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研究生:李致瑋
研究生(外文):Chih-Wei Lee
論文名稱:111In-VNB-liposome結合各種分子影像技術在帶有雙報導基因之人類大腸直腸癌HT-29/tk-luc動物模式之臨床前療效評估
論文名稱(外文):Precilincal therapeutic efficacy evaluation of 111In-VNB-liposome by molecular imaging on HT-29/tk-luc human colorectal cancer xenografts
指導教授:黃正仲黃正仲引用關係
指導教授(外文):Jeng-Jong Hwang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物醫學影像暨放射科學系暨研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:77
中文關鍵詞:大腸直腸癌微脂體藥物動力生物分佈生物冷光影像微正子斷層掃描
外文關鍵詞:Colorectal cancerPEG-liposomepharmacokineticsbiodistributionbioluminescencemicro positron emission tomography
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大腸直腸癌(colorectal cancer)是工業化國家中發生率排名第三且世界腫瘤致死率排行第四之常見惡性腫瘤。臨床上大腸直腸癌病人仍以手術切除為主,也會合併化學治療、放射治療或是免疫療法,然而病人之五年存活率卻只有40-60%,傳統的化療以及放射療法都存在著標靶性低這個缺點,造成較大的副作用因此,發展新的治療方法使病人可以獲得更好的治療效果,有其必要性。
利用微脂體包裹化療藥物在臨床已被證實,可以使藥物在體內穩定性增加,提高藥物在血液中較長的循環時間,並累積在新生血管眾多的腫瘤部位,進一步的減低化療藥物對正常組織器官之副作用。
而本研究之目的在於以0.9莫耳百分比聚乙二醇 (PEG)修飾之微脂體發展新的放射化學療法,以人類大腸直腸癌細胞株(HT-29),轉染單純疱疹病毒第一型胸腺激素及螢火蟲的螢光酵素之雙重報導基因,並篩選出穩定表現此雙重報導基因的人類大腸直腸癌細胞 (HT-29/tk-luc)以建立腫瘤動物模式,並探討111In-VNB-liposome於NOD/SCID免疫缺乏小鼠體內之藥物動力學、生物分布,最大容忍劑量、所接受之輻射劑量及藥物毒性,再結合基因及分子影像技術來評估治療大腸直腸癌HT-29的效果。
藥物動力實驗中,以微脂體攜帶放療藥物其在血液中的半衰期遠
大於沒有包覆的組別(13.70小時:0.038小時),且曲線下面積(AUC)為對照組的16.3倍;當腫瘤生長到100mm3大小左右進行藥物生物分佈實驗,結果指出腫瘤/肌肉的比值在注射後1, 4, 24, 48以及 72小時分別為 0.70, 1.68, 3.56, 12.73, 8.57, 平面伽瑪影像及自動放射顯影術也證明此微脂體 (NanoX-liposome) 具有被動標靶特性的實驗結果;而利用卡尺所追蹤腫瘤生長情形可以看出化療放療合併組與對照組比較的腫瘤生長明顯受到抑制(p<0.05),以公式計算其合併係數(combination index)約等於1,推論此化療放療藥物對腫瘤的控制效率為1+1=2的協同作用;124I-FIAU以及18F-FEAU此兩種核醫藥物使用方便不但在帶有單純疱疹病毒第一型胸腺激脢基因的腫瘤照影上表現高專一性,且能清楚的看到大腫瘤中心壞死的部位。
我們的結果證實微脂體(NanoX)是治療癌症時理想的藥物載體,微脂體同時攜帶化療與放療藥物確實提高分別使用的療效,而利用非侵入式的活體分子影像的生物冷光(BLI)、核醫影像如微正子斷層掃描(μPET)不但能追蹤腫瘤生長情形,間接證明111In-VNB-liposome確實具有療效,同時確定此HT-29/tk-luc的小鼠動物模式技術在評估藥物對腫瘤控制上為一大利器。
Colorectal cancer (CRC) is the third most common malignancy and the fourth most frequent cause of cancer deaths worldwide. The five-year overall survival rate for all stages is 40-60% based on surgery, adjuvant chemotherapy and radiotherapy. Therefore, newly developed and/or more effective adjuvant therapies are needed to prevent the recurrence and spreading of this disease.
