups. In the third portion study, there was no significant difference among groups in the amount of lung nodules.

Conclusion: Combining pulsed-wave ultrasound hyperthermia and OK-432 may not be able to produce sufficient immune responses against rechallenge tumor and tumor metastasis. In addition, the outcomes were different in treating different cancer tumors.

中文摘要……………………………………………………………………………i
英文摘要……………………………………………………………………………iii
目錄………………………………………………………………………………v
圖目錄……………………………………………………………………………vii
表目錄……………………………………………………………………………ix
第一章 緒論…………………………………………………………………………1
1.1 腫瘤……………………………………………………………………1
1.2 小鼠腫瘤轉移模式………………………………………………………2
1.3 超音波治療……………………………………………………………3
1.4 腫瘤細胞株………………………………………………………………4
1.5 OK-432免疫刺激劑……………………………………………………4
1.6 研究目的………………………………………………………………5
第二章 材料與方法………………………………………………………………6
2.1 腫瘤細胞株………………………………………………………………6
2.2 實驗動物…………………………………………………………………6
2.3 OK-432 A群3型溶血性鏈球菌乾燥冷凍製劑………………………7
2.4 超音波系統與參數設定…………………………………………………7
2.5 超音波治療熱電耦溫度測量……………………………………………8
2.6 腫瘤組織切片……………………………………………………………8
2.7 肺臟染色…………………………………………………………………8
2.8 再發腫瘤模式…………………………………………………………9
2.9 動物實驗…………………………………………………………………9
2.10 腫瘤轉移評估…………………………………………………………13
2.11 統計分析方法…………………………………………………………14
第三章 結果………………………………………………………………………15
3.1 熱電耦溫升量測……………………………………………………15
3.2 第一部份再發腫瘤模式………………………………………………16
3.2.1 體重變化…………………………………………………………16
3.2.2 CT26原位腫瘤治療……………………………………………17
3.2.3 CT26再發腫瘤模式………………………………………………20
3.2.4 4T1原位腫瘤治療………………………………………………22
3.2.5 4T1再發腫瘤模式…………………………………………………25
3.2.6 存活率分析…………………………………………………………27
3.3 第二部分再發腫瘤模式………………………………………………29
3.3.1 體重變化……………………………………………………………29
3.3.2 CT26原位腫瘤治療……………………………………………….30
3.3.3 CT26再發腫瘤模式…………………………………………31
3.4 第三部分再發腫瘤模式………………………………………………32
3.4.1 體重變化……………………………………………………………32
3.4.2 4T1原位腫瘤治療…………………………………………………33
3.4.3 再發腫瘤之肺臟結節數……………………………………………34
第四章 討論與結論…………………………………………………………………36
第五章 參考文獻……………………………………………………………………41

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