Alpha Camp PTT的問題，透過圖書和論文來找解法和答案更準確安心。 我們找到下列懶人包和總整理

轉錄因子HBP1調節糖質新生作用

為了解決Alpha Camp PTT的問題，作者張晉銘 這樣論述：

目錄謝誌 ··························································································· 1中文摘要 ····················································································· 2英文摘要 ····················································································· 4目錄 ··············

············································································· 6圖目錄 ······················································································· 11第一章、前言 ·············································································· 14第二章、文獻探討 ······························

·········································· 16一、糖質新生 (Gluconeogenesis) ···················································· 16I. 糖質新生與血糖恆定 ····························································· 16II. 糖質新生作用 ····································································· 17III. 肝臟、腎臟與糖質新生

························································ 19二、激素調控糖質新生訊息傳遞 ···················································· 21I. 簡介 ·················································································· 21II. 胰島素 / PI3K / AKT / FOXO1 ················································ 2

1III. 升糖素 ············································································· 23IV. 糖皮質激素 ······································································· 25三、糖質新生與糖尿病 ································································ 27四、轉錄因子 HBP1 (HMG box-containing protein 1) ·····

······················ 28I. 簡介 ·················································································· 28II. 生理功能 ··········································································· 29第三章、研究動機與目的 ······························································· 31一、實驗動機 ············

································································ 31二、實驗目的與設計 ··································································· 33I. 確認在不同代謝生理狀況下，HBP1 與糖質新生關鍵酵素 PEPCK G6Pase 的表達一起受到調節。 ·················································· 33II. 確認 HBP1 調控 PEPCK 及 G6Pase 基因表達 ··········

··············· 34III. 確認 HBP1 調控糖質新生刺激因子所誘導的 PEPCK 及 G6Pase 表達 ························································································ 35IV. HBP1 調節葡萄糖產出 ························································· 36V. 以 HBP1 基因剔除小鼠模式驗證 ··········································· 37第

四章、材料與方法 ····································································· 38一、實驗藥品與儀器 ··································································· 38I. 實驗藥品 ············································································ 38II. 實驗儀器 ···········································

································ 40二、細胞培養 (Cell culture) ·························································· 41I. 細胞株與細胞培養條件 ·························································· 41II. 試劑配置 ··········································································· 41III. 繼代培養方法 ········

···························································· 42IV. 細胞凍存 ·········································································· 42V. 細胞計數 ··········································································· 43三、轉染 (Transfection) ·············································

·················· 44I. 原理 ·················································································· 44II. 質體 (Plasmid) ··································································· 44III. 實驗步驟 ·········································································· 44四、RNA干擾術 (RNA

interference; RNAi) ······································ 45I. 原理 ·················································································· 45II. 使用的siRNA ····································································· 45III. 實驗步驟 ·····················································

····················· 45五、蛋白質萃取 ········································································· 46I. 原理 ·················································································· 46II. 試劑配置 ··········································································· 46III. 實驗步驟 ····

······································································ 46六、蛋白質定量 ········································································· 47I. 原理 ·················································································· 47II. 標準曲線(Standard curve)製備··························

······················· 47III. 實驗步驟 ·········································································· 47七、西方墨點法 (Western blotting) ················································· 48I. 原理 ·················································································· 48II. 試劑配製 ········

··································································· 48III. 實驗步驟 ·········································································· 52八、RNA萃取 ··········································································· 53I. 原理 ···················································

······························· 53II. 試劑配製 ··········································································· 53III. 實驗步驟 ·········································································· 54九、Reverse Transcription-Real-Time Polymerase Chain Reaction (RT qPCR) ······················

········································································ 55I. 原理 ·················································································· 55II. 試劑配製 ··········································································· 55III. 實驗步驟 ·····································

····································· 56十、葡萄糖產出 ········································································· 58I. 原理 ·················································································· 58II. 試劑配製 ···········································································

58III. 實驗步驟 ·········································································· 58十一、HBP1基因剃除小鼠 (HBP1 knockout mice model) ····················· 59I. 來源 ·················································································· 59II. 原理 ················································