Chemotherapy combined with liposome has been developed to improve the clinical treatment of cancer by obtaining high accumulation of drugs in tumor tissues but limited accumulation in normal organs.
The aim of this study is to develop novel chemoradiotherapy and to determine the pharmacokinetics, biodistribution, maximum tolerated dose, radiation, and toxicity of nanoliposome conjugated with Vinorelbine (VNB) and 111In in NOD/SCID mice.
In the pharmacokinetics experiment, show the AUC (area under curve) of PEGylated liposome is about 17 folds higher than the group of free radioisotope. Biodistribution of the drug was performed when tumor size reached 100mm3. The tumor/muscle ratios of 111In-VNB-liposome were 0.70, 3.67, 3.22, 18.16, 8.07, respectively, at 1, 4, 24, 48 and 72 h postinjection, gammascinitigraphy also confirmed this results.
The combination therapy inhibit tumor growth as tracked by caliper measurement (p<0.05), and we exhibited an additive effect (combination index = 1.045), show that the use of 0.9 mol% PEG 111In-VNB- liposomes for passively targeted tumor therapy.
We have demonstrated a non-invasive imaging technique with a luc and HSV1-tk reporter gene of BLI and μPET for evaluation of tumor treatment efficacy in vivo. The NOD/SCID mice bearing HT-29/tk-luc xenografts treated with 111In-VNB-liposome were shown with tumor reduction by this technique.
目錄:
論文摘要………………………………………………………………………4
一、
前言
1.1 大腸直腸癌…………………………………………………………9 1.2 滅癌平 (Vinorelbine,VNB) …………………………………9 1.3 銦-111(111In)…………………………………………………10 1.4 微脂體(liposome) ……………………………………………11 1.5 報導基因(reporter gene) ……………………………………14 1.6 研究目的…………………………………………………………15
二、
材料與方法
實驗材料………………………………………………………………17 實驗流程………………………………………………………………18 2.1 腫瘤細胞株的培養………………………………………………19 2.2 小鼠動物模式的建立……………………………………………19 2.3 轉染雙報導基因tk-luc進入人類大腸直腸癌細胞株…………19 2.4 微脂體(NanoX-liposome)的合成……………………………20 2.5 111In-NanoX-liposome /111In-VNB-liposome之放射化學合成21
2.6 評估以NanoX-liposomes 包覆抗癌藥物在HT-29/tk-luc 動 物模式下的藥物動力以及生物分佈情形………………………..23
2.7 小鼠生物冷光影像追蹤…………………………………………24 2.8 單光子放射電腦斷層造影(SPECT)……………………………25 2.9 小鼠全身冷凍切片………………………………………………25 2.10 實驗設計、追踪腫瘤大小、動物體重變化……………………26 2.11 微正子電腦斷層照影(μPET)…………………………………26
2.12 血液血清生化分析………………………………………………27 2.13 小鼠病理切片……………………………………………………28 2.14 統計方法………………………………………………………30
三、
結果 3.1 評估HT-29/tk-luc 報導基因之穩定表現………………31 3.2 111In-VNB-liposome 在NOD/SCID 小鼠體內之藥物動力……31 3.3 111In-VNB-liposome在 HT-29/tk-luc 之NOD/SCID小鼠動物模式 下的生物分佈……………………………………………………32 3.4 以γ-imaging 驗證微脂體腫瘤被動標靶的效果………………32 3.5 小鼠冷凍切片之自動放射顯影…………………………………33 3.6 111In-VNB-liposome 在NOD/SCID小鼠之最大耐受劑量………33 3.7 療效評估之體重以及腫瘤大小追蹤 (單一劑量給藥) ………33 3.8 療效評估之體重以及腫瘤大小追蹤 (多劑量給藥) …………34
3.9 生物冷光影像追蹤……………………………………………34
3.10 【124I-FIAU】之微正子電腦斷層影像於療效之評估………35 3.11 【18F-FEAU】之微正子電腦斷層影像於療效之評估…………35 3.12 血液血清生化分析………………………………………………36
四、
圖及表………………………………………………………………37
五、
討論…………………………………………………………………63
六、
結論…………………………………………………………………67
七、
參考文獻……………………………………………………………68
附錄………………………………………………………………………72
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