臺灣博碩士論文加值系統

異三元體鳥糞嘌呤核酸結合蛋白(Heterotrimeric guanine nucleotide binding proteins, G proein)，位於細胞雙層膜內部，負責接收膜上受體(receptor)活化之訊號，並活化作用體(effector)，將訊號傳遞(signal transduction)至下游，而G蛋白為訊號傳遞路徑之樞紐，於細胞生長、分化(differentiation)及發育(development)上極具重要性。而G蛋白是由α、β和γ次單元(subunit)組合而成，目前共發現27種α次單元、5種β次單元以及14種γ次單元，G蛋白α次單元具有GTPase活性，能夠與GTP結合並釋放GDP，共可分為四大家族：Gs、Gi/o、Gq/11和G12/13。本研究選用人類慢性骨髓性白血病細胞株(chronic myeloid leukemia cell line, CML)K562細胞作為細胞模式，利用黃耆(Huangqi)、Hemin及HMBA誘導細胞進行分化，以合成β-球蛋白(globin)或γ-球蛋白作為分析指標。利用退化性引子擴增G蛋白，以觀察誘導過程中不同G蛋白之表現變化，從中共選殖到四種基因為GNAS isoform(GNASL和GNASS)、Gαi2、G11 pseudo gene。於誘導後表現不盡相同，共歸納出三點：(1)經黃耆誘導後可增加GNASL並同時抑制GNASS之表現量，Hemin和HMBA則皆無特別明顯之變化。(2)Gαi2於誘導後皆有較明顯之表現。(3)本研究所選殖之G11 pseudo gene，經由限制酵素XhoI剪切後，發現主要表現於經黃耆誘導後之K562細胞當中。以上不同之G蛋白其訊號傳遞調控機制尚未明瞭，須更進一步探討，尤其中藥黃耆對於血液疾病之治療具有極大助益，若能完全了解黃耆誘導分化機制，或許患者可藉此得到治癒或減緩病情之機會。

Heterotrimeric G proteins have been demonstrated to play integral role in the transduction of extracellular signals from cell membrane receptor to intracellular effectors proteins. G proteins regulate critical processes such as cell growth, differentiation and development. G proteins compose of α、β and γ subunits. To date, 27 different Gα, 5 Gβ and 14 Gγ subunits have been described. The α subunit have GTPase activities, hydrolyze GTP, return to the GDP-bound state, and reassociate with βγ-complex to form inactive heterotrimers. Gα subunits have been divided into four families (Gs、Gi / o、Gq / 11 and G12 / 13) based on homology at the amino acid level and function. K562 cells are human chronic myeloid leukemia cell line (CML). When K562 cells exposed to Huangqi, Hemin and HMBA, it markedly increase the β-globin or γ-globin expression and cell differentiation along the erythoid pathway. Studied expression of G proteins in differential drug induced erythoid differentiation of K562 cells by degenerate-primer PCR. The experiment clones are GNAS isoform (GNASL and GNASS), Gαi2 and Gα11 pseudo gene. The result of different drug induced of K562 cells has three conclusions: (1) It can increase the expression of GNASL and decrease the expression of GNASS by Huangqi-induced. (2) Gαi2 all has more obvious difference by durg. (3) G11 pseudo gene displays in Huangqi-indudced mainly. The above different that the mechanism of the signal transduction has not been clear yet, must enter a cloth discussion even more, especially the Huangqi treatment of the blood disease of traditional Chinese medicine is benefitted greatly, if can totally understand that Huangqi leads the mechanism of differentiation, perhaps the chance that the blood cancer patient can slow down the condition and cure.

封面內頁
簽名頁
授權書.............................................................iii
中文摘要............................................................iv
英文摘要............................................................vi
誌謝...............................................................vii
目錄..............................................................viii
圖目錄.............................................................xii
表目錄.............................................................xiv

