臺灣博碩士論文加值系統

黃芩苷是黃酮類化合物，它萃取自黃芩。黃酮類化合物具有抗氧化、抗菌、抗細胞凋亡、抗發炎。以前報告發現在長波紫外線(UVA)暴露後黃芩苷可減少活性氧(ROS)、超氧化物(O2−)、一氧化氮(NO●)的產生。本研究旨在探討黃芩苷合併中波紫外線處理後人類皮膚角質細胞之細胞殘留分率及釐清其作用機制。
合併處理黃芩苷 (50、100g/ml)以及紫外線的細胞存活率比單獨輻射組增加20%及30%。細胞週期分析UVB與黃芩苷合併處理比較，黃芩苷合併處理與單獨處理相比，其在Sub-G1期比率減少1.1倍。在西方墨點法實驗中，加入黃芩苷處理後與單獨UVB相比可以減少細胞蛋白酶-3與增加MCL-1之活性。
黃芩苷具有輻射保護效應，明確的可以降低經由中波紫外線照射後的細胞死亡以及細胞凋亡率。
發酵的小麥胚芽萃取物 (FWGE)為癌症病人常用的營養補充品。目前有少數的文獻指出其兩種含苯醌成分的成份2-methoxy benzoquinone 和 2,6-dimethoxybenzquinone有抗癌細胞增生的效能、抗轉移和調節免疫能力的效果。這些體外抗腫瘤的實驗數據，使我們進一步評估在HaCaT細胞中FWGE單獨處理以及合併5-FU或順鉑。結果顯示，與FWGE（250、500g/ml）和5-FU（100M）合併處理上是沒有作用的。 FWGE（250、500g/ml）合併順鉑（30M）處理在結果中發現有劑量的依賴性的，細胞週期停留在S期。西方墨點法的實驗中， 合併處理的caspase-3和caspase-8的活性是被抑制的。前處理FWGE能減少人類角質細胞因順鉑造成的細胞死亡和凋亡。

Baicalin is a flavonid glycoside isolate from Scutellaria baicalensis. The flavones have the multiple potentials in exerting antioxidant, anti-bacteral, anti-apoptotic, anti-inflammatory. In previous studies, Baicalin were reported with radioprotection mediating by decreases of Reactive oxygen species (ROS), super-oxide anion(O2−), and Nitogen oxide (NO●) on photodamage after ultraviolet A (UVA) radiation. The aims of this study were to analyze survival fractions and toinvestigate the mechanisms of Baicalin and ultraviolet B(UVB) to human keratinocytes cell.
In combined groups of baicalin (50、100g/ml) and UVB, cell viabilities were increased 20% and 30％ than UVB alone respectively. On cell cycle distribution, the Sub-G1 stage reduce 1.1 fold. In the western blot, the combined group decrease the expression of caspase-3 and increase the MCL-1 expreesion than UVB alone.
The Baicalin pretreatment have radioprotective effects, it reduces cell death and apoptosis rate in the human keratinocyte cell after UVB exposure.
Fermented wheat germ extract (FWGE) is currently used as nutrition supplement for cancer patients. Limited recent data has suggested antiproliferative, antimetastatic and immunological effects which were at least in part exerted by two quinones, 2-methoxy benzoquinone and 2,6-dimethoxybenzquinone as ingredients of FWGE. These activity data prompted us to further evaluate the in vitro anti-tumor activity of FWGE alone or in combination with 5-FU and cisplatin in HaCaT cell.
The results showed that the combined treatments with FWGE(250、500g/ml) and 5-FU(100M) is ineffective. The combined group of FWGE(250、500g/ml) and CDDP(30M) have dose dependent effects. Combined effects present enhancement of S arrest. In the western blotting, the combined group decrease the expression of caspase-3 and caspase-8. The FWGE pretreatment have protective effects, it reduces cell death and apoptosis rate in the human keratinocyte cell after CDDP damage.