································· 59III. HBP1 KO小鼠繁殖 ····························································· 61IV. 動物飼養 ·········································································· 61V. 動物犧牲與樣品收集 ···························································· 61VI. 丙酮酸耐受測試 Pyruvate

Tolerance Test (PTT) ·························· 62十二、細胞免疫染 ······································································ 63十三、統計分析 ········································································· 64第五章、結果 ·············································································

· 65一、不同代謝生理狀況下，轉錄因子 HBP1 與糖質新生關鍵酵素 PEPCK及 G6Pase 的表達。 ·································································· 65二、HBP1 表現量影響 PEPCK 及 G6Pase 基因的表達 ····················· 66三、HBP1 調控糖質新生刺激因子誘導的 PEPCK 及 G6Pase 表達 ······ 68四、HBP1 調節葡萄糖產出 ·······················································

··· 69五、以 HBP1 基因剔除小鼠模式驗證 HBP1/PEPCK/G6Pase 的關係 ····· 70第六章、討論 ·············································································· 91一、人類肝癌細胞株 HepG2 及人胚胎腎臟上皮細胞株 HEK-293T 分別作為研究肝臟與腎臟糖質新生的細胞模式: ······································· 91二、轉錄因子 HBP1 在糖質新生中扮演的角色: ······························

· 92三、HBP1 基因敲除小鼠模式於葡萄糖產出以及糖尿病治療的應用： ···· 93七、結論 ···················································································· 94參考文獻 ···················································································· 95圖目錄圖2-1. 胰島素和升糖素相互調節恆定血糖 ...............................................

........ 16圖2-2. 糖質新生調節的主要酵素和代謝物。 ................................................... 18圖2-3. 禁食狀態後葡萄糖釋放到循環中的機制 ............................................... 20圖2-4. FOXO1與PGC-1α在胰島素、升糖素及糖皮質激素調節的糖質新生角色 ................................................................................................

................ 21圖2-5. FOXO1核質轉移影響糖質新生 .............................................................. 22圖2-6. CREB對PGC-1啟動子的轉錄調控 ....................................................... 24圖2-7. 升糖素透過GCGR增加糖質新生的基因的表達的途徑 ...................... 24圖2-8. GC通過GC/GR/KLF9/PGC1α訊息傳遞路徑促進肝臟糖質新生 ....... 26圖2-9

. HBP1之結構 ............................................................................................. 28圖3-1. 研究設計 ................................................................................................... 32圖3-2. 實驗設計I .....................................................................

............................ 33圖3-3. 實驗設計II ............................................................................................... 34圖3-4.實驗設計III ................................................................................................ 35圖3-5. 實驗設計IV .................................

............................................................. 36圖3-6. 實驗設計V ............................................................................................... 37圖4-1. 利用CRISPR/Cas 9技術於HBP1基因的exon 2插入early stop codon .......................................................................

................................................. 60圖4-2. 利用PCR配合BclI限制酶鑑定HBP1 KO小鼠 .................................. 60圖5-1. HepG2 及 HEK-293T 細胞中糖質新生關鍵酵素表現。 .................... 72圖5-2. 不同血清與胰島素濃度對 HBP1 及糖質新生關鍵酵素表現之影響。 ...............................................................................

......................................... 73圖5-3. 不同血清與葡萄糖濃度對 HBP1 及糖質新生關鍵酵素表現之影響。 ........................................................................................................................ 74圖5-4. 減低 HBP1表現對肝臟及腎臟細胞中糖質新生相關酵素的蛋白質表現影響。 ....................................................

.................................................... 75圖5-5. 過度表現 HBP1 對肝臟及腎臟細胞中糖質新生相關酵素的蛋白質表現影響。 ........................................................................................................ 76圖5-6. 減低 HBP1 表現對肝臟細胞中糖質新生相關酵素的 mRNA 表現影響。 ....................................................

