臺灣博碩士論文加值系統

人類熱穩定鹼性磷酯包含兩個相關基因所轉錄的同功：胎盤型鹼性磷酯（PAP），特別存在於胎盤組織；生殖細胞型鹼性磷酯（GCAP）主要表現於生殖型細胞中。在人類TMK-1胃癌細胞株中，糖皮質固醇Dexamethasone（Dex）會誘發胎盤型鹼性磷酯活性及mRNA表現，經由mRNA的穩定性及run on轉錄分析，證明Dex在TMK-1細胞誘導鹼性磷酯表現主要是經由增加PAP基因轉錄活性。為了分析研究糖皮質固醇活化之機轉，將胎盤型及生殖細胞型鹼性磷酯啟動區轉殖到螢光表現載體，轉染至TMK-1細胞中，結果顯示， Dex會誘發胎盤型鹼性磷酯啟動區活性約6至10倍，雖然兩者基因啟動區有約86％之相同之核酸序列，但生殖細胞型鹼性磷酯啟動區幾乎不受糖皮質固醇調控。我們確認受Dex調控之胎盤型鹼性磷酯啟動區為-280至-375，將此片段插入含GCAP及CMV基因啟動區與只有TATA box之螢光（luciferase）報告載體中，並共轉染糖皮質固醇表現質體以觀察Dex對其誘導之情形。另外並將PAP啟動區-331至-296刪除片段及GCAP啟動區轉染至四種人類細胞株，觀察Dex對其之誘導情形。經由分析PAP基因啟動區序列發現可能有數個一半的糖皮質固醇反應序列（GRE）存在，分別在-374、-308、-302、-292及+33的位置，為了鑑定這些可能的GRE序列，將PAP啟動區-375至-280不同序列之片段插入只有TATA box之luciferase報告載體，實驗結果顯示任何單一的GRE無法有足夠能力造成糖皮質固醇反應，可能還有其它區域參與。在凝膠位移分析使用老鼠rGR-DBD當蛋白質萃取液，發現只有GREs位置在-374、-302及+33有顯著結合能力。由以上結果證明，在TMK-1細胞中Dex增加PAP基因轉錄活性是藉由GR與PAP基因啟動區上數個不完美之一半GRE的交互作用，此外，在PAP基因啟動區上可能還有其他因子參與Dex對其之專一調控。

Human heat stable alkaline phosphatases are encoded by two closely related genes: the placental alkaline phosphatase (PAP), which specifies the term placental isozyme, and the germ cell alkaline phosphatase GCAP, which is expressed primarily in germ cells. In the human gastric cancer cell line TMK-1 cells, glucocorticoid exhibits an marked induction in the enzyme activity and accumulation of the mRNA of PAP. Analysis of mRNA stability and run on transcription demonstrated that glucocorticoid induction of PAP gene epxression occurs primarily at the level of transcription. In order to study the molecular mechanism underlying the glucocorticoid induction, PAP and GCAP promoters were fused to luciferase gene and transfection were carried out in TMK-1 cells. Dexamethasone confers about 6 to 10 fold of induction in luciferase activity in PAP promoter. Despite that the PAP and GCAP promoters share about 86% in sequence identity, the GCAP promoter directs a very low level of response to dexamethasone. The dexamethasone response region in PAP promoter was identified to be located in between -280 to -375. Insertion of this region into GCAP, CMV promoters, and TATA-luciferase reporter enabled these promoter responsive to dexamethasone when cotransfected with the glucocorticoid receptor (GR) expression plasmid pRS-hGR. Further studies indicated that the region from -296 to -331 is critical for glucocorticoid induction in all four different human cell lines assayed. Sequence analysis of the PAP promoter identified several potential half-glucocorticoid response elements (GRE) at -374, -307, -302, -292, and +33, respectively. In order to identify which of these potential GREs that is required for PAP promoter activity, DNA oligonucleotides encompass various length in between -375 to -280 were inserted into TATA-luciferase reporter. Transfection studies indicated that any one of the half-GREs alone is not enough to confer full glucocortcoid response and other regions may be required in the glucocorticoid-mediated induciton. In gel mobility shift assays using rat GR DNA binding domain (GR-DBD), only the GREs at -374, -302, and +33 were shown to bind GR-DBD efficiently. These results demonstrated that in TMK-1 cells, the increased transcription of PAP gene in the presence of glucocorticoid is mediated in part by interaction of the GR with the imperfect half-GREs in the gene promoter. In addition, other potential elements in PAP promoter may be involved in conferring the full and specific transcription activity mediated by glucocorticoid.

