TRIB3 involves in Doxorubicin - induced apoptosis in gastric cancer cells

Abstract

TRIB3基因存在於果蠅及哺乳類動物細胞中，目前有研究證實TRIB3參與許多細胞內的調控，包含新陳代謝、發炎反應以及促進腫瘤細胞轉移等。我們運用TRIB3低、中、高表現量的七種胃癌細胞，以化學治療藥物Doxorubicin處理後，探討TRIB3在細胞內扮演的角色。在低表現量的胃癌細胞AGS中，我們超量表現TRIB3，讓細胞大量表現TRIB3後加入Doxorubicin，顯微鏡下觀察其細胞生長情況較為良好，利用MTS分析細胞存活率為61.4%(Vector存活率為51.2%)，流式細胞儀檢測及細胞凋亡訊號結果都顯示細胞凋亡的比例降低。相反的，我們利用表現量較高的胃癌細胞TMK並降低TRIB3的表現，再加入Doxorubicin後顯微鏡下觀察其細胞生長情況較差，MTS分析細胞存率TMK siTRIB3 57%(TMK siControl:74%)，流式細胞儀檢測及細胞凋亡訊號結果也都顯示細胞凋亡的比例增加。由上述結果我們推測TRIB3在胃癌細胞加入抗癌藥物後是扮演著保護細胞的角色，當TRIB3存在的情況下細胞死亡情形降低，相反的TRIB3被抑制的同時細胞死亡的情形也跟著增加，此一相關的機轉與細胞路徑目前仍不清楚，有待日後再探討。TRIB3 gene exists in the cells of drosophila and mammalian. It has proven so far that TRIB3 has taken parts in regulating metabolism and inflammnatory responses in cells, as well as promoting cancer cell migration. In this study, we applied chemotherapy medicine Doxorubicin to seven distinct gastric cancer cells with different levels of TRIB3 to explore the role of TRIB3 in cells responding to Doxorubicin. Overexpression of TRIB3 improved cell survival and lowered apoptosis in gastric cancer cell AGS, which expresses the lower TRIB3 protein as compared to other gastric cancer cell lines, upon Doxorubicin treatment. On the contrary, knock-down of TRIB3 expression in TMK, which expresses the higher TRIB3 compared to AGS, attenuated cell growth and increased apoptosis under Doxorubicin treatment. In conjunction with the results we concluded that TRIB3 protects cells from Doxorubicin-induced apoptosis. Further experiments will be required to study the molecular events in TRIB3 against Doxorubicin-induced apoptosis.目錄 中文摘要…..……………………………………………………………………..…..…..i 英文摘要…..…......………………………………………………………………..….…ii 壹、緒論………………………………………………………………………..…..…...1 1. TRIB3………………………..…………….……..….…………….……........….1 2. 阿黴素(Doxorubicin)……………………………....………………….…...........1 3. 細胞凋亡(Apoptosis)………………………….……….………….….……..….....2 4. 研究目的………………………....………...…………………………………....4 貳、實驗材料與方法………………………………………………………..…..….….5 1. 細胞培養及繼代培養(Cell culture and subculture)...……..………..…..….…...5 2. 細胞凍存與解凍(Cell cryopreservation and recovery)..………………..…....…5 3. 細胞存活率分析(Cell proliferation assay)…………………………............…...6 4. 細胞凋亡測定(Detection of cell apoptosis)…………………………….………6 5. 活性氧化物分析(Detection of intracellular reactive oxygen species concentration)………………………………..………………………………...7 6. 蛋白質定量(Quantitative measurement of protein)…………………….....7 7. 西方墨點轉漬法(Western blot analysis)…………………….….....…7 8. RNA萃取(RNA extraction)…………………………………………….....8 9. 逆轉錄聚合酶連鎖反應(Reverse transcription-polymerase chain reaction)……………………………………………………………..………....9 10. 細胞轉染(Transfection)………………………………………………………10 11. RNA interference……………………………………………………………..…10 12. 抗體(Antibodies)………………………………………………………..10 叁、實驗結果…………………………………………………………………….…..12 1. 探討TRIB3 protein在胃癌細胞中的表現量..….……………………..…..…..12 2. 探討TRIB3是否參與Doxorubicin藥物反應機轉…..…………………….…12 3. 探討TRIB3對於細胞加入Doxorubicin後扮演的角色…………………..….13 4. 探討TRIB3對於細胞加入Doxorubicin後死亡的形式……………………...15 肆、討論……………..………………………………...…………………………….18 伍、參考文獻…………………………………………….………………………….20 陸、結果圖表………………………………………………………………………..2