臺灣博碩士論文加值系統

亨氏舞蹈症 (Huntington’s disease, HD) 是一種顯性遺傳的神經退化性疾病。致病原因主要是由於Hntingtin (HTT) 基因在exon 1的位置，出現CAG三核苷酸重複序列異常擴增的現象，造成HTT突變基因所產生的蛋白質具有細胞毒性，最後導致神經細胞的死亡；同時，臨床診斷中發現，在HD病患的基底核其紋狀體區域神經萎縮的現象會比大腦皮質嚴重。SPT4是轉錄延伸因子，可以協助RNA polymerase II 在DNA模板上的轉錄穩定性。先前，實驗室研究發現，在酵母菌模式系統中剔除SPT4，會讓帶有過長CAG重複序列基因的表現量減少，但並不影響帶有短的CAG重複序列基因，顯示SPT4在轉錄層級上會有選擇性的調控。為了進一步探討SUPT4H (SPT4之同源基因)在人類神經細胞中對於HD致病基因表現的影響，我們以HD病患的誘導型多能幹細胞 (induced pluripotent stem cell, iPSC)建構基因改造之穩定細胞株，以Tet-on轉錄誘導系統進行SUPT4H默化，然後進行分析。結果發現在HD-iPSCs以及HD-iPSCs所分化的GABAergic neurons中，SUPT4H默化會導致HTT突變基因的表現量下降。並且在某些HD-iPSCs所分化的GABAergic neurons中，SUPT4H默化對於HTT突變基因相較於正常的HTT基因有更大的影響力。

Huntington’s disease (HD) is caused by abnormal expansion of CAG tri-nucleotide repeats in the first exon of Huntingtin (HTT) gene, which produces gene products with detrimental effects on neurons. HD, a dominantly progressive neurodegenerative disorder, is clinically associated with movement, cognitive, and emotional impairments. The pathological hallmark of disease is the atrophy of striatum, which is consist of more than 95% GABAergic medium spiny neurons (MSNs). The transcription elongation factor, Spt4, is involved in the process of genes transcription by enhancement of RNA polymerase II processivity on DNA templates. Our previous study, using yeast as a model system, showed that SPT4 inactivation results in a preferential transcription reduction of genes containing a long stretch of CAG tri-nucleotide repeats, while it only marginally affects the expression of genes with short or no CAG repeats. In order to investigate the effect of SUPT4H, the ortholog of yeast SPT4, on the expression of HTT alleles in HD, we introduced a shRNA specifically against SUPT4H under the control of Tet-on system in induced pluripotent stem cells (iPSCs) that are derived from HD patients. Using the engineered iPSCs and GABAergic neurons differentiated from the iPSCs, we demonstrated that the expression of mutant HTT is reduced upon the knockdown of SUPT4H. More importantly, in HD-iPSCs and their differentiated GABAergic neurons, mutant HTT is affected in a greater extent than its wild-type counterpart. These results suggest that preferential reduction of mutant HTT in GABAergic neurons of HD patients is achievable through SUPT4H knockdown.
 

致謝…………………………I
中文摘要…………………………II
Abstract…………………………III
contents…………………………IV
Introduction…………………………1
1.1. General introduction of Huntington’s disease…………………………1
1.2. The function of SUPT4H in transcription regulation…………………………1
1.3. Induced pluripotent stem cells (iPSCs)…………………………2
1.4. iPSCs of Huntington’s disease (HD-iPSCs)…………………………2
1.5. Differentiation of GABAergic neurons from iPSCs…………………………3
1.6. Tet-on system…………………………4
1.7. The aim of this study…………………………4
Material and methods
2.1 iPSCs maintenance…………………………6
2.2 Establishment of stable cell clones…………………………6
2.3 Clone screening by PCR and Western blot analysis…………………………6
2.4 Neural differentiation of HD-iPSCs…………………………7
2.5 SUPT4H knockdown by Tet-on inducible system…………………………8
2.6 Immunocytochemistry (ICC)…………………………8
2.7 Reverse transcription polymerase chain reaction (RT-PCR)…………………………9
2.8 Western blot analysis…………………………9
2.9 Statistical analysis…………………………10
RESULTS
3.1 Strategy of SUPT4H genetic knockdown in human HD-iPSCs…………………………11
3.2 Establishment of Tet-on inducible system for shRNA expression in GM23225 and iHDA1…………………………12
3.2.1 Establishment of genetically engineered clones using iHDA1 (43/17)…………………………12
3.2.2 Establishment of genetically engineered clones using GM23225 (72/17)…………………………12
3.3 Characterization of stemness property of Tet-on inducible stable clones…………………………13
3.4 SUPT4H knockdown in iPSC stable clones…………………………13
3.4.1 SUPT4H knockdown in 43Q-iPSCs (C221, C226 and L36)…………………………13
3.4.2 SUPT4H knockdown in 72Q-iPSCs (C219 and L1)…………………………14
3.5 Differentiation of GABAergic neurons from iPSC stable clones…………………………14
3.6 Change of HTT and TetR expression upon the process of neuron differentiation from HD-iPSCs…………………………15
3.7 SUPT4H knockdown in GABAergic neurons derived from iPSC clones…………………………16
3.7.1 SUPT4H knockdown in GABAergic neurons derived from 43Q-iPSC (C221, C226 and L36)…………………………16
3.7.2 SUPT4H knockdown in GABAergic neurons derived from 72Q-iPSC (C219 and L1)…………………………17
3.8 The degree of HTT down-regulation by SUPT4H knockdown in iPSC stable clones and GABAergic neurons derived from iPSC stable clones…………………………17
Discussion…………………………19
References…………………………23
Figure…………………………26
Table…………………………41

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