Effect of liposome formula and size in contrast medium induced nephropathy prevention and lesion malignancy diagnosis with computed tomography

運用電腦斷層掃瞄良、惡性腫瘤是目前廣泛診斷方式，但受限於顯影劑無法有效累積至惡性腫瘤內，降低周圍造影程度而不易呈現不規則形狀特徵，容易誤判為良性腫瘤而增加罹癌死亡的風險。 含碘顯影劑注射和飲用能增加X-光吸收度，方便醫護人員觀察病患器官或組織功能和狀況，然而傳統顯影劑以腎臟過濾形成尿液而排出病患體外。對於老年人、糖尿病和腎臟功能不全患者，容易受大量顯影劑累積至腎臟而引發急性腎衰竭(CIN)狀況發生。 本實驗採用長效型微脂體配方(DSPC : soyPC : Chol : DSPE-PEG 2k = 54 : 27 : 16 : 6 mole ratio)包覆顯影劑，以100和200nm微脂體粒徑注射攜帶腫瘤裸鼠體內，在動態時間-密度圖形中，微脂體能經由EPR效應滲透並累積於惡性腫瘤內部，造影強度明顯高於良性腫瘤，兩者相差約1.5倍(60分鐘內)，而惡性腫瘤周圍累積顯影量可達到33小時，說明微脂體具備良好穩定性。 DPSC微脂體包覆顯影劑能預防顯影劑誘發急性腎衰竭的機率，主要增加血液循環時間和改變代謝路徑至肝膽系統，降低腎臟代謝負擔。以200nm微脂體(70%微脂體和30%純顯影劑混合液)注射至糖尿病大鼠體內，分析48小時血清肌酐酸和尿素氮數值，無明顯上升趨勢(顯影劑注射:上升27%)，表示腎臟不受微脂體影響其功能。在裸鼠造影影像說明微脂體經由肝膽路徑形成糞便而排出體外，微脂體長期累積至肝臟，而造成顯影維持一定的強度。 結合以上結果，微脂體包覆顯影劑能有效區分良、惡性腫瘤動態顯影趨勢，提供醫療人員診斷的臨床指標，此外微脂體能預防CIN發生，改善傳統顯影劑所帶來的副作用。Computed tomography is extensively diagnostic equipment on benign and malignant screening, but contrast medium is difficulty accumulate in tumor, reducing contrasted intensity on tumor rim to analysis irregular shapes , easily regards as benign tumors and increases the risk of death by malignant. Injected and drank iodine contrast medium can increase X-ray absorption, easy to observe patient organ or tissue function and situation by medical staff, but the conventional contrast medium filtered urine by kidney. For the elderly, diabetes and renal dysfunction patient, a large number of contrast medium accumulate on kidneys and easily induced acute renal failure (CIN). The aim of the research is preparation of contrast medium encapsulated long circulation liposomes(DSPC : soyPC : Chol : DSPE-PEG 2k = 54 : 27 : 16 : 6 mole ratio), injected 100 and 200nm liposome into implanted tumor NU/NU mice, liposome can permeate and accumulate in malignant tumor by EPR effect, the contrasted intensity is significantly higher than benign on Dynamic time-density pattern, the difference of both contrasted about 1.5 times (60 minutes), and the contrast medium accumulated in malignant for 33 hours, indicating liposome has stability property. Contrast medium encapsulated DPSC liposomal can prevent the risk of CIN, the reason is liposome can remain long-term blood circulation and alter the metabolic path to the hepatobiliary system, reduce the burden of kidney. Injected 200nm liposome(70%liposome and 30% contrast medium mixture) into diabetic rats, analyzed serum creatinine and urea nitrogen values for injected 48hrs , no significant rising trend (contrast medium: increased by 27% ), indicated liposome did not affect kidney function. NU/NU mice contrasted imaging was observed liposome accumulate to the liver and contrasted intensity maintain a certain strength, indicated liposome through hepatobiliary path formed to feces and excreted. The preliminary results indicated that contrast medium encapsulated liposomal can effectively distinguish between benign and malignant dynamic contrasted trend, provide clinical indices to medical staff, furthermore liposome can prevent the risk of CIN, improve the side effects of conventional contrast medium.第一章 緒論-1 1.1. 以DCE-MDCT判讀腫瘤良、惡性的優勢和挑戰-1 1.2. 顯影劑誘發急性腎衰竭-4 1.3. 研究背景和目的-5 1.3.1. 背景-5 1.3.2. 目的-6 第二章 文獻回顧-7 2.1. 顯影劑誘發腎病變(CIN)-7 2.2. 微脂體種類與運用 9 2.3. 微脂體粒徑和配方對腫瘤良、惡性篩選的影響-14 第三章 實驗材料與方法-19 3.1. 實驗設計架構-19 3.2. 儀器設備-21 3.3. 材料和藥品-22 3.4. 藥品配製-24 3.4.1. 細胞培養液-24 3.4.2. 磷酸緩衝溶液-25 3.4.3. MTT檢測溶液(暗房配製)-25 3.4.4. Streptozotocin(STZ)誘發溶液-25 3.4.5. Ammonia ferrothiocyante檢測溶液-26 3.5. 微脂體配製步驟-26 3.6. 微脂體包覆率分析-29 3.7. 微脂體穩定檢測-30 3.8. 微脂體脂質含量檢測-30 3.9. 顯影劑濃度對細胞活性檢測-31 3.9.1. MTT檢測-32 3.9.2. 細胞計數檢測-32 3.10. 微脂體濃度對細胞活性檢測-33 3.11. 微脂體包覆顯影劑保護評估-34 3.12. 注射微脂體判斷良惡性腫瘤-35 3.13. 大鼠誘發急性腎衰竭實驗-36 3.14. 利用微脂體預防急性腎衰竭實驗-36 第四章 實驗結果-37 4.1. 微脂體包覆率檢測-37 4.2. 微脂體粒徑大小分析(DLS)-38 4.3. 微脂體安定性分析-40 4.4. 顯影劑濃度對細胞活性檢測-44 4.5. 微脂體包覆顯影劑保護評估-47 4.6. 微脂體包覆對良、惡性腫瘤顯影之影響 -49 4.6.1. 含碘顯影劑判讀良、惡性腫瘤特徵-49 4.6.2. 微脂體包覆顯影劑分析良、惡性腫瘤造影程度與半生期-50 4.7. 微脂體包覆對顯影劑生理分佈之影響-53 4.8. 微脂體包覆對DM SD大鼠腎臟顯影劑累積的影響-60 4.9. 微脂體包覆對DM SD大鼠誘發CIN的影響-63 4.10. Matlab分析大鼠腎臟含碘濃度分佈情形-64 4.11. 微脂體延長循環時間證明(大鼠外觀、血液)-65 第五章 結論-67 第六章 參考文獻-6