臺灣博碩士論文加值系統

背景：SGLT1是主要表現於腸道負責主動吸收葡萄糖之糖轉運蛋白。之前我們已經證實人參皂苷Compound K及Rg1可以分別增加或減少人類腸道細胞的葡萄糖吸收。主要是因為Compound K和Rg1增加或減少Sodium-dependent glucose cotransporter (SGLT-1) 的基因表現之故。 此處，我們想探討SGLT-1 基因表現的調控機制。材料與方法：(一)以分化的人類腸細胞Caco-2為研究模式，建構WT和mutant SGLT-1 基因啟動子區報告載體，再轉染 (transient transfection)至Caco-2及HEK293細胞株中，分別觀察Compound K、Rg1以及同時處理signal pathway inhibitors時SGLT-1 gene promoter的活性。(二) 利用[14C]標定的α-methylglucopyranoside (AMG) 測定各種條件下對於糖吸收的影響及SGLT-1動力學常數之變化。(三) 利用西方點墨法及定量PCR觀察各種條件下對於腸道細胞內SGLT-1表現之影響及參與信息路徑。此外，(四) 利用CHIP方法分析並鑑定與CK及Rg1或EGF等因子調節SGLT-1基因表現之轉錄因子。結果：(一) SGLT-1動力學研究顯示：CK或Rg1對Km無影響，但改變反應的Vmax；顯示CK或Rg1導致的增加或減少葡萄糖吸收效應與SGLT-1之表現量有關。(二) 發現Compound K 和Rg1會影響 CREB 因子磷酸化，其磷酸化程度與SGLT-1基因表現活化有密切關係。(三) 在Caco-2及HEK293細胞株中PKC、PI3K、EGF-EGFR等信息路徑與 Compound K增加SGLT-1基因表現有關；PKA信息路徑與Rg1抑制SGLT-1基因表現有關。(四) CHIP分析結果顯示Compound K會增加，而Rg1則會抑制CREB因子與SGLT-1啟動子的結合。(五)發現在已分化的Caco-2中EGF及其受體EGFR之表現可受CK或Rg1調節。EGF可提高SGLT-1 Vmax，促進糖吸收，誘導SGLT-1基因之mRNA及蛋白質表現量，促進CREB因子與SGLT-1啟動子區結合。結論：我們發現人參皂苷會影響腸道細胞的表皮生長因子的表現量，進而影響CREB 因子磷酸化與SGLT-1之表現；經由研究天然物之作用，有助瞭解影響細胞糖吸收及生長之機轉。

In our previous study, the purified ginsenoside Compound K (CK) and Rg1 were found to enhance and inhibit the steady-state glucose uptake, respectively, in differentiated human intestinal Caco-2 cells. The effect was attributed to altered SGLT-1 gene expression in the cells treated with CK and Rg1. In this study, the specific substrate of SGLT-1, [14C]-α-methyl glucopyranoside (AMG), was used to investigate the kinetic parameters of SGLT-1 protein in the presence of CK or Rg1. We showed that the Km value did not change in cells treated by either compound, but the Vmax value markedly increased or decreased in CK- or Rg1-treated cells, respectively. These results demonstrated that the effect of CK and Rg1 on glucose uptake was due to increase or decrease expression level of SGLT-1 gene rather than to alter the substrate binding affinity of SGLT-1 protein. To elucidate the regulatory mechanism involved in SGLT-1 expression, transient transfection was performed using WT and mutated SGLT-1 gene promoters. Specific kinase inhibitors were also used in transient transfection, western and Q-PCR analysis to find out the signaling pathways involved in the action of these compounds. The effect of Rg1 was found to be reversed by PKA inhibitor and adenyl cyclase inhibitor, suggesting the involvement of PKA pathway in Rg1-mediated inhibition of SGLT-1 gene expression. On the other hand, the PKC, PI3K, an EGF receptor kinase signaling pathways were found to be involved in CK-mediated activation of SGLT-1 gene expression in Caco-2 cells. Our results also demonstrated that the effect of compound K and Rg1 on SGLT-1 gene expression appears to be correlated to the extent of CREB (cAMP response element binding factor) phosphorylation. The essential role of CREB in CK- or Rg1-mediated alteration of SGLT-1 gene expression was further demonstrated by CHIP assays. In addition to CK and Rg1, epidermal growth factor (EGF) was also shown to induce glucose uptake and SGLT-1 gene expression in Caco-2 cells in a time and concentration-dependent manner. The EGF-mediated induction was also found to correlate with the extent of phosphorylation and association of CREB to SGLT-1 gene promoter. Furthermore, CK was shown to enhance and Rg1 to decrease the EGF expression levels in these cells; however, both compounds increased the expression level of EGF receptor (EGFR). Taken together, our results demonstrated the essential role of EGF-EGFR pathway and CREB in the regulation of SGLT-1 gene expression in human Caco-2 cells.