............................................................ 77圖5-7. 減低 HBP1 表現對腎臟細胞中糖質新生相關酵素的 mRNA 表現影響。 ................................................................................................................ 78圖5-8. 過度表現 HBP1 對肝臟細胞中糖質新生相關酵素的 mRNA 表現影響。 ....................................

............................................................................ 79圖5-9. 過度表現 HBP1 對腎臟細胞中糖質新生相關酵素的 mRNA 表現影響。 ................................................................................................................ 80圖5-10. HBP1對 PEPCK 的調節不一定需要透過 DNA binding。 ............... 81圖5

-11. Glucagon 誘導肝臟及腎臟細胞中 HBP1、PEPCK 及 G6Pase之表現。 ................................................................................................................ 82圖5-12. 糖質新生刺激因子 cAMP 及 DEX 對 HBP1、PEPCK 及 G6Pase之表現影響。 .................................................................................

............... 83圖5-13. 糖質新生刺激因子 cAMP/DEX 誘導下相關酵素的mRNA表現。 84圖5-14. HBP1 對糖質新生刺激因子誘導的相關酵素表現之影響。 .............. 85圖5-15. HBP1 調節肝臟及腎臟細胞的葡萄糖生成。 ...................................... 86圖5-16. 不同代謝情況對小鼠肝臟 HBP1 及糖質新生相關酵素的表現影響。 .............................................................................

................................... 8713圖5-17. HBP1 基因剔除對肝臟糖質新生相關酵素的表現影響。 .................. 88圖5-18. HBP1 基因剔除對禁食後血糖值的影響。 .......................................... 89圖5-19. HBP1 基因剔除對丙酮酸耐受試驗的影響。 ...................................... 90

植物成份curcumin及alisolBacetate對細胞生長與凋亡之作用暨可能之抗動脈硬化機轉

為了解決Alpha Camp PTT的問題，作者陳惠文 這樣論述：

本篇論文主要在體外以大鼠血管平滑肌細胞(A7r5 cells)和人類淋巴癌細胞(CEM lymphocytes)，研究薑黃的主成分─curcumin (diferuloylmethane)，和澤瀉主成份─alisol B acetate，具有抗細胞增生(antiproliferation)與導引細胞凋亡(apoptosis)作用，並探討其分子作用機轉。並進一步以高脂血兔子為動脈粥狀硬化的動物模式，來研究curcumin在活體動物對動脈粥狀硬化病變的藥理作用。此外，我們以離體的兔子血管環來測定curcumin清除自由基的能力；並且在大鼠心肌細胞(H9c2 cells)觀察curcumin和fu

llerenols對過氧化氫(H2O2)導引細胞凋亡的保護作用。 我們以細胞的[3H]-thymidine攝入量來測定細胞中DNA生成量，並以此表示細胞的增生。薑黃素(curcumin)及其它相關之化學物質 (esculetin，baicalein，ferulic acid，和epigallocatechin)在10-6至10-4 M濃度範圍對於胎牛血清引起的血管平滑肌細胞(包括:大鼠血管平滑肌細胞株A7r5 cell line和培養的兔子血管平滑肌細胞)增生皆具有濃度相依性(concentration-dependent)的抑制作用。其中以curcumin抑制作用最大，而

ferulic acid的作用最小。我們利用trypan blue dye exclusion method來測定細胞存活率(cell viability)，10-6至10-5 M濃度之curcumin對於A7r5細胞的存活率沒有影響。而10-4 M curcumin使細胞存活率有意義的降低到少於基準值(2105 cells)。 為了更進一步探討藥物對細胞週期的影響，我們先以細胞的[3H]-thymidine攝入量來分析細胞週期中的不同時期的進行過程，以每三小時測一次細胞的[3H]-thymidine攝入量來觀察細胞週期(cell cycle)的進行。A7r5 cells

