中文摘要
5-Aminolevulinic acid（ALA）應用在光化學動力療法（photodynamic therapy, PDT）用以治療腫瘤，於過去10年內引起相當廣泛之討論及研究。而以ALA-PDT治療黑色素瘤以外的皮膚癌（如superficial squamous cell carcinoma，sSCC）時，能得到約90 %以上的治療效果；但是如用在治療皮膚基底細胞瘤（basal cell carcinoma, BCC）時，無論是初次治療或是復發率方面，皆不如其他的皮膚癌有效，如對nodular basal cell carcinoma僅有約10 ~ 50%之療效，且復發率高達100 %。且過去對於基底細胞瘤之研究主要著重於臨床治療，並無直接關於ALA用於皮膚基底細胞瘤細胞株的研究報告。
過去本實驗室以正常之裸鼠皮膚作為研究之模式，發現不論是在表皮或是真皮內PpIX的體內蓄積量隨著體外ALA經皮穿透速率之增加而漸趨飽和，顯示PpIX之蓄積量主要受限於細胞內將ALA代謝為PpIX之能力。
因此本研究之目的乃以細胞層級來探討ALA本身對BCC細胞株及人類株化角質細胞HaCaT的毒性（cytotocixity）、細胞產生PpIX之動力學、照光後細胞的存活率及鐵離子螯合劑對於兩細胞株內PpIX生成的影響。期望能藉由本研究的結果提供一最適當之ALA給藥劑量、光照時間及劑量用於BCC之治療。
研究結果顯示，投予0.2 ~ 5 mM ALA 24小時後，BCC仍保有約50%的存活率，而ALA對於HaCaT則較不具毒性。ALA培養 24小時內，兩種細胞所產生的PpIX量，除了5 mM之外，皆會隨著給藥濃度及時間而增加，而以BCC相對於HaCaT所產生之最大比例值在各濃度分別為：0.2 mM為24小時，0.5 mM為2小時，而1、2、5 mM為0.5小時，且最大之比例值出現在0.2 mM培養24小時。接著評估分別以ALA培養2 及4小時除藥後之PpIX動力學時，發現產生最大PpIX量及兩種細胞間最大之PpIX比值的時間，會隨著所投予的藥品濃度增加而增加，而最大之比值則出現在以0.5 mM ALA培養4小時後4小時。雖然BCC細胞株達到PpIX最高量的時間比HaCaT細胞株慢，但是就PpIX生成之最高量（PpIXmax）及AUC值來看，BCC細胞株皆大於HaCaT細胞株，顯示BCC細胞株生合成PpIX之能力大於HaCaT細胞株。
當兩細胞株分別以0.2至5 mM ALA培養4小時後，再以不同劑量LED光源（波長630 nm）於不同時間照射後，可得到BCC相對於HaCaT之存活率最低出現於以0.5 mM和1 mM ALA培養後4小時施以60 J/cm2之照光，且以0.5 mM不影響HaCaT之存活率。此結果亦顯示細胞內PpIX之含量為影響照光後存活率主要之因素。
繼續探討鐵離子螯合劑desferrioxamine （DFO）對兩細胞株PpIX生成之影響，研究結果顯示，兩細胞株皆會因DFO作用之故，而有PpIX蓄積及減緩排除的現象，間接指出兩者可能都有ferrochelatase酵素的存在，而以HaCaT細胞株較易受到DFO的作用而使細胞內PpIX含量較快達到飽和。但添加DFO後，BCC細胞株相對於HaCaT細胞株之PpIX含量比值則降低，顯示DFO反而不利於應用ALA-PDT對於BCC之選擇性。