第一章 前言...........................................................1
第二章 文獻回顧.......................................................3
2.1 人類血癌細胞株K562細胞...........................................3
2.2 細胞增生與癌細胞之形成...........................................4
2.3 細胞訊號傳遞....................................................6
2.3.1 細胞外刺激..................................................6
2.3.2 細胞膜上受體................................................7
2.3.3 細胞膜上表面蛋白G蛋白........................................7
2.3.4 胞內作用體..................................................8
2.4 G蛋白之簡介.....................................................8
2.4.1 G蛋白α次單元之Gs家族.......................................10
2.4.2 G蛋白α次單元之Gi/o家族.....................................10
2.4.3 G蛋白α次單元之Gq/11家族....................................11
2.4.4 G蛋白α次單元之G12/13家族...................................12
2.4.5 G蛋白相關疾病..............................................13
2.5 G蛋白於K562細胞間之表現.........................................14
2.6 研究動機......................................................15
2.6.1 血液相關遺傳疾病...........................................15
2.6.2 分化療法..................................................16
第三章 研究方法......................................................18
3.1 材料..........................................................18
3.1.1 菌種及細胞株...............................................18
3.1.2 培養基....................................................19
3.1.3 誘導劑....................................................19
3.1.4 染劑......................................................19
3.1.5 載體......................................................20
3.2 方法..........................................................20
3.2.1 培養基之配製...............................................20
3.2.1.1 RPMI 1640培養基.......................................20
3.2.1.1.1 K562細胞之培養基...................................20
3.2.1.1.2 H358細胞之培養基...................................21
3.2.1.2 DMEM培養基............................................21
3.2.1.2.1 293T細胞之培養基...................................21
3.2.1.2.2 Hep3B細胞之培養基..................................22
3.2.1.3 L-15培養基............................................22
3.2.2 細胞計數及其繼代培養........................................22
3.2.3 誘導K562細胞分化...........................................23
3.2.3.1 誘導劑製備.............................................23
3.2.3.2 誘導劑之添加...........................................23
3.2.3.3 細胞分化之判定.........................................24
3.2.3.4 MTT assay............................................24
3.2.4 細胞RNA萃取與cDNA製備......................................25
3.2.4.1 RNA萃取...............................................25
3.2.4.2 反轉錄聚合酶連鎖反應....................................25
3.2.4.3 洋菜膠體電泳...........................................26
3.2.5 球蛋白於K562細胞中之表現....................................27
3.2.6 細胞膜上蛋白G蛋白α次單元基因片段之放大分析....................27
3.2.6.1 放射性同位素引子之標定..................................27
3.2.6.2 管柱製備與放射性同位素引子之純化.........................28
3.2.6.3 退化性聚合酶連鎖反應....................................28
3.2.6.4 聚丙醯胺凝膠配製及其電泳分析.............................29
3.2.6.5 流洗、Phenol/CHCl3萃取沉澱作用..........................29
3.2.6.6 限制酵素剪切作用.......................................30
3.2.6.7 Adaptor/Linker 製備...................................30
3.2.6.8 接合聚合酶連鎖反應......................................31
3.2.6.9 小片段核酸之純化.......................................31
3.2.6.10 核酸接合作用..........................................32
3.2.6.11 勝任細胞製備及其質體選殖之轉形作用.......................32
3.2.6.12 質體DNA小量備製.......................................33
3.2.7 細胞膜上蛋白G蛋白α次單元基因片段之聚合酶連鎖反應分析............34
3.2.7.1 刺激性G 蛋白(Gs)基因之5’RACE........................34
3.2.7.2 刺激性G 蛋白(Gs)基因之Nest PCR......................35
3.2.7.3 細胞膜上蛋白G蛋白α次單元基因全長序列之聚合酶連鎖反應.....35
3.2.7.4 細胞膜上蛋白G蛋白α次單元基因於K62細胞及人類組織癌症細胞株
中之專一性表現......................................36
3.2.8 統計分析...............................................36
第四章 結果..........................................................38
4.1 誘導K562細胞進行終點分化........................................38
4.1.1 細胞誘導之最佳條件..........................................38
4.1.2 細胞分化後之血紅素表現......................................38
4.2 誘導K562細胞分化後G蛋白α次單元基因之表現分析......................39
4.2.1 於K562細胞中放大分析G蛋白α次單元基因.........................39
4.2.2 擴增於K562細胞內G蛋白α次單元基因不同表現之全長片段.............40
4.2.3 G蛋白α次單元基因於K562細胞內之表現情形.......................40
第五章 討論..........................................................42
第六章 結論..........................................................45
參考文獻............................................................87
附錄................................................................94

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