目錄
頁
目 錄…………………………………………….………………Ⅰ
縮 寫 表……………………………………………………………Ⅲ
圖表目錄…………………………………………….…………… Ⅳ
中文摘要………………………………………………………....Ⅵ
英文摘要…………………………………………………………….Ⅶ
緒 論…………………………………………………………….1
材料與方法………………………………………………………….5
一、主要儀器及藥品試劑………………………………..………5
二、小量質體的製備…….………………………….…………....6
三、核酸限制反應 ……………………………………………7
四、DNA片段的回收……………………………………………7
五、DNA連結反應……………………………………………….8
六、勝任細胞的製備………………….…… …..………………..8
七、轉形作用………………………….………….………………9
八、質體的構築……………………….……….…………………9
九、DNA序列的確認………………….…………………………10
十、大量質體的製備………………….………………………….12
十一、細胞培養………………………………….……………….13
十二、細胞轉染………………………………….……………….16
十三、b-galactosidase活性分析…………………………………18
十四、b-galactosidase染色………………………………………19
十五、蛋白質濃度的測定………………………….…………….19
十六、相對的螢光（luciferase）活性分析………..…………20
十七、SDS-聚丙烯醯酸胺電泳及染色………………………….21
十八、從大腸桿菌表現DBD：IPTG誘導、rifampicine處理…24
十九、細菌蛋白質的萃取………………………………………..25
二十、凝膠位移分析（gel shift assay）……………….………..26
結 果…… ………………………………………………………34
一、人類胃癌細胞TMK-1鹼性磷酯，活性表現之探討…34
二、人類TMK-1細胞GCAP及PAP啟動區（promoter）刪除片段報告質體之構築及受Dex誘導轉錄活性序列之探討..34
三、KATO細胞GCAPP及PAPP質體之構築及受Dex誘導轉錄活性序列之探討……….…………………………………36
四、Dex對不同長度PAPP活性序列之探討……………………37
五、PAPP序列的5'端-375至-296 序列之構築……………….37
六、Dex對包含PAPP -375/-280之不同報告載體其轉錄活性之探討……………………………………………………….38
七、PAPP序列的5'端-375至-296 bp刪除片段之構築…………38
八、PAPP -375/-296 bp間GRE之分析.……….…………………39
九、Dex對共轉染糖皮質固醇表現載體（RS-hGR）之各個PAPP載體之活性探討……………………………...………………39
十、在不同細胞中Dex對PAPP-375/-280各刪除片段及GCAPP之誘導轉錄活性之分析..…………………………………….41
十一、PAPP與GCAPP-375/-280間各片段之螢光報告載體之構築載體……………………………….……………….41
十二、Dex對PAPP-375/-280間不同片段的報告載體轉染活性之分析…………………………………….……………….42
十三、以凝膠位移法分析細菌蛋白萃取液…...…………………43
討 論………….…………………………………………………46
結 論………………………………………………………….…50
表…………………………………………………….………………51
圖………………………………………………….…………………52
參考文獻…………………………………………………………….90
圖表目錄
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表一、比較人類PAP之GRE與一些其他的GREs…………………………51
圖一、人類PAP及GCAP基因5'-flanking region之比對…………..……52
圖二、構築人類PAPP之luciferase報告質體……………………………..53
圖三、構築人類GCAPP之luciferase報告質體...………………………….54
圖四、比較不同細胞來源的PAPP及GCAPP，在TMK-1細胞的活性……55