Abstract
5-Aminolevulinic acid (ALA)-based photodynamic therapy to treat cancers has been widely discussed and investigated in the last decade. The application of ALA-PDT for the treatment of non-melanoma skin cancers, such as superficial squamous cell carcinoma, resulted in satisfied, complete response. However, the therapeutic outcomes of ALA-PDT in basal cell carcinoma (BCC) were not as good as in other skin cancers. Despite extensive clinical experience and reports on ALA-PDT for the treatment of BCCs, none has addressed the PpIX generation kinetics and phototoxicity in cultured BCC cells. Previous study has demonstrated that saturable correlations exist between PpIX accumulation in both the epidermis and dermis in vivo and its transdermal flux in vitro, indicating PpIX accumulation is mainly limited by metabolic capacity of the skin.
The objectives of the study were to investigate ALA toxicity, protoprophyrin IX (PpIX) generation kinetics, cell survival ratio after light irradiation, and the influences of iron chelator, desferrioxamine (DFO) in PpIX generation following ALA incubation in basal cell carcinoma cells (BCC/KMC-1) and human immortalized keratinocytes (HaCaT). The ultimate goal was to optimize ALA-PDT in BCC.
Cultured BCC and HaCaT were incubated with 0.2 to 5 mM ALA for various lengths of time. After 24 hours incubation of ALA, only 50 % BCC survived, but HaCaT were not affected. In general, the PpIX content in both cells increased with incubation time at all ALA concentration except 5 mM. The ratio of PpIX content in BCC vs. HaCaT decreased with increasing concentration during 24 hours ALA incubation. The time of maximal ratio was 24, 2, 0.5, 0.5 and 0.5 h from 0.2 to 5 mM, respectively, with the greatest ratio occurring at 24 h incubation 0.2 mM ALA. The time to reach peak PpIX content post 2 h and 4 h ALA-incubations in both cells and the time of maximal ratio increased with ALA concentration. The greatest ratio appeared at 4 h post 4 h-incubation of 0.5 mM ALA. Although the time to reach peak PpIX content in BCC was slower than HaCaT, the peak PpIX content and area under the PpIX content-concentration curve were greater in BCC than HaCaT, indicating higher biosynthetic capacity of PpIX in BCC than HaCaT.
When both cells were irradiated by different doses of LED light at 0-24 h post 4 h-incubation of 0.2-5 mM ALA, the results demonstrated that the relative survival ratio of BCC vs. HaCaT was the lowest at 4 h post incubation of 0.5 and 1 mM ALA with light irradiation of 60 J/cm2 at 630 nm, with HaCaT survival not influenced by 0.5 mM ALA. The results also suggested cellular PpIX content as the major determinant for cytotoxity in both cells.
Finally, the addition of iron chelator, desferrioxamine (DFO), resulted in enhanced PpIX accumulation and delayed clearance, suggesting the presence of ferrochelatase in both cells. The cellular PpIX content has reached plateau in HaCaT at a lower DFO concentration than BCC. However, the ratio of PpIX content in BCC vs. HaCaT was decreased with DFO in comparison with ALA only, indicating lower selectivity of ALA-PDT in BCC vs. HaCaT.

目錄
中文摘要 i
英文摘要 iv
致謝 vi
目錄 vii
表目錄 ix
圖目錄 xi
縮寫表 xiii
第壹章 緒論 1
第貳章 文獻回顧 2
第一節光化學動力療法簡介 2
第二節光感前趨物質ALA之特性 9
第三節PpIX生合成機轉及作用 13
第四節調控PpIX生成之酵素及相關因素 16
第五節光化學動力療法應用於基底細胞瘤之臨床治療 18
第參章 研究目的 22
第肆章 研究方法 24
第一節實驗細胞 24
第二節藥品、儀器裝置及分析原理 26
第三節ALA對細胞之毒性試驗 45
第四節ALA培養期間細胞內PpIX含量之經時變化 47
第五節ALA培養後細胞內PpIX含量之經時變化 51
第六節光照對ALA培養後細胞之影響 53
第七節鐵離子螯合劑對ALA培養後細胞內PpIX含量 56
經時變化之影響
第伍章 研究結果 62
第一節ALA對細胞之毒性試驗 62
第二節ALA培養期間細胞內PpIX含量之經時變化 69
第三節ALA培養後細胞內PpIX含量之經時變化 77
第四節光照對ALA培養後細胞之影響 87
第五節鐵離子螯合劑對ALA培養後細胞內PpIX含量 113
經時變化之影響
第六章 討論 130
第一節ALA對細胞的毒性影響與培養期間細胞內PpIX 130
經時變化之相互關係
第二節ALA培養後細胞內PpIX含量之經時變化 132
第三節細胞內之PpIX含量與光照對ALA培養後細胞之影響 138
第四節鐵離子螯合劑對ALA培養後細胞內PpIX動力學研究 141
第五節結論 148
參考文獻 149
自述 156