在靜止生長48小時後，以10 %胎牛血清刺激後9到12小時DNA生成開始明顯增加，此階段稱為 G1/S phase，是細胞由靜止階段進入細胞分裂的一個重要分界點﹔在15到18小時達到一個高原期，此階段稱為 S phase，而兔子的血管平滑肌細胞在12到15小時達到G1/S phase﹔在21到24小時達到S phase。Curcumin及其它相關化學物質(esculetin，baicalein，ferulic acid，和epigallocatechin)在10-6至10-4 M濃度範圍皆能濃度相依性的在G1/S phase和S phase抑制細胞之[3H]-thymidine攝入量。我們更進

一步利用流式細胞儀(flow cytometry)，以propidium iodide (PI)來染細胞核內的DNA，以確認curcumin及其它相關化學物質(esculetin，baicalein，ferulic acid，和epigallocatechin)對細胞週期中G0/G1，S，G2/M phases分布情形之影響。結果顯示curcumin確實能使靜止生長的血管平滑肌細胞繼續停留在G0/G1 phase，並減少細胞在S phase的百分比。流式細胞儀的分析發現，curcumin 在10-4 M的濃度下會導引細胞凋亡。我們的結果顯示，curcumin會使細胞停止生長並導引細胞凋亡而減少

細胞對[3H]-thymidine攝入量。再以蘇木紫-依紅(hematoxylin-eosin)染色法、細胞凋亡分析法(TdT-mediated dUTP nick end labeling assay；TUNEL) 、及DNA電泳來分析斷裂DNA片段所形成之DNA階梯(DNA laddering) …等方法，進一步證實了10-4 M curcumin會造成細胞萎縮、染色質濃染、DNA斷裂…等細胞進行凋亡時典型的現象。證明curcumin會導引血管平滑肌細胞凋亡(apoptosis; programmed cell death)。 研究curcumin抑制細胞增生及導引細胞

凋亡的可能作用機轉，curcumin在10-6至10-4 M濃度範圍能濃度相依性的抑制細胞膜上蛋白質酪氨酸激酉每 (protein tyrosine kinase)的活性，在西方墨點分析法中亦證明curcumin在10-6至10-4 M濃度範圍可濃度相依性的抑制蛋白質酪氨酸激酉每 受質(大約180 kDa)的磷酸化。另外，只有在10-4 M curcumin才會明顯的抑制蛋白質激酉每 C (protein kinase C)的活性，在西方墨點分析法中，curcumin在10-6至10-4 M濃度範圍可以濃度相依性的抑制protein kinas C的異構酉每 (PKC-)的translo

cation，而對於PKC-及PKC-的translocation則只有在10-4 M curcumin才會有明顯的抑制作用。利用反轉錄-聚合酉每 連鎖反應(RT-PCR)我們發現curcumin會影響一些與細胞凋亡相關的基因表現，其中，curcumin可抑制c-myc和bcl-2mRNA表現，而對p53mRNA表現則無明顯之影響。我們也用北方墨點法來確定此作用。我們的結果指出，curcumin可能經由抑制一些protein tyrosine kinase之訊息傳遞途徑，或 c-myc基因表現來停止細胞週期的進行，以抑制細胞增生；而經由抑制與protein tyrosine kinase，

protein kinase C之訊息傳遞途徑，或c-myc，及bcl-2之基因表現，來導引細胞凋亡。 我們也利用hyperlipidemic cholesterol-fed rabbit及hyperlipidemic cholesterol-fed alloxan-diabetic rabbit兩種高脂血動物模式，來研究curcumin在活體動物之作用，包括：血中總膽固醇(total cholesterol)、總三酸甘油酯(total triglyceride)、血液凝固、及血管心臟組織中platelet-derived growth factor-A (PDGF-A)