目錄
中文摘要-------------------------------------------------------------------I

英文摘要-----------------------------------------------------------------III

目錄----------------------------------------------------------------------V

圖目錄-----------------------------------------------------------------VIII

表目錄------------------------------------------------------------------XII

第一章 前言--------------------------------------------------------------1

第二章 文獻回顧-------------------------------------------------------3
HaCaT人類角質細胞------------------------------------------------------3
黃芩苷介紹-------------------------------------------------------------3
紫外線-----------------------------------------------------------------5
小麥胚芽萃取物----------------------------------------------------------7
順式-二氯二氨合鉑(II) --------------------------------------------------8
5-氟尿嘧啶-------------------------------------------------------------9
細胞凋亡---------------------------------------------------------------11
細胞週期---------------------------------------------------------------12

第三章 材料與方法 --------------------------------------------------------13

第一節 實驗材料 ----------------------------------------------------------13
一. 細胞來源 ------------------------------------------------------------13
二. 藥品試劑 ------------------------------------------------------------13
三. 儀器設備、器材 -------------------------------------------------------16

第二節 實驗方法 ----------------------------------------------------------19
一.藥品配製以及紫外線照射--------------------------------------------------19
二. 細胞培養-------------------------------------------------------------22
三. 細胞存活率分析-------------------------------------------------27
四. 流式細胞儀分析測定---------------------------------------------30
五. 細胞蛋白質之萃取-----------------------------------------------33
六. SDS-PAGE----------------------------------------------------37
七. 西方墨點法----------------------------------------------------41
八. 統計分析------------------------------------------------------44

第四章 實驗結果 ----------------------------------------------------------45
第一節 黃芩苷於人類角質細胞在中波紫外線之輻射保護作用------------------45
第二節 小麥胚芽萃取物合併化療藥物之效應-------------------------------53

第五章 討論---------------------------------------------------------------80
第一節 黃芩苷於人類角質細胞在中波紫外線之輻射保護作用--------------------------80
第二節 小麥胚芽萃取物合併化療藥物之效應--------------------------------------83
第六章 結論---------------------------------------------------------------87

