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前腦興奮性神經元中缺損Disc1基因之小鼠的表現型鑑定

為了解決CTRL + C terminal的問題，作者蕭佩芬 這樣論述：

思覺失調症是一種多致病因子的慢性精神疾病，患者的病徵是具有幻聽幻覺、行為異常、無法辨別幻覺與現實等。先前研究發現，許多基因與思覺失調症的發病機理有關，而其中一種基因是Disrupted-in-Schizophrenia 1（DISC1）。先前有研究發現在一群具有神經認知障礙的台灣思覺失調症患者身上，有一類極短型的DISC1被過度表達，而這樣的情形可能與患者的持續性專注力受損有關。在本篇研究中，為了研究極短型的DISC1對大腦的結構和功能有什麼影響，我們使用Cre / loxP系統使得小鼠前腦興奮性神經元中Disc1基因4至13的外顯子在Cre的作用下被剔除。我們利用前腦興奮性神經元中Disc

1被剔除的基因突變鼠（FbDisc1 KO mice），進行了一系列的行為鑑定與大腦組織的分析。研究中，我們使用了8-12週齡大的公鼠做實驗，以純合子（Homo，Disc1Δ4-13/ Δ4-13; Emx1-Cre）和異型合子（Het，Disc1Δ4-13/ +; Emx1-Cre）的FbDisc1 KO 小鼠與年齡、性別匹配的對照組（Ctrl，Emx1-Cre）作比較。首先，我們發現Homo和Het的FbDisc1 KO小鼠的外表與Ctrl組相比並沒有明顯的異常。此外，FbDisc1 KO小鼠在開放場地（open field test）、高架型迷宮（elevated plus maze t

est）、Y字型迷宮（Y maze test）、辨別新物體（novel object recognition test）和前脈衝抑制（prepulse inhibition test）的測試中皆表現正常。然而，Homo小鼠在強迫游泳試驗中表現出異常的應激反應。囓齒動物的內側前額葉皮層（mPFC）參與應激應對行為，於是我們進一步檢查了mPFC中神經元的組織學特徵。我們發現FbDisc1 KO小鼠大腦mPFC的第二、三層中的錐狀神經元，其樹突（dendrite）複雜程度並沒有改變，但是樹突棘（dendritic spine）的密度在Homo小鼠中卻顯著降低。另外，思覺失調症患者的前額葉皮質被證實存

在抑制性神經元的缺損，因此我們利用組織免疫染色法來觀察FbDisc1 KO小鼠的抑制性神經元。我們發現Het和Homo小鼠的mPFC中，parvalbumin抑制性神經元的密度與Ctrl組相比並無差異；然而與Ctrl組相比，calbindin抑制性神經元的密度在Het和Homo組均顯著下降。在維持專注力時，需要皮質與紋狀體互相投射的神經網絡所調控，而前額葉皮質在維持專注力扮演重要的角色。根據行為與組織分析的結果，我們認為極短型DISC 1藉由影響前額葉的功能，導致病人出現持續性專注力受損的問題。因此，我們的Homo小鼠是一種可以模擬具有極短型DISC1的思覺失調症患者的認知功能受損的動物模式。

運用CRISPR/Cas9基因編輯與多能幹細胞發展治療法布瑞氏症心肌病變之新穎策略

為了解決CTRL + C terminal的問題，作者宋惠詠 這樣論述：

Chinese Abstract iEnglish Abstract iiiContents VList of Figures ViiList of Tables XAbbreviations XiIntroduction 1Epidemiology of Fabry Disease 2Pathophysiology of Fabry Disease 3Late-onset mutation IVS4 + 919G>A in Fabry Disease 5Therapeutic strategy for Fabry disease

