臺灣博碩士論文加值系統

Actinodaphnine誘導細胞內nitric oxide、reactive oxygen species及降低NF-kB活性導致人類肝癌Mahlavu細胞株計畫性死亡的研究

中文摘要

從台灣山肉桂中所萃取出來的一種生物鹼Actinodaphnine 在一些腫瘤中具有生物毒性，並且會使人類肝癌細胞造成細胞凋亡（apoptosis），但是其機轉至今尚未清楚，我們利用actinodaphnine處理hepatoma Mahlavu 細胞，使其造成細胞凋亡並且探討其機轉。
首先分別對Mahlavu 細胞處理不同濃度的actinodaphnine，發現細胞內的nitric oxiden (NO)以及reactive oxygen species (ROS)會隨著濃度的增加而增加，且actinodaphnine會誘導粒線體膜電位下降與caspase 3/7活化，實驗結果還指出actinodaphnine會降低nuclear factor kB (NF-kB)的活性。此外actinodaphnine所造成Mahlavu cells產生細胞凋亡的反應卻可以被dexsamethasone（NO inhibitor）, N-acetylcysteine (NAC)（antioxidant）, and Boc-Asp(OMe)-fmk（broad caspases inhibitor）所抑制，這些結果推論actinodaphnine會經由NO 或 ROS的增加以及caspases-dependent pathway而誘導細胞凋亡。
總而言之，actinodaphnine處理Mahlavu 細胞之後會因為NO 或 ROS增加而造成粒線體膜電位下降還有caspase被活化，進而使Mahlavu 細胞造成細胞凋亡。

The study of actinodaphnine induces apoptosis through increased nitric oxide, reactive oxygen species and down-regulation of NF-kB signaling in human hepatoma Mahlavu cells

Abstract

Actinodaphnine, extracted from Cinnamomum insularimontanum (Lauraceae), possesses cytotoxicity in some cancers, but the mechanism by which actinodaphnine induces apoptosis in human hepatoma cells remains poorly understood. In this study, we investigated the mechanisms of apoptosis induced by actinodaphnine in human hepatoma Mahlavu cells. Treatment with actinodaphnine dose-dependently induced apoptosis in Mahlavu cells that correlated with increased intracellular nitric oxide (NO) and reactive oxygen species (ROS), disruptive mitochondrial transmembrane potential (DYm), and activation of caspase 3/7. Our data also demonstrated that actinodaphnine down-regulated activity of nuclear factor kB (NF-kB). The apoptotic response to actinodaphnine was markedly decreased in Mahlavu cells pretreated with dexsamethasone, a NO inhibitor, N-acetylcysteine (NAC), an antioxidant, and Boc-Asp(OMe)-fmk, a broad caspases inhibitor. These results suggested that actinodaphnine-induced apoptosis is initially mediated through the NO and/or ROS increase and caspases-dependent pathway. In conclusion, our results indicate that an increase of ROS and/or NO is the initial essential event that results in the decrease of DYm and the activation of caspases that commits the cells to the apoptotic pathway in actinodaphnine-treated hepatoma Mahlavu cells.

目 錄

誌謝 -------------------------------------------------- i
中文摘要 -------------------------------------------------- iv
英文摘要 -------------------------------------------------- vi
目錄 -------------------------------------------------- viii
圖目錄 -------------------------------------------------- x
縮寫表 -------------------------------------------------- xi
第一章 文獻探討------------------------------------------ 1
第一節 前言---------------------------------------------- 1
第二節 實驗目的----------------------------------------- 2
第三節 細胞凋亡（Apoptosis）----------------------------- 3
第四節 ROS（reactive oxygen species） ---------------------- 6
第五節 NO（nitric oxide）--------------------------------- 7
第六節 NO 與apoptosis------------------------------------ 7
第七節 粒線體與apoptosis的關係-------------------------- 8
第八節 p53---------------------------------------------- 9
第九節 NF-kappa B--------------------------------------- 10
第二章 研究方法------------------------------------------ 11
第一節 材料---------------------------------------------- 11
第二節 方法---------------------------------------------- 14
一 細胞培養------------------------------------------ 14
二 細胞週期分析Cell cycle analysis---------------------- 14
三 利用TUNEL assay觀察apoptosis的DNA fragmentation 15
四 細胞內ROS的分析--------------------------------- 16
五 細胞內NO的分析---------------------------------- 16
六 細胞內粒線體膜電位分析DYm----------------------- 17
七 利用flow cytometry分析細胞內caspase 3/7的表現情形 18
八 利用flow cytometry分析細胞內caspase 8, and caspase9的表現情形------------------------------------------ 18
九 利用flow cytometry分析細胞內p53的表現情形-------- 19
十 利用Electrophoretic mobility shift assay (EMSA)觀察細胞內NF-kB的表現------------------------------------ 20
第三章 實驗結果------------------------------------------ 23
第一節 觀察actinodaphnine造成Mahlavu 細胞DNA的損壞程度 23
第二節 利用TUNEL assay觀察actinodaphnine所造成Mahlavu 細胞的細胞凋亡------------------------------------ 24
第三節 探討actinodaphnine造成Mahlavu 細胞內的reactive oxygen species（ROS）之影響------------------------ 25
第四節 探討actinodaphnine造成Mahlavu 細胞內的nitric oxid（NO）之影響。----------------------------------- 26
第五節 觀察actinodaphnine對Mahlavu cells粒線體膜電位的影響 27
第六節 觀察actinodaphnine對Mahlavu細胞caspase 3/7, 8, 9的活性影響-------------------------------------------- 28
第七節 觀察actinodaphnine影響Mahlavu cells中p53的表現------ 29
第八節 Actinodaphnine對Mahlavu細胞NF-kB的活性調節----- 29
第九節 探討actinodaphnine誘導Mahlavu細胞 apoptosis的途徑 30
第四章 討論---------------------------------------------- 32
參考文獻 -------------------------------------------------- 36


圖 目 錄

圖一 Actinodaphnine的化學結構------------------------------- 45
圖二 Actinodaphnine對Mahlavu cell line細胞週期的影響---------------- 46
圖三 觀察Actinodaphnine所造成Mahlavu 細胞的細胞凋亡-------- 47
圖四 評估actinodaphnine處理Mahlavu 細胞之後細胞內ROS的含量----------------------------------------------------- 48
圖五 評估actinodaphnine處理Mahlavu 細胞之後，細胞內NO的含量----------------------------------------------------- 49
圖六 觀察actinodaphnine處理Mahlavu 細胞之後粒線體膜電位的改變----------------------------------------------------- 50
圖七 觀察actinodaphnine處理Mahlavu 細胞之後caspase 3/7, 8, 9的活性表現------------------------------------------------- 51
圖八 觀察actinodaphnine處理Mahlavu 細胞之後p53的活性表現-- 52
圖九 觀察actinodaphnine處理Mahlavu 細胞之後NF-κB的活性表現 53
圖十 探討actinodaphnine誘導Mahlavu細胞 apoptosis的途徑----- 54
圖十一 Actinodaphnine造成apoptosis之機轉圖-------------------- 55

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