臺灣博碩士論文加值系統

血漿中脂蛋白(a) [lipoprotein (a)；Lp(a)] 的濃度可視為檢測心血管疾病及中風的一項風險因子，脂蛋白元(a) [apolipoprotein；Apo(a)] 是組成脂蛋白(a) 的其中一個分子，是一個以114個胺基酸 (被稱為kringle) 為單位重複15-50次的蛋白；在基因序列上，每342個核苷酸之間夾有一內插子 (本實驗中稱之intron C)，而在每個 kringle 內又有另一內插子 (本實驗中稱之intron A/B) 加以分隔。在臨床統計上，血漿中 脂蛋白(a) 的濃度與脂蛋白元(a) 的kringle 重複次數成反比，因此我們提出一個假設，在此重複單位內的內插子序列可能扮演調降脂蛋白元(a) 啟動子 (promoter) 的活性，在過去啟動子活性分析 (promoter assay) 的實驗中，我們證實了intron A,B,C的確具有調降啟動子的活性，因此我們利用電泳移動偏移分析 (Electrophoretic mobility shift assay；EMSA) 及DNA親合性沉澱試驗 (DNA affinity precipitation assay；DAPA) 的實驗來尋找可能參與的順式作用序列 (cis element) 及反式作用因子 (trans factor)。利用軟體分析這些內插子序列，我們發現脂蛋白元(a) 的內插子序列中含有數個類似 GC-rich binding factor (GCF；GCGGGGC) 及silencing factor A (SFA；GGAGCAGGA) 結合的位置。電泳移動偏移分析的結果顯示，當內插子序列與HepG2肝癌細胞株的核蛋白抽出物 (nuclear extract；NE) 作用以後，產生明顯偏移 (shift) 的現象，接著我們以人工的方式合成 GCF 及 SFA 的結合序列作為 EMSA 實驗中的探針 (probe) 及競爭抑制試驗 (competition assay) 中的競爭者 (competitor)，實驗結果顯示，脂蛋白元(a) 內插子序列中的 GCF 結合序列能夠專一性結合來自HepG2核抽出物中所含的特定蛋白，然 SFA 則不具有此能力。利用streptavidin agarose beads 沉澱結合在 GCF 序列上的蛋白，接著進行十二烷基磺酸鈉-聚丙烯醯胺膠體 (SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel) 電泳，發現數個蛋白結合在 GCF 結合序列上，以基質輔助雷射解析電離飛行時間質譜 (MALDI-TOF/MS) 鑑定的結果，這些蛋白分別是β-actin、heterogeneous nuclear ribonucleoprotein (hnRNP) C isoform b 及dystonin，這些蛋白與脂蛋白元(a) 內插子之間的關係仍需要進一步的實驗證明。統整上述，在脂蛋白元(a) 重複單位的內插子序列內的GCF結合序列能夠結合HepG2 NE內特定的核蛋白，而這些核蛋白可能是作為調降脂蛋白元(a) 啟動子活性的反式作用因子。

Elevated blood lipoprotein (a) [Lp(a)] concentration is an independent risk factor for cardiovascular disease and stroke. Apolipoprotein (a) [Apo(a)] is a component of Lp(a) and contains 15-50 114 amino acid repeats. Each 342 nucleotide repeat is separated by an intron between repeat (designated as Intron C) and an intron inside the repeat (designated as intron A/B). As blood Lp(a) level is inversely related to the repeat number of Apo(a), we hypothesize that the intron sequences of the repeat may play role in downregulating apo(a) promoter activity. Our previous studies revealed a down regulation of apo(a) promoter by intron A, B, C. We further investigated what cis element or trans factors were involved by Electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA), respectively. The intron sequences were scanned for known transcription silencer sequences. These are GC-rich binding factor (GCF) binding site with a consensus of GCGGGGC and silencing factor A (SFA) binding sequence with a consensus of GGAGCAGGA. Several similar motifs are found in the apo(a) introns. The gel shift assays showed bindings of the DNA fragment containing the multiple GCF or SFA homolog (with 1-2 base mismatch) with the HepG2 nuclear extract (NE). GCF homolog was a better competitor than the SFA homolog. We further used the multimer of GCF homolog as a probe and observed gel retardation and competition in the presence of HepG2 NE. Using streptavidin agarose to capture biotinylated GCF homolog plus HepG2 NE, we observed several protein bands in the elute by SDS-PAGE. The proteins were identified by MALDI-TOF experiment as members of heterogeneous nuclear ribonucleoprotein (hnRNP) family, β-actin and actin binding protein, dystonin tentatively. The relationship of these proteins with the apo(a) intron sequence needs further investigation. In conclusion, the apo(a) introns in the repeats contain the known transcription downregulator GCF that can interact with HepG2 NE and this may play role in downregulating apo(a) promoter activity.

壹. 英文摘要______________________________________________2
貳. 中文摘要______________________________________________4
參. 導論__________________________________________________6
肆. 附圖_________________________________________________16
伍. 材料與方法___________________________________________21
陸. 結果_________________________________________________29
柒. 結論與討論___________________________________________53
捌. 附表_________________________________________________58
玖. 參考文獻_____________________________________________62

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