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研究生:張雅慧
研究生(外文):Ya-Hui Chang
論文名稱:Solasodine促進腫瘤壞死因子-a�n(TNF-a)所誘導細胞凋亡之研究
論文名稱(外文):ENHANCED TUMOR NECROSIS FACTOR-a INDUCED APOPTOSIS BY SOLASODINE
指導教授:戴達英戴達英引用關係何錦玟
指導教授(外文):Dar-in TaiChin-wen Ho
學位類別:碩士
校院名稱:大同大學
系所名稱:生物工程學系(所)
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:2004
畢業學年度:92
語文別:英文
論文頁數:40
中文關鍵詞:腫瘤壞死因子細胞凋亡
外文關鍵詞:ApoptosissolasodineTNF
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Solasodine存在於台灣本土植物黃水茄,具有抗病毒、抗癌之作用。同類的類固醇生物鹼solamargine能誘導腫瘤壞死因子受體-I (TNFR-I) 之表達,因此能引發細胞凋亡。由於C型肝炎病毒核蛋白可能藉調控TNFR-I 訊息傳導途徑來造成持續性感染,且solasodine比solamargine含量多易取得。因此探討solasodine在腫瘤壞死因子-�� (TNF-��)誘導之細胞凋亡的作用與機制。
以 tetrazolium (MTT) 來測量不同濃度之solasodine 對HepG2 細胞之活存率之影響。然後HepG2肝腫瘤細胞分別或一齊加入特定濃度之TNF-�� 與solasodine,再測量Caspase 3 活性,以定量細胞凋亡狀況。核因子kappaB (NF-�羠) 在抑制性蛋白質kappaB (I�羠) 被磷酸化以及解離後,進入細胞核活化釵h基因:可調控細胞凋亡。以西方墨點法測量抑制性蛋白質kappaB (I�羠) 磷酸化以及被解離過程,再以電泳移動改變測量細胞核內活性NF-�羠之含量,可了解solasodine促進細胞凋亡之作用與TNF-�� 訊息傳遞間的關係。
處理2-15 μg/ml solasodine,均明顯的降低HepG2肝腫瘤細胞存活率。Caspase 3 活性在一齊加入40 ng/ml TNF-�� 與10 μg/ml solasodine後6小時顯著升高,而單獨使用40 ng/ml TNF-�� 要到16小時後才升高。只用solasodine及未加入任何藥物之控制組皆未測出Caspase 3 活性。顯示solasodine 強化經由TNF引起的細胞凋亡。HepG2肝腫瘤細胞在加入 TNF-�� �n���n分鐘後,�n即測量出磷酸化之I�羠。TNF-�� �n�n與solasodine一齊加入並未影響I�羠之磷酸化。而單獨加入solasodine 在80 分時出現微弱的I�羠磷酸化之作用。同時使用TNF-�� 與solasodine在40分鐘出現略微增強的NF-�羠電泳移動改變。
在HepG2肝腫瘤細胞中,一齊加入TNF-�� 與solasodine強化了細胞凋亡,其機制不是阻斷了NF-�羠 的訊息傳導途徑。可能是solasodine破壞細胞膜的完整或solasodine尚有其它弁鄏傢騿C
Solasodine, a native plant product of Taiwan, was found to have anti-viral and anti-cancer effects. Solamargine, a similar steroid alkaloid compound, was reported to enhance tumor necrosis factor receptor-I (TNFR-I) expression and induce apoptosis to tumor cells. Hepatitis C virus core protein may bind to cytoplasmic tail of TNFR-I thus may regulate TNF signal transduction pathway. This action may favor for persistent HCV infection. Solasodine is more abundant than solamargine in plant, so we examined the effect of solasodine in tumor necrosis factor (TNF) induced apoptosis.
HepG2 cells were incubated with different doses of solasodine. Cell viability was measured by colorimetric tetrazolium (MTT) assay. Apoptosis was studied by measuring Caspase 3 activity from cell lysate treated by solasodine with or without combination of TNF-��. I�羠 phosphorylation western blotting and nuclear factor kappaB (NF-�羠) electrophoretic motility shift assay (EMSA) were done to understand the interaction of solasodine with TNF-ωignal transactivation pathway.
Solasodine (2 to 15 μg/ml) significantly decreased HepG2 cell viability in MTT assay. Enhanced apoptosis was found by elevation of Caspase 3 activity six hours after coincubation of solasodine and TNF, whereas incubation with TNF-� alone showed elevation of Caspase 3 activity at 16 hour. Incubation with solasodine or without any treatment in HepG2 cells did not showed elevation of Caspase 3 activity for up to 16 hours. Incubation of HepG2 cells with TNF-�, with or without solasodine, induced IκB phosphorylation at 3 min after treatment. Incubation with solasodine alone in HepG2 cell showed a weak IκB phosphorylation at 80 min after treatment. Coincubation of solasodine and TNF-� slightly increased nuclear NF-κB activity in gel shift assay at 40 min.
The mechanism for solasodine to enhance TNF-�-induced apoptosis is not through blocking of NF-κB signal transduction pathway. Disrupt membrane integrity or other unrecognized function of solasodine may be involved in TNF-�-induced apoptosis.
中文摘要………………………………………………………………………….. i
ABSTRACT………………………………………………………………………. iii
LIST OF TABLE AND FIGURES……………………………………………….. iv
ABBREVIATIONS……………………………………………………………….. v
1. INTRODUCTION……………………………………………...……………… 1
2. MATERIALS AND METHODS………………………………………………. 16
2.1 Cell line and Culture…………………………………………………..……. 16
2.2 Cell proliferation/viability assay…………………………………….…..….. 16
2.3 Assessment of TNF- –induced apoptosis…………………………………... 17
2.4 Semiquantification for TNFR-I…………………………………………….. 18
2.4.1. Extraction of total RNA………………………………………………... 18
2.4.2. Reverse transcription polymerase chain reaction………………..…….. 18
2.4.3. Western blotting for TNFR-I………………………...………...…….…. 19
2.5 Examination of I�羠 phosphorylation……………………………………….. 19
2.5.1. Preparation of cytosolic and nuclear proteins………………………….. 19
2.5.2. Western blotting for non-phosphorylated I�羠 and phosphorylated I�羠.. 20
2.6 Electrophoretic Mobility Shift Assay (EMSA) study………………………. 21

3. RESULTS……………………………………………………………………… 23
3.1 Cell viability assay……………………………………………………...…... 23
3.2 Apoptosis Assay……………………………………………………….……. 24
3.3 Detection of TNFR-I mRNA and protein after incubation with solasodine... �n����
3.4 Western blotting for non-phosphorylated I�羠 and phosphorylated I�羠…… 26
3.5 EMSA study………………………………………………………………… 29
4. DISCUSSION………………………………………………………………….. 30
5. CONCLUSION………………………………………………………………… 34
6. REFERENCES…………………………………………………………...……. 35
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