圖五、鑑定人類PAPP之糖皮質固醇反應序列區域……………………….56
圖六、構築人類PAPP-375/-280片段插入TATA luciferase報告質體…..…57
圖七、構築人類PAPP-375/-280片段插入GCAPP-556/+51-luciferase報告質體…………………………………………………………………58
圖八、構築人類PAPP-375/-280片段插入GCAPP-281/+51-luciferase報告質體…………………………………………………………………59
圖九、構築人類PAPP-375/-280片段插入CMV啟動區-luciferase報告質體…………………………………………………………..…………..60
圖十、人類PAPP-375/-280片段在pTZ-ATL0載體在TMK-1細胞的活性………………………………………………….…………………...61
圖十一、人類PAP基因啟動區-375至-280片段插入GCAP啟動區在TMK-1細胞的活性……………………………………………………………62
圖十二、人類PAPP-375/-280片段插入CMV啟動區在TMK-1細胞的活性.63
圖十三、構築人類PAPP-375/-280各個刪除片段之luciferase報告質體……64
圖十四、人類PAPP-375/-280各個刪除片段在TMK-1細胞的活性…………65
圖十五、共轉染RS-hGR對不同細胞的PAPP一些刪除片段在TMK-1細胞的活性…….…………………………………………………..…….66
圖十六、在TMK-1細胞中，共轉染不同濃度RS-hGR的PAPP-567/+54序列誘導活性……………………………...…………………………….67
圖十七、共轉染RS-hGR對人類GCAPP各個刪除片段在TMK-1細胞的活性……………………………………………………………………68
圖十八、共轉染RS-hGR對人類PAPP-375/-280片段在TMK-1細胞的活性………………………………………………………………………69
圖十九、共轉染RS-hGR對人類PAPP-375/-280片段插入GCAPP在TMK-1細胞的活性………………….………………………………………...70
圖二十、共轉染RS-hGR對人類PAPP-375/-280片段插入CMV啟動區在TMK-1細胞的活性…………………………………………...………71
圖二十一、共轉染RS-hGR對人類PAPP-375/-280各個刪除片段在TMK-1細胞的活性…………………………………………………………....72
圖二十二、人類PAPP各個刪除片段及GCAPP在HeLa細胞的活性…………73
圖二十三、人類PAPP各個刪除片段及GCAPP在MCF-7細胞的活性……….74
圖二十四、人類PAPP各個刪除片段及GCAPP在BC-M1細胞的活性……….75
圖二十五、構築人類GCAPP一些片段之luciferase報告質體…………………76
圖二十六、構築人類PAPP一些片段之luciferase報告質體……………………77
圖二十七、構築人類PAPP一些片段之luciferase報告質體……………………78
圖二十八、共轉染RS-hGR對GCAPP一些片段在TMK-1細胞之活性………79
圖二十九、共轉染RS-hGR對PAPP之一些片段在TMK-1細胞之活性……….80
圖三十、老鼠及人類GR-DBD於大腸桿菌之過量表現………………………81
圖三十一、Consensus GRE與人類hGR-DBD的凝膠位移分析………………..82
圖三十二、Consensus GRE與老鼠rGR-DBD的凝膠位移分析…………………83
圖三十三、Consensus GRE及一些PAPP及GCAPP之片段與老鼠rGR-DBD的凝膠位移分析……………………….……………………………...84
圖三十四、人類PAPP不含GRE區域之DNA片段不會與老鼠rGR-DBD專一結合…………………………………………..……………………..85
圖三十五GCAPP-313/-279片段與老鼠rGR-DBD的凝膠位移分析…………86
圖三十六、PAPP-335/-295片段與老鼠rGR-DBD的凝膠位移分析……………87
圖三十七、PAPP-320/-295片段與老鼠rGR-DBD的凝膠位移分析……………88
圖三十八、PAPP-335/-295片段與位置相當之GCAPP-313至-279片段與TMK-1核萃取液有不同之凝膠位移分析………….………………..89

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