表目錄

表2 - 1細胞株之比較25
表2 - 2細胞培養用物品27
表2 - 3分析用化學試藥30
表2 - 4測量用儀器設備31
表5 — 1ALA水溶液之pH值64
表5 - 2BCC以ALA培養24小時期間之存活率65
表5 - 3HaCaT以ALA培養24小時期間之存活率65
表5 - 4以ALA培養24小時期間BCC相對於HaCaT之存活率比值67
表5 - 5BCC細胞株以ALA培養24小時期間內PpIX含量變化73
表5 - 6HaCaT細胞株以ALA培養24小時期間內PpIX含量變化73
表5 - 7ALA培養24小時期間細胞株內PpIX含量之比較: BCC相對於HaCaT75
表5 - 8BCC細胞株以ALA培養2小時後PpIX含量經時變化79
表5 - 9HaCaT細胞株以ALA培養2小時後PpIX含量經時變化79
表5 - 10以ALA培養2小時後細胞內PpIX含量之比較: BCC相對於HaCaT81
表5 - 11BCC細胞株以ALA培養4小時後PpIX含量之經時變化83
表5 - 12HaCaT細胞株以ALA培養4小時後PpIX含量之經時變化83
表5 - 13以ALA培養4小時後細胞內PpIX含量之比較: BCC相對於HaCaT85
表5 - 14細胞在僅受照光下之存活率92
表5 - 150.2 mM ALA培養4小時後接受不同之光照劑量之細胞存活率94
表5 — 160.5 mM ALA培養4小時後接受不同之光照劑量之細胞存活率96
表5 — 171 mM ALA培養4小時後接受不同之光照劑量之細胞存活率98
表5 — 182 mM ALA培養4小時後接受不同之光照劑量之細胞存活率100
表5 — 195 mM ALA培養4小時後接受不同之光照劑量之細胞存活率102
表5 — 20細胞以0.2 mM ALA培養後照光之存活率比較: BCC相對於HaCaT104
表5 — 21細胞以0.5 mM ALA培養後照光之存活率比較: BCC相對於HaCaT104
表5 — 22細胞以1 mM ALA培養後照光之存活率比較: BCC相對於HaCaT106
表5 — 23細胞以2 mM ALA培養後照光之存活率比較: BCC相對於HaCaT106
表5 — 24細胞以5 mM ALA培養後照光之存活率比較: BCC相對於HaCaT108
表5 — 25BCC及HaCaT細胞株照光後存活率排序 110
表5 — 26細胞存活率比值排序112
表5 — 27DFO溶液於加ALA前後之pH值變化117
表5 — 28不同濃度之DFO對細胞株的存活率影響118
表5 — 29不同濃度之DFO對細胞株內PpIX含量之影響120
表5 — 30不同濃度之DFO對細胞株內PpIX含量之比較: BCC相對於HaCaT122
表5 — 31以不同濃度DFO培養後細胞株內PpIX含量相較於單獨以ALA培養時增加之比值122
表5 — 32以1 mM DFO及ALA培養後細胞內之PpIX含量經時變化124
表5 — 33以1 mM DFO及ALA培養後細胞內PpIX含量之比較: BCC相對於HaCaT126
表5 — 34細胞株以不同濃度DFO培養後接受照光之細胞存活率128
表6 — 1細胞株以ALA培養2或4小時所得之PpIXmax, Tmax, AUC 134
表6 - 2細胞株以ALA及DFO培養4小時所得之PpIXmax, Tmax, AUC144