目錄 I

表目錄 V

圖目錄 VI

縮寫表 VIII

中文摘要 X

英文摘要 XII

第一章、緒言 1
第二章、材料與方法
一、主要儀器、藥品試劑、及抑制劑 10
二、細胞培養（Cell culture） 13
三、蛋白質濃度測定(Protein assay) 16
四、西方點墨法（Western blotting） 17
五、萃取細胞RNA(RNA extraction) 24
六、半定量反轉錄聚合酶連鎖反應(Semi-quantitative RT-PCR) 26
七、質體構築（Plasmid construction） 27
八、勝任細胞製作(Preparation of Competent Cells) 29
九、轉形作用(Transformation) 30
十、小量質體製備(mini-preparation) 32
十一、大量質體製備(maxi-preparation) 34
十二、細胞轉染( Transfection) 36
十三、樣本收集(Cell collection) 39
十四、相對螢光酶活性分析(Luciferase assay) 40
十五、-gal活性分析 41
十六、-galatosidase染色法 42
十七、染色質免疫沈澱法（ChIP assay） 43
十八、葡萄糖轉運分析（Glucose transport assay） 49
第三章、結果
第一部分：人參皂苷之作用
壹、人參皂苷對人類腸道細胞吸收葡萄糖的動力學之分析
一、Compound K對人類腸道細胞吸收葡萄糖的動力學分析 51
二、Rg1對人類腸道細胞吸收葡萄糖的動力學分析 52
貳、人參皂苷影響腸道細胞SGLT-1基因啟動子的信息路徑
一、構築SGLT-1上游啟動子(-500/+22)之載體 52
二、Compound K對SGLT-1上游啟動子誘導作用的信息路徑 53
三、Rg1對SGLT-1上游啟動子抑制作用的信息路徑 53
参、人參皂苷對人類腸道細胞SGLT-1 mRNA表現之影響
一、Compound K對人類腸道細胞SGLT-1 mRNA的表現分析 54
二、Rg1對人類腸道細胞SGLT-1 mRNA的表現分析 55
肆、人參皂苷調節SGLT-1基因表現之轉錄因子研究分析
一、構築突變的SGLT-1上游啟動子(-500/+22)之載體 56
二、突變HNF-1、CRE保留序列的SGLT-1上游啟動子的作用分
析 56
三、人參皂苷對於突變CRE保留序列的SGLT-1上游啟動子的
作用分析 57
伍、人參皂苷調節CREB影響SGLT-1蛋白質表現之分析
一、Compound K誘導CREB磷酸化增加SGLT-1蛋白質表現之分
析 58
二、Rg1弱化CREB磷酸化減少SGLT-1蛋白質表現之分析 58
陸、染色質免疫沉澱法分析人參皂苷影響SGLT-1基因表現之轉錄因子CREB 59
第二章部分：表皮生長因子之作用
壹、表皮生長因子影響人類腸道細胞SGLT-1基因啟動子的信息路徑研究
一、表皮生長因子對SGLT-1上游啟動子誘導作用的信息路
徑 59
貳、表皮生長因子對人類腸道細胞吸收葡萄糖的動力學分析 60
参、表皮生長因子對人類腸道細胞SGLT-1 基因表現之影響
一、EGF對人類腸道細胞SGLT-1 mRNA的表現分析 61
二、不同濃度下，表皮生長因子對SGLT-1蛋白質表現之分
析 62
三、不同時間下，表皮生長因子對SGLT-1蛋白質表現之分
析 62
四、表皮生長因子對SGLT-1蛋白質表現的機轉分析 62
肆、探討人參皂苷對EGF-EGFR mRNA的表現分析
一、Compound K對EGF及EGFR mRNA的作用分析 63
二、Rg1對EGF及EGFR mRNA的作用分析 63
伍、表皮生長因子調節SGLT-1基因表現之轉錄因子研究分析
一、 表皮生長因子誘導CREB磷酸化增加SGLT-1蛋白質表現之分析 64
二、 染色質免疫沉澱法分析表皮生長因子影響SGLT-1基因表現之轉錄因子CREB 64
第四章、討論
一、 藉由Compound K 探討人類腸道細胞SGLT-1基因表現
機轉 66
二、藉由Rg1探討人類腸道細胞SGLT-1基因表現機轉 68
三、EGF對人類腸道細胞SGLT-1基因表現機轉 68
第五章、結論
第六章、參考文獻 98
表目錄
頁次
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