和transforming growth factor-1 (TGF-1)基因表現的變化，進而評估curcumin對活體動脈粥狀硬化病變之影響。我們將動物分為五組：第一組, 正常組(normal)：餵食正常兔用標準飼料。第二組, 餵食高膽固醇飼料組(CHOL)：餵食含1 % cholesterol及20 % corn oil的飼料。第三組, 餵食高膽固醇糖尿病組(CHOL+DM)：餵食含1 % cholesterol及20 % corn oil的飼料之外，加上alloxan (60 mg/kg i.v.)引起糖尿病。第四組, Curcumin治療之餵食高膽固醇飼料組(curcumin tr

eated；CHOL+curcumin)：CHOL的兔子連續施打curcumin (5 mg/kg/day, i.m.)五星期。第五組, Curcumin治療之餵食高膽固醇糖尿病組(curcumin treated；CHOL+DM+curcumin)：CHOL+DM的兔子連續施打curcumin (5 mg/kg/day, i.m.)五星期。初步實驗結果顯示，血中總膽固醇及總三酸甘油酯，CHOL組及CHOL+DM組均可使血中總膽固醇及總三酸甘油酯濃度上升，CHOL+DM組在第三週之後更為明顯，甚至total cholesterol可達到1,377.7±354 mg/dl，而total trig

lyceride也會高到1,633±246 mg/dl。而在CHOL+curcumin組中，curcumin對total triglyceride有明顯的降低作用，但對total cholesterol則影響並不明顯。而在CHOL+DM+curcumin組可見到明顯的降低total cholesterol及total triglyceride。在血液凝固測定方面，凝血酵素原時間(prothrombin time；PT)及部分血栓時間(partial thromboplastin time；PTT)均無顯著影響。利用光學顯微鏡觀察不同位置的血管心臟形態變化，發現正常組的兔子在血管方面不論主動脈弓

、胸主動脈或腹主動脈，血管管腔皆平滑，內皮細胞排列規則。心臟方面，左心室沒有冠狀動脈硬化及心肌細胞肥厚現象。CHOL+DM 組的兔子血管方面其主動脈弓、胸主動脈明顯的有動脈硬化斑(atherosclerosis plaques)，血管內膜有許多泡沫細胞堆積，且可見平滑肌細胞增生的現象，腹主動脈則病變部位較少。心臟方面並沒有明顯的病變發生。Curcumin處理過的兔子(CHOL+DM+curcumin組)會減少在主動脈弓、胸主動脈及腹主動脈所發生的動脈硬化斑、泡沫細胞及平滑肌細胞的增生，而心臟方面並沒有明顯影響。以反轉錄-聚合酉每 連鎖反應(RT-PCR)方法分析PDGF-A和TGF- mR

NA表現，CHOL+DM組在主動脈弓及胸主動脈增加PDGF-A mRNA的表現，在腹主動脈則增加TGF-β1 mRNA表現，左心室則有增加的傾向，但不具統計意義。而CHOL+DM+curcumin組可明顯的降低主動脈弓、胸主動脈PDGF-A mRNA的表現，及降低腹主動脈、左心室TGF-β1 mRNA的表現。 此外，在分離兔子胸部主動脈的體外實驗中，我們也發現curcumin能濃度相依性的抑制alloxan所引起superoxide anion的產生，這個結果顯示，curcumin在血管具有清除自由基(free radical scavenger)的性質。

綜合以上結果顯示，curcumin對高脂血兔子的作用，不但能降低高脂血兔子血中總膽固醇、總三酸甘油酯濃度。更能改善高脂血兔子主動脈弓、胸主動脈之動脈硬化形態上之病變。並降低CHOL+DM之兔子主動脈弓、胸主動脈PDGF-A之基因表現，以及降低腹主動脈、左心室TGF-β1之基因表現。此外，curcumin在血管中具有free radical scavenger的活性。Curcumin這些保護動脈硬化病變作用可能經由其降低血脂、free-radical scavenger之特性、及抑制血管平滑肌細胞增生而來。因此，薑黃素(curcumin) 可作為發展新一類藥物之模式(template)，可能

應用於預防與研究動脈粥狀硬化(atherosclerosis)，及血管成形術再阻塞(post-angioplasty restenosis)的血管病變。 減少心肌梗塞造成的缺血區域(myocardial ischemic area)的最有效的方法是再灌流(reperfusion) ，但是再灌流會使得自由基增加(包括, superoxide和hydroxyl radicals)，而造成組織的傷害(包括, necrosis和apoptosis)。在兔子的心臟缺血/再灌流動物模式中，我們發現由缺血/再灌流區域的心肌細胞所抽取的DNA，會在水平電泳膠上呈現出大約180 bp的DNA

