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研究生:林祐毅
研究生(外文):Yu-Yi Lin
論文名稱:探討內界膜剝離手術之染劑毒性
論文名稱(外文):A toxicity study of dyes used in the inner limiting membrane peeling procedure
指導教授:詹家泰詹家泰引用關係
指導教授(外文):Chia-Tai Chan
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生物醫學工程學系
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:65
中文關鍵詞:視網膜毒性黃斑部破孔內界膜剝離術染劑
外文關鍵詞:retina toxicitymacular holeILM peelingdye
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治療黃斑部破孔的內界膜剝離手術中,會透過輔助染劑indocyanine green (ICG)、brilliant blue G (BBG),trypan blue (TB) 來進行手術,然而這些染劑對於視網膜有毒性疑慮。本實驗利用人類視網膜色素上皮細胞株(ARPE-19),搭配現行手術之清洗液balanced salt solution (BSS)及降低毒性之清洗液fetal bovine serum (FBS),搭配前處理及後處理兩種清洗方式,透過細胞存活率、生長速率曲線、細胞內氧化壓力、粒線體膜電位變化、粒線體質量變化,來進行毒性及解毒實驗探討。
實驗結果顯示,ICG染劑濃度小於2.5 mg/ml,BBG及TB皆無細胞毒性反應。在細胞生長速率曲線測試,當ICG及BBG濃度大於1 mg/ml會有顯著之細胞生長停滯的現象,TB在所有的測試濃度下,皆無顯著之細胞生長停滯現象。在細胞內氧化壓力測試,ICG、BBG 、TB在各種實驗濃度下皆有顯著之氧化壓力上升,但是在FBS介入及清洗液前處理皆會有顯著之氧化壓力緩解 。
本研究中發現,(1)內界膜剝離術中所使用的染劑ICG、BBG,TB皆會對視網膜細胞造成負面影響;(2)FBS具有比BSS更好的緩解效果;(3)沖洗液前處理對於染劑所造成之負面效果有較好之舒緩效果。透過本研究結果,可應用於未來臨床上進行內界膜剝離術之參考。
Aims:
To investigate and compare the cytotoxicity of indocyanine green (ICG)、brilliant blue G (BBG) and trypan blue (TB) using ARPE-19 cells that have been pre-treated/post-treated with balanced salt solution (BSS) or fetal bovine serum (FBS).
Methods:
The cultured human retina pigment epithelium (RPE) ARPE-19 cells were pre-treated/post-treated with BSS or FBS (represent the autologous serum in clinic) in parallel with cells being soaked with various concentrations of ICG, BBG, and TB. Cells were then assessed for viability, growth rate, reactive oxygen species (ROS) level, mitochondrial (MT) membrane potential (Δψ) and MT mass as cytotoxic indices. For the FBS pre-treated cells, only ROS was examined.
Results:
Using the MTT assay, cytotoxicity seemed to appear when the dye concentration was above 2.5mg/ml for ICG but no cytotoxicity for BBG and TB at tested concentrations used. Cell growth was arrested at a concentration 1mg/ml when ICG or BBG were present but no arrest at any of the tested concentrations was found for TB with the cell-growth curve was slowest for ICG. Cellular ROS levels increased at all concentrations of all dyes, but the increasing slopes were decreased after FBS post-treatment washout.
Conclusions:
As a rinse buffer FBS performs much better than BSS in terms of cell rescue, which agrees with a clinical report when autologous whole blood was applied to macular hole surgery. However, FBS pre-treatment seems to be much better than FBS use as washout buffer in post-treatment.
致謝 i
目錄 ii
中文摘要 vi
英文摘要 vii
第一章 簡介 1
1.1 研究背景與動機 1
1.2 研究方法 3
1.3 研究假設 4
1.4 研究重要性 4
第二章 文獻回顧 5
2.1染劑應用於黃斑部破孔 5
2.2染劑毒性 5
2.3替代染劑 6
2.4解毒洗清液 7
2.5毒性原理 8
第三章 研究材料方法 9
3.1實驗溶液材料配製 9
3.1.1 Phosphate Buffered Saline (PBS)配製 9
3.1.2 Bovine serum albumin (BSA)配製 9
3.1.3DCF配製 10
3.1.4MTT配製 10
3.1.5培養基配製 10
3.2細胞培養 11
3.2.1細胞培養(cellculture) 11
3.2.2細胞繼代培養(subculture) 11
3.2.3實驗細胞種植 12
3.3實驗染劑 13
3.3.1染劑ICG 13
3.3.2染劑BBG 13
3.3.3染劑TB 13
3.3.4染劑使用步驟 13
3.3.5細胞染色前處理及後處理 14
3.4 研究方法 15
3.4.1 MTT毒性測試 15
3.4.2生長曲線 15
3.4.3細胞氧化壓力測試 16
3.4.4粒線體質量及電位測試 16
3.4.5殘留染劑吸收光譜量測 17
3.4.6染色能力測試 18
3.4.7細胞型態觀測 18
3.4.8白蛋白影響效益 19
3.4.9細胞吞噬能力測試 19
3.4.10系統性文獻回顧方法 19
3.4.11統計方法 20
第四章 結果 21
4.1 MTT毒性測試 21
4.2生長曲線 21
4.3 ROS分析 22
4.4 粒線體質量及電位測試 23
4.5殘餘染料測量 23
4.6細胞形態學檢查 24
4.7白蛋白效應 25
4.8 細胞吞噬能力測定 25
4.9染色能見度測試 26
第五章 討論 27
5.1染劑使用現況 27
5.2研究結果 27
5.3研究比較 28
5.4研究限制 29
5.5角色定位 29
5.6其他降低毒性方法 29
5.7未來發展 30
第六章 結論 31
第七章 圖表 32
圖一、細胞毒性 32
圖二、生長曲線 33
圖三、氧化壓力變化 34
圖四、粒線體膜電位及粒線體質量 35
圖五、染劑殘留光譜 36
圖六、ICG處理之細胞型態 37
圖七、BBG處理之細胞型態 38
圖八、TB處理之細胞型態 39
圖九、BSA對於氧化壓力之變化效果 40
圖十,吞噬能力測試 41
圖十一、染色能力測試 42
表一、染色能力比較表 43
表二、文獻分析 44
文獻參考 47
附件:發表論文 54
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