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研究生:余主文
研究生(外文):Ju Wen Yu
論文名稱:小蘗鹼、甘草素和棉酚對人類淋巴癌細胞Jurkat細胞株MAPK, Sirt1, Sirt2和環氧合酶2表現之影響
論文名稱(外文):Effects of Berberine, Glycyrrhizin and Gossypol on the Expression of MAPK, Sirt1, Sirt2, and Cyclooxygenase-2 in Jurkat Cells
指導教授:王錫五王錫五引用關係
指導教授(外文):S. W. Wang
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
論文頁數:111
中文關鍵詞:甘草素小蘗鹼棉酚
外文關鍵詞:GlycyrrhizinBerberineGossypol
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甘草素和黃連素為已使用2500年之中國傳統中草藥。研究指出,甘草素、黃連素和棉酚具有抗癌,鎮痛,消炎,退熱藥,降血脂,抗腫瘤及血管舒張的作用。此外,這些藥劑會抑制細菌感染,從體外細菌與核DNA相互作用破壞利什曼原蟲的多醣體,調節免疫系統,及葡萄醣代謝。環氧合酶(COX)1、2和前列腺素PGE2已被證明存在於白血球細胞中,且為NF-κB訊息路徑的下游產物。從我們實驗室以往的研究指出,這些中草藥透過降低MAPK的磷酸化抑制IL-2分泌。然而,這些藥物在Jurkat細胞中是否影響MAPK和COX2的表現仍未完全明瞭。許多研究指出Sirtuin與細胞的細胞週期、基因體穩定、能量代謝、抗發炎和抗老化有關。Regucalcin會促進發炎因子產生和調節能量代謝。我們假設Glycyrrhizin、Berberine和Gossypol可能透過MAPK、Sirtuin、COX2和Regucalcin的表現和細胞週期的調控影響Jurkat細胞分泌IL-2。研究結果顯示:(1) 加入甘草素和PHA後,Jurkat細胞中磷酸化的ERK1/2(p-ERK1/2)、p-p38、Sirt1及regucalcin的表現會增加;(2) 加入黃連素與PHA會使Jurkat細胞中的p-JNK、COX2、Sirt2和regucalcin表現增加;(3) 加入棉酚與PHA會使Jurkat細胞中的Sirt2表現增加,但沒有顯著性的變化;(4) 甘草素(0.1及0.5 μM)促使細胞停滯在G0/G1;黃連素於20 μg時會使細胞停滯於G2/M;加入棉酚(10-6M)及PHA(1 μg)時會促使細胞停滯於G0/G1。綜合以上結果顯示這些藥物抑制Jurkat細胞分泌IL-2的原因如下:(1) 甘草素經由Sirt1的增加,抑制MAPK的表現進而抑制PHA引起Jurkat細胞分泌IL-2;(2) 小蘗鹼透過減少磷酸化的JNK、COX2、Sirt2及regucalin的表現抑制PHA誘導Jurkat細胞分泌IL-2;(3) 棉酚經由Sirt2的增加降低PHA在Jurkat細胞中所引起的IL-2分泌作用。
Glycyrrhizin and berberine have been used as Chinese traditional herb medicine for 2500 years. Studies indicated that glycyrrhizin, berberine and gossypol possess as anticancer, analgesic, anti-inflammatory, febrifuge, hypolipidemic, anti-cancer, and vasodilation effect. In addition, these materials inhibit bacterial infection, destroy the Leishmania donovani by interaction in vitro with nuclear DNA from this bacterial, regulate immune system, and glucose metabolism. Cyclooxygenase (COX) 1, 2 and prostaglandin (PG)-E2 have been demonstrated to be existed in leukocytes, and they were the downstream products of the NF-κB signaling pathway. Previous studies from our laboratory showed these herbs inhibit the IL-2 secretion from Jurkat cells in part through the attenuated MAPK phosphorylation. However, it is still unknown the mechanism of these herbs on decreased MAPK and COX2 expression from Jurkat cells. Studies indicate that sirtuins have more relavant with cell cycle, stable of the genomic, energy metabolism, anti-inflammatory and anti-aging. Regucalcin can be pro-inflammatory factor and regulates the energy metabolism. Our hypothesis is that glycyrrhizin, berberine and gossypol may affect the IL-2 secretion from Jurkat cells through the expression of COX2, MAPK, sirtuins, regucalcin, and the cell cycle regulation. Our results showed that: (1) increased expression of phosphorylated ERK1/2 (p-ERK1/2), p-p38, SIRT1, and regucalcin proteins in Jurkat cells treated with glycyrrhizin and PHA were found; (2) berberine inhibited the expression of p-JNK, COX2, SIRT2, and regucalcin proteins in Jurkat cells treated with PHA; (3) gossypol increased SIRT2 expression, not significant, in Jurkat cells treated with PHA; (4) glycyrrhizin (0.1 and 0.5 M) promotes G0/G1 arrest; 20 g/ml berberine results in G2/M arrest; G0/G1 arrest was found in Jurkat cells treated with gossypol (10-6 M) and PHA (1 g). Taken together, these results indicated that: (1) the inhibition of PHA-induced IL-2 secretion from Jurkat cells by glycyrrhizin is in part via an inhibited expression of MAPK through the increased SIRT1; (2) berberine inhibited the PHA-induced IL-2 secretion through the attenuated expression of p-JNK, SIRT1, and regucalcin; (3) decreased secretion of PHA-induced IL-2 from Jurkat cells treated with gossypol is through the increased SIRT2.
指導教授推薦書
口試委員審定書
誌謝 III
中文摘要 IV
Abstract VI
目錄 VII
圖表目錄 X
第一章、前言 1
1.1、小蘗鹼(Berberine) 1
1.1.1小蘗鹼的研究背景 1
1.1.2小蘗鹼的藥理作用: 3
第二節、棉酚 (Gossypol) 7
1.2.1棉酚的研究背景 7
1.2.2棉酚的藥理作用 9
第三節、甘草素(Glycyrrhizin) 12
1.3.1甘草素的研究背景 12
1.3.2甘草素的藥理作用: 14
第四節、PHA及相關蛋白質簡介 17
1.4.1 致裂殖原植物凝集素Phytohemagglutinin(PHA) 17
1.4.2 MAPK 17
1.4.3 Sirtuin 18
1.4.4 Regucalcin(RGN) 21
第五節、研究動機 24
第六節、實驗假說 25
第七節、研究目標: 26
第二章、實驗材料與方法 28
第一節、實驗材料 28
第二節、實驗方法: 29
2.1 Jurkat cell的培養製備 29
2.2 Western blot 29
2.3 RT-PCR(reverse transcription polymerase-chain reaction) 31
2.4 IL-2之(Enzyme-Linked Immunosorbent Assay(ELISA)測定 35
2.5 Flow cytometry 36
2.6 統計分析 36
第三章、實驗結果 37
PHA對Jurkat細胞MAP kinase之影響 37
PHA對Jurkat細胞中的蛋白質COX2、IL-2及Sirt1/2之影響 37
加入Glycyrrhizin後對於Jurkat細胞受到PHA刺激之影響 38
加入Berberine後對於Jurkat細胞受到PHA刺激之影響 39
加入Gossypol後對於Jurkat細胞受到PHA刺激之影響 40
PHA對Jurkat細胞之COX2 mRNA的影響 40
第四章、實驗討論 42
總結: 47
參考文獻(References) 50
圖表與附錄 59
附錄一、Western blot電泳膠片與溶液之配方 93
附錄二、實驗儀器 95

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