階梯片段，這結果顯示此區域心肌細胞受到缺血/再灌流的傷害，發生細胞凋亡。在實驗室中用過氧化氫(hydrogen peroxide, H2O2)當做產生自由基的來源，我們發現過氧化氫在培養的大鼠心肌細胞(rat cardiac myoblast cell line, H9c2 cells)會導引細胞凋亡，而這作用會因為加入抗氧化劑，curcumin和芙醇fullerenols而減輕。Curcumin (10-5 M)可能除了以其free radicals scavenger的特性直接減少過氧化氫對心肌細胞的傷害外，也可能經由抑制細胞凋亡相關之訊息傳遞或基因之表現，而減少細胞凋亡的產生。

最近，我們發現中藥材澤瀉(Alisma rhizoma)的主成分之一alisol B acetate (alisol B-23-monoacetate) ，具有抑制細胞增生，和導引細胞凋亡的作用。因此，我們使用大鼠的血管平滑肌細胞(A7r5 cells)和人類淋巴癌細胞(CEM cells)來研究alisol B對於細胞增生或凋亡現象的作用。Alisol B在10-7至10-4 M濃度範圍會隨時間增加(24-48小時)而濃度相依性的抑制細胞受血清刺激所增加之[3H]-thymidine攝入量。經過48小時藥物處理之後，alisol B在A7r5細胞的IC50是(4.0±0.8)×1

0-6 M；而在CEM細胞的IC50是(2.5±1.2)×10-6 M。由細胞存活率的分析和細胞外型的觀察可見alisol B在10-4 M的濃度下會造成細胞死亡。利用流式細胞儀(flow cytometry)、抽取細胞DNA跑電泳、及TUNEL的分析，我們發現在加入10-4 M alisol B，4小時後便可觀察到細胞凋亡(apoptosis)現象的發生，並隨著時間的增加而更趨明顯，經過48小時後，從顯微鏡可觀察到細胞萎縮(cell shrinkage)、染色質濃染(chromatin condensation)、DNA斷裂(DNA fragmentation) 、和凋亡小體(apoptot

ic bodies)的產生、並且在DNA電泳上形成DNA階梯(DNA ladder)。但是在10-5 M 濃度(48 h)時作用則不太明顯，而小於10-5 M濃度的alisol B即使經過48小時仍不會引起細胞凋亡。由於alisol B與glucocorticoid的化學結構相似，因此我們比較alisol B 與dexamethasone (合成的glucocorticoid)導引細胞凋亡的作用和其機轉。由流式細胞儀的分析結果發現，細胞經過48小時dexamethasone (3×10-7 M)的處理會導引17.3±5.1 %的A7r5細胞凋亡，25.3±2.2 % 的CEM細胞凋亡。研究al

isol B誘導細胞凋亡的分子機轉，我們使用RT-PCR法來測定alisol B或dexamethasone對mRNA基因表現的影響。結果顯示，alisol B (10-5 M, 24 h)會造成c-myc的表現增加，dexamethasone則否；而對p53的表現則也只有alisol B增加其mRNA表現。由實驗結果得知，alisol B對於細胞增生的的抑制作用主要來自導引細胞凋亡的產生，而alisol B對於c-myc和p53基因表現的調節，可能在導引細胞凋亡過程中扮演部分的角色。但與alisol B結構相似的glucocorticoids類化合物的作用機轉，則其所影響的訊息傳遞路徑不同。

根據實驗結果，alisol B可用於發展藥物之模板(template)，來預防或治療因血管平滑肌細胞過度增生所引起知心血管疾病(atherosclerosis、restenosis…等)、也可能用於抑制癌症、抑制免疫發炎細胞、或用於研究細胞凋亡。