臺灣博碩士論文加值系統

亨氏舞蹈症（HD）是一種顯性遺傳的腦神經退化性疾病。致病原因主要歸因於Huntingtin（Htt）基因在exon 1的CAG重複序列有異常擴增的現象，造成致病的基因產物被生成，進而導致神經細胞死亡。正常Htt在細胞中具有極為重要的功能，因此，如何選擇性地降低突變Htt的基因表現，而不影響正常Htt表現是研發HD藥物的最終目標。
實驗室先前發現抑制Supt4表現或活性可以選擇性的降低突變Htt，但不影響正常Htt基因的表現。在細胞中Supt4與Supt5的NGN domain結合後，可與RNA polymerase II相互作用，增加其在模板上的轉錄持續力。因此，我們建立BiFC篩選平台系統找尋抑制Supt4h與Supt5h-NGN結合的小分子藥物，利用螢光強度觀察Supt4h與Supt5h-NGN在細胞中結合狀態的改變。首先，我們確認此作業平台在加入競爭物而降低Supt4h與Supt5h-NGN結合機率時，螢光強度會顯著下降；之後與國家衛生研究院合作，執行小分子藥物高通量篩選，尋找到七個可能的抑制物。並進一步確認其中六個小分子，可以降低BiFC的螢光強度，但是對分析系統中的蛋白質表現並無顯著影響。
上述結果顯示新建立的篩藥平台，能有效並迅速地進行高通量篩選。目前，已找到六個小分子藥物，可以影響Supt4h與Supt5h-NGN的結合。在未來，將驗證此平台所篩選到的小分子抑制物，是否具備選擇性的降低突變Htt基因表現。

Huntington’s disease (HD) is an inherited neurological disorder caused by abnormal expansion of CAG repeats, which encode a long stretch of polyglutamine, in Huntingtin (HTT) gene. While the expression of mutant HTT contributes a gain-of-function for pathogenesis of HD, the biological activity of normal HTT is essential for neuron development and survival. For this reason, it is an optimal goal having HD treatments that inhibit the expression of HTT alleles in a selective manner. Currently, effective treatment is not available for this disorder and the development of therapeutic compounds is urgently needed.
In our previous study, we demonstrated that Supt4h down-regulation can selectively reduce mutant HTT gene expression without an overt impact on the expression of wild-type HTT. Furthermore, the function of Supt4h in supporting RNA polymerase II transcribing through CAG expansion region is dependent on the formation of Supt4h/5h complex.
Here, based on Bimolecular Fluorescence Complementation (BiFC), we established a cell-based assay system to monitor the interaction between Supt4h and Supt5h, and also applied this system to identify small chemical compounds that could interfere with the Supt4h/Supt5h complex formation. Among 60,000 compounds screened, 7 hits were identified with an inhibitory effect on the fluorescence signal of BiFC. These compounds were subjected to further characterization to assure their activities on Supt4h/5h protein-protein interaction.

目錄
致謝 i
摘要 ii
Abstract iii
縮寫對照表 iv
緒論 1
一、亨氏舞蹈症（HD） 1
二、目前對於亨氏舞蹈症的治療趨勢 2
三、Transcription elongation 的調控機制 3
四、Supt4h 與Supt5h 的結構與功能 4
五、Supt4h 與HD 5
六、Bimolecular Fluorescent Complementation（BiFC） 6
七、Tet‐on system 轉錄誘導系統 6
實驗目的 7
材料與方法 8
材料 8
一、載體的建立 8
二、細胞株與細胞培養液 10
三、細菌培養液與培養基 10
四、抗體 11
五、Supt4h shRNA 11
方法 12
一、大腸桿菌載體的轉型與抽取 12
二、Stable clone 細胞株建立 12
三、細胞影像與螢光強度的定量 13
四、細胞蛋白質的萃取 13
ii
五、西方墨點法 13
六、質體的轉殖(Transfection) 14
七、Supt4h 基因的默化 ( Konckdown ) 15
八、免疫沉澱 (Co‐immunoprecipitation, Co‐IP) 15
結果 17
一、BiFC 可用於偵測細胞中，Supt4h 與Supt5h‐NGN 兩蛋白質之間結合
狀態的改變 17
二、BiFC 的螢光強度會受到外來競爭蛋白質的影響 19
三、建構可穩定表現Supt4h‐VC 與Supt5h‐NGN‐VN 的細胞株2‐pn4 與對
照組細胞株21‐vs 19
四、利用2‐pn4 及21‐vs 細胞株篩選到七個可能抑制Supt4h 與Supt5h‐NGN
結合的小分子化合物 20
五、六個候選小分子抑制物，可能影響Supt4h 與Supt5h‐NGN 的結合 21
(1) H 22
(2) 6CR 22
(3) 549 22
(4) 717 23
(5) 373 23
(6) 459 23
(7) 893 24
討論 25
圖表 29
Figures 29
Figure 1. The interaction between Supt4h and Supt5h‐NGN can be
detected by BiFC. 29
Figure 2. BiFC generated from the expression of pTRE‐Supt4h‐VC‐NGN‐VN
is Doxycyclin‐dependent. 31
Figure 3. The BiFC of Supt4h and NGN‐VN is sensitive to polypeptides that
interfere with the Supt4h/5h complex formation. 32
Figure 4. A Stable cell line 2‐pn4 with BiFC of Supt4h‐VC and NGN‐VN and
21‐vs control cell line that expresses Venus protein is generated
individually. 33
Figure 5. Stable cell line 2‐pn4 can detect the inhibitory effect of
competitive peptide that interferes with the complex formation of Supt4h
and Supt5h‐NGN. 34
Figure 6. Stable cell line 2‐pn4 can detect the change of protein‐protein
interaction between Supt4h‐VC and Supt5h‐NGN. 36
Figure 7. Compound H interferes with the expression of reporter genes in
both 2‐pn4 and 21‐vs cells. 38
Figure 8. 6CR is able to inhibit the BiFC of Supt4h and Supt5h‐NGN. 40
Figure 9. 549 inhibits the BiFC of 2‐pn4 cells, but also reduce the
fluorescence of 21‐vs at high concentration. 41
Figure 10. 717 inhibits the BiFC of Supt4h and Supt5h‐NGN specifically. 42
Figure 11. 373 inhibits the BiFC of 2‐pn4 cells, but also shows a
moderately inhibitory effect on the fluorescence signal of 21‐VS cells in
high doses. 43
Figure 12. 459 inhibits the BiFC of Supt4h and Supt5h‐NGN specifically. 44
Figure 13. 893 inhibits the BiFC of 2‐pn4 cells, but also shows a
moderately inhibitory effect on the fluorescence signal of 21‐VS cells in 10
μM. 45
Tables 46
Table1.The summary and proposed future works of seven hit compounds
identified in this study. 46
Table2. The targeting nucleotide sequence of Supt4h shRNAs 47
Table3. Oligonucleotide primers used in plasmid constructs 47
參考文獻 49
附件 52

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