圖目錄

圖2 — 1PpIX與血紅素生合成路徑21
圖5 — 1BCC以ALA培養24小時期間之細胞存活率66
圖5 — 2HaCaT以ALA培養24小時期間之細胞存活率66
圖5 — 3以ALA培養24小時期間BCC相對於HaCaT之存活率比值圖68
圖5 — 4PpIX校正曲線71
圖5 — 5蛋白質校正曲線72
圖5 — 6BCC以ALA培養24小時期間細胞內PpIX含量之經時變化74
圖5 — 7HaCaT以ALA培養24小時期間細胞內PpIX含量之經時變化74
圖5 — 8以ALA培養24小時期間BCC相對於HaCaT之細胞內PpIX含量比值76
圖5 — 9BCC以ALA培養2小時後細胞內之PpIX含量經時變化80
圖5 — 10HaCaT以ALA培養2小時後細胞內之PpIX含量經時變化80
圖5 — 11以ALA培養2小時後BCC相對於HaCaT之細胞內PpIX含量比值82
圖5 — 12BCC以ALA培養4小時後細胞內之PpIX含量經時變化84
圖5 — 13HaCaT以ALA培養4小時後細胞內之PpIX含量經時變化84
圖5 — 14以ALA培養4小時後BCC相對於HaCaT之細胞內PpIX含量比值86
圖5 — 15BCC在僅受照光下之細胞存活率93
圖5 — 16HaCaT在僅受照光下之細胞存活率93
圖5 — 17光照對以0.2 mM ALA培養後之BCC細胞株存活率之變化95
圖5 — 18光照對以0.2 mM ALA培養後之HaCaT細胞株存活率之變化95
圖5 — 19光照對以0.5 mM ALA培養後之BCC細胞株存活率之變化97
圖5 — 20光照對以0.5 mM ALA培養後之HaCaT細胞株存活率之變化97
圖5 — 21光照對以1 mM ALA培養後之BCC細胞株存活率之變化99
圖5 — 22光照對以1 mM ALA培養後之HaCaT細胞株存活率之變化99
圖5 — 23光照對以2 mM ALA培養後之BCC細胞株存活率之變化101
圖5 — 24光照對以2 mM ALA培養後之HaCaT細胞株存活率之變化101
圖5 — 25光照對以5 mM ALA培養後之BCC細胞株存活率之變103
圖5 — 26光照對以5 mM ALA培養後之HaCaT細胞株存活率之變化103
圖5 — 27照光對以ALA培養後之細胞存活率比值：BCC相對於HaCaT105
圖5 — 28照光對以ALA培養後之細胞存活率比值：BCC相對於HaCaT105
圖5 — 29照光對以ALA培養後之細胞存活率比值：BCC相對於HaCaT107
圖5 — 30照光對以ALA培養後之細胞存活率比值：BCC相對於HaCaT107
圖5 — 31照光對以ALA培養後之細胞存活率比值：BCC相對於HaCaT109
圖5 — 32BCC以DFO培養24小時期間內存活率經時變化119
圖5 — 33HaCaT以DFO培養24小時期間內存活率經時變化119
圖5 — 34不同濃度DFO及0.5 mM ALA培養後之細胞PpIX含量變化121
圖5 — 35BCC與HaCaT以DFO培養後細胞內PpIX含量增加之比值123
圖5 — 36以不同濃度DFO及 0.5 mM ALA培養後BCC相對於HaCaT之細胞內PpIX含量比值123
圖5 — 37BCC以1mM DFO及不同濃度ALA培養後之PpIX經時變化125
圖5 — 38HaCaT以1mM DFO及不同濃度ALA培養後之PpIX經時變化125
圖5 — 39以1mM DFO及不同濃度ALA培養後BCC相對於HaCaT之細胞內PpIX含量比值127
圖5 — 40BCC細胞株在以不同濃度DFO培養後接受照光之存活率129
圖5 — 41HaCaT細胞株在以不同濃度DFO培養後接受照光之存活率129
圖6 — 1ALA濃度與細胞株產生最大PpIX量（PpIXmax）之關係圖136
圖6 — 2ALA濃度與細胞株產生最大PpIX量時間點（Tmax）之關係圖136
圖6 — 3ALA濃度與兩細胞株間PpIX AUC值之關係圖137
圖6 — 4ALA濃度與細胞株產生最大PpIX量（PpIXmax）之關係圖146
圖6 — 5ALA濃度與細胞株產生最大PpIX量時間點（Tmax）之關係圖146
圖6 — 6ALA濃度與兩細胞株間PpIX AUC值之關係圖147

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