臺灣博碩士論文加值系統

肌陣攣性癲癇發作伴破碎紅纖維病變 (Myoclonic epilepsy associated with ragged-red fibers , MERRF)是一種母性遺傳的粒線體腦肌疾病，主要的症狀會有肌陣孿 (Myoclonus)、.癲癇 (Generalized epilepsy) 、運動失調 (Ataxia)等臨床表徵。致病的原因是因為在粒線體中負責攜帶賴氨酸(Lysine)的tRNA基因上產生了A8344G的粒線體核苷酸序列(mtDNA)點突變，佔了80%以上。從先前實驗室的研究結果已知在帶有A8344G粒線體DNA點突變的融合細胞株中，碳酸酐酶八號(carbonic anhydrase VIII, CA8)在mRNA和蛋白質表現上，皆顯著低於在正常的融合細胞株中，因此猜測CA8在兩株融合細胞株中表現量的差異，很可能是來自CA8基因轉錄活性的差異。然而，在MERRF突變融合細胞株中使得核內CA8轉錄活性降低的分子機制目前還未可知。首先，我們建立了人類CA8基因啟動子報導載體，包含了在CA8基因5’側區分析而得的GC-box、AP-2和TATA-binding element。。將建立好的hCA8啟動子報導載體送到正常和突變的融合細胞株中進行冷光活性分析，結果發現在MERRF細胞株中CA8啟動子的活性顯著低於在正常的細胞株當中。我們也利用一系列突變的人類CA8啟動子報導載體進一步去分析，發現到重要的轉錄因子會作用在CA8啟動子近端的GC-box序列去調控CA8基因的轉錄，而我們所熟知的轉錄因子Sp1和其他Sp家族中的成員是主要會辨識這段GC-box的序列去活化下游的基因表達。然而，我們卻偵測不到在正常和MERRF疾病的融合細胞株中，內生性Sp1的蛋白表現量有明顯的差異。在進一步去確定Sp1的角色後，結果發現當我們外送大量的eGFP-Sp1到融合細胞株當中，可以顯著的增加人類CA8啟動子活性，但是並無法偵測到明顯內生性CA8蛋白表現量的增加。進一步，利用序列專一性的shRNA去knockdown細胞株中內生性的Sp1，也無法觀察到CA8的蛋白表現量有顯著的降低。令我們意外的是，當改用攜帶較小標定蛋白的Flag-Sp1大量表達在HEK-293T細胞中，卻可以顯著地增加內生性CA8蛋白的表達，而先前報導系統的分析已知道hCA8基因在293T細胞中有較強的啟動子活性。進一步證明，當表達Sp1的顯性抑制蛋白後，會使hCA8啟動子活性明顯地下降，顯示了GC box-binding protein對於hCA8轉錄活性的重要性。藉由染色體免疫沉澱法也證實，在D5-1融合細胞株中可以偵測到內生性Sp1結合到近端hCA8啟動子的訊號。總結目前的研究結果，不管是in vitro或是in vivo的分析都顯示Sp1確實是透過hCA8啟動子近端的GC box序列來啟動CA8的轉錄活性。然而，mtDNA的突變到底是透過什麼樣的分子機制影響了哪個轉錄因子，而使得核內CA8基因轉錄活性的下降，還需要更進一步的分析。

Myoclonic epilepsy with ragged-red fibers (MERRF) is a maternally inherited mitochondrial neuromuscular disease, which is pathologically characterized by myoclonus, generalized epilepsy and ataxia. The most common mutation in MERRF syndrome is an A-to-G transition mutation at nucleotide pair (np) 8,344 in tRNALys gene of mitochondrial DNA (mtDNA). Our previous study reported a significant decrease of carbonic anhydrase VIII (CA8) in mRNA and protein levels in cybrids harboring A8344G mtDNA point mutation. However, the regulatory mechanisms underlying low transcriptional activity of human nuclear CA8 (hCA8) gene in MERRF disease are still unknown. We have established a luciferase gene-carrying hCA8 promoter reporter construct containing several putative transcription factor-binding sites, including GC-box, AP-2 and TATA-binding element in the 5’flanking region of the hCA8 gene. The reporter system showed a significant decrease of the promoter activity in the mutant MERRF cellular model. Using a series of mutated hCA8 promoter constructs, we demonstrated that GC-box, recognized by Sp1 and the Sp family members, may be a key cis-element functioning at the proximal region of the promoter. However, there is no detectable difference in the endogenous expression of Sp1 between wild-type and mutant cybrids. In further confirming the role of Sp1, a significant increase of the hCA8 promoter activity was observed in cybrids with over-expression of eGFP-Sp1, but there is no detectable increase on the CA8 protein and RNA expression. Moreover, knockdown of endogenous Sp1 by the sequence-specific shRNA did not result in any decrease of the endogenous CA8 protein level. Unexpectedly, over-expression of Flag-Sp1, compared to eGFP-Sp1, significantly increased endogenous CA8 protein expression observed in neuron-like HEK-293T cells, where a strong hCA8 promoter activity was identified in the luciferase reporter assay. As expected, expression of dominant negative Sp1 to compete with all GC-box binding activities resulted in a decreased hCA8 promoter activity in 293T cells. To confirm the role of Sp1 in vivo, chromatin immunoprecipitation (ChIP) assay was performed and the results demonstrated the binding ability of endogenous Sp1 on hCA8 promoter in wild-type cybrid cells. Taken together, these results suggest that Sp1 transactivates hCA8 gene through the proximal GC box element on the promoter region both in vitro and in vivo. However, the key modulator-responsive factor to the mtDNA mutation and how it may affect nuclear hCA8 gene transcription still need further investigation.

致謝 …………………………………………………1

中文摘要 …………………………………………………2-3

英文摘要 ………………………………………………4-5

前言 …………………………………………………6-13

材料與方法……………………………………………14-22

結果 …………………………………………………23-33

討論 …………………………………………………34-49

文獻 …………………………………………………50-57

圖目 …………………………………………………58-85

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