參考文獻 -----------------------------------------------------------------88


圖目錄

圖2-1. 黃芩甘的結構形狀---------------------------------------------------------------5
圖2-2. 順鉑（Cisplatin）的結構式------------------------------------------------------9
圖 2-3. 5-氟尿嘧啶（5- fluorouracil）的結構形狀---------------------------------------11
圖 3-1 細胞計數盤--------------------------------------------------------------------23
圖3-2 轉漬夾排序組成-----------------------------------------------------------------44
圖4-1 HaCaT在不同濃度時間的黃芩苷處理之細胞存活率--------------------------------------45
圖4-2 HaCaT在不同劑量中波紫外線暴露後所測得的細胞存活率---------------------------------46
圖4-3 HaCaT在不同濃度的黃芩苷與的中波紫外線處理所測得的細胞存活率-------------------------47
圖4-4使用顯微鏡(10X40)直接觀察HaCaT在不同濃度的黃芩苷與中波紫外線處理所觀察之細胞型態------48
圖4-5使用流式細胞儀測得24小時黃芩苷與紫外線細胞週期量化之結果----------------------------49
圖4-6使用流式細胞儀測得24小時黃芩苷與紫外線細胞週期統計之結果----------------------------50
圖4-7在不同濃度的黃芩苷與同一劑量的中波紫外線處理所測得之Sub-G1--------------------------51
圖4-8西方墨點法來測得黃芩苷與中波紫外線之蛋白濃度---------------------------------------52
圖4-9在不同濃度時間的FWGE以及不同的細胞存活率------------------------------------------53
圖4-10HaCaT在不同濃度的5-FU苷以及時間下的細胞存活率------------------------------------54
圖4-11HaCaT在不同濃度的CDDP以及時間下的細胞存活率--------------------------------------55
圖4-12 HaCaT在不同濃度的FWG與5-FU合併處理12小時---------------------------------------56
圖4-13 HaCaT在不同濃度的FWGE與5-FU合併處理24小時--------------------------------------57
圖4-14 HaCaT在不同濃度的FWGE與5-FU合併處理48小時--------------------------------------58
圖4-15 HaCaT在不同濃度的FWGE與CDDP 合併處理12小時-------------------------------------59
圖4-16 HaCaT在不同濃度的FWGE與CDDP合併處理24小時--------------------------------------60
圖4-17 HaCaT在不同濃度的FWGE與CDDP合併處理48小時--------------------------------------61
圖4-18用顯微鏡直接觀察HaCaT在不同濃度的FWGE與CDDP合併處理12小時-------------------------62
圖4-19用顯微鏡直接觀察HaCaT在不同濃度的FWGE與CDDP合併處理24小時-------------------------63
圖4-20用顯微鏡直接觀察HaCaT在不同濃度的FWGE與CDDP合併處理48小時-------------------------64
圖4-21使用流式細胞儀測得24小時FWGE與5-FU細胞週期量化之結果------------------------------65
圖4-22使用流式細胞儀測得24小時FWGE與5-FU細胞週期統計之結果------------------------------66
圖4-23使用流式細胞儀測得24小時FWGE與5-FU Sub-G1統計之結果------------------------------67
圖4-24使用流式細胞儀測得12小時FWGE與CDDP細胞週期量化之結果------------------------------68
圖4-25使用流式細胞儀測得24小時FWGE與CDDP細胞週期統計之結果------------------------------69
圖4-26使用流式細胞儀測得12小時FWGE與CDDP Sub-G1統計之結果------------------------------70
圖4-27使用流式細胞儀測得24小時FWGE與CDDP細胞週期量化之結果------------------------------71
圖4-28使用流式細胞儀測得24小時FWGE與CDDP細胞週期統計之結果------------------------------72
圖4-29使用流式細胞儀測得24小時FWGE與CDDP Sub-G1統計之結果------------------------------73
圖4-30使用流式細胞儀測得48小時FWGE與5-FU細胞週期量化之結果------------------------------74
圖4-31使用流式細胞儀測得48小時FWGE與CDDP細胞週期量化之結果------------------------------75
圖4-32使用流式細胞儀測得48小時FWGE與CDDP細胞週期統計之結果------------------------------76
圖4-33西方墨點法來測得FWGE與CDDP24小時蛋白濃度表現之結果--------------------------------77
圖4-34西方墨點法來測得FWGE合併處理CDDP 24小時其蛋白濃度表現定量之結果---------------------79

表目錄
表3-1.小麥胚芽萃取物（FWGE）之配製---------------------------------19
表3-2.黃芩甘（Baicalin）之配製------------------------------------20
表 3-3.順鉑（CDDP）之配製-----------------------------------------20
表 3-4. 5-FU之配製-----------------------------------------------21
表 3-5. UVB照射--------------------------------------------------22
表3-6.不同的培養容器所需的細胞量-----------------------------------25
表3-7. PI（Propidium iodide）染劑之組成---------------------------31
表3-8. 裂解液之組成----------------------------------------------35
表3-9. 蛋白質標準品之配製------------------------------------------36
表3-10. SDS-PAGE下層膠（Separation gel）之配製---------------------37
表3-11. SDS-PAGE上層膠（Stacking gel）之配製-----------------------38
表3-12. Tris-base(4℃)-------------------------------------------38
表3-13. Solution B之配製(4℃) ------------------------------------38
表3-14. Solution C之配製(4℃) ------------------------------------39
表3-15. Running buffer之配製--------------------------------------39
表3-16. Transfer buffer配製---------------------------------------42
表3-17. 10 X TBS-T配製--------------------------------------------42
表3-18. Blocking buffer配製---------------------------------------42

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