6Chaperone small molecular 9Protease inhibitor 10Glycosylation modified in lysosomal enzyme 11In vivo Fabry disease model: GLA-knockout Mouse 12GLA knockout transgenic mouse aortic endothelial cells 13GLA-null Accelerated atherogenesis 13GLA-null Impaired vasoreactivity 14In

vitro Fabry disease models 14Induced pluripotent stem cells (iPSCs) derived from Fabry patients 15CRISPRs/Cas9 genome editing 17Impact of lysosome status on exosomes content and release 19Shear Stress 20Autophagic flux impairment in Fabry disease 21Vesicle turnover impairment in F

abry disease 22Inflammasome associated with autophagic impairment 23Statement of the problem 25Materials and Methods 30Result 1.Graphic Abstract 441-1 Aim 451-2 Specific Material & Methods 461-3 Results 471-4 Discussion 541-5 Figure legends 58Result 2.Graphic Abstract

732-1 Aim 742-2 Specific Material & Methods 752-3 Results 762-4 Discussion 812-5 Figure legends and Table lists 85Result 3.Graphic Abstract 1043-1 Aim 1053-2 Specific Material & Methods 1053-3 Results 1073-4 Discussion 1153-5 Figure legends and Table lists 119Discu

ssion 148Future works 159Publication lists 167Reference 168Figure 1-1. Synthesis and degradation of some globo-series GSLs 4Figure 1-2. Identification of the mutant a-Gal A cDNA 6Figure 1-3. Illustration of Current and Emerging Treatment Strategies for Fabry Disease 8Figure 1-4.

Genetic perturbations enabled by engineered CRISPR/Cas9 systems 19Figure 2-1 Validated efficiency of transfected CRISPR/Cas9 plasmids in HEK293T cells 58Figure 2-2 CRISPR/Cas9-mediated genome disruption of human GLA result in cell lacking detectable GLA expression 59Figure 2-3 Screen and s

elect the CRISPRs introduced clones 60Figure 2-4 The GLA expression and enzyme activity in selected CRISPRs transduced clones.. 61Figure 2-5 Enzyme activity and protein expression of rhα-GLA in the GLA-null cell lines 62Figure 2-6 Time schedule determining the intracellular pharmacokinetics

of rhα-GLA was illustrated 63Figure 2-7 Cytotoxicity of proteasome inhibitors MG132 in the GLA-null cells were determined by WST-8 assay 64Figure 2-8 Co-administration of rhα-GLA and MG132 increases the enzyme activity and protein stability of rhα-GLA in vitro 65Figure 2-9 Subcellular loca

lization of the rhα-GLA and the clearance of the lysosomal accumulated Gb3 in Fabry patient-derived fibroblasts co-treating with Fabrazyme and MG132 66Figure 2-10 Lysosomal accumulated Gb3 was determined by immunofluorescence staining 67Figure 2-11 GLA protein expression treated with Protease

or Protease inhibitors 68Figure 2-12 The flowchart of producing and purifying the rhGLA by using Bac-to-Bac Baculovirus expression system 69Figure 2-13 Purification of rhGLA protein by using baculovirus expression system 70Figure 2-14 CRISPR/Cas9 mediated gene editing of GLA effectively and

completely ablated endogenous GLA protein expression in human cells... 71Figure 3-1 CRISPR/Cas9-mediated knockout of expression of GLA in hESCs 85Figure 3-2 Sanger sequencing analysis confirming the selected clones sequence 86Figure 3-3 T7E1 digestion assay validating the absence of CRSIPR

/Cas9-induced mutations in the predicted off-target genes 87Figure 3-4 Characterization of CRISPR/Cas9-edited GLA-null hESC clones 88Figure 3-5 Characterization of differentiated capability in CRISPR/Cas9-edited GLA-null hESC clones 89Figure 3-6 Deficiency GLA expression in GLA-null cardiom

yocytes 90Figure 3-7 Gb3 accumulated in GLA-null cardiomyocytes... 91Figure 3-8 Recapitulation of FD-specific cardiac abnormalities in GLA-null cardiomyocytes 92Figure 3-9 Proteomic analysis of GLA-null CMs 93Figure 3-10 Validation of protein expression from Proteomic analysis of GL

A-null CMs 94Figure 3-11 Characterization of CM-derived exosomes 95Figure 3-12 Validated the number of exosomes in the GLA-null CMs cultured medium 96Figure 3-13 Vesicle turnover impairment induces cardiotoxicity in GLA-null CMs 97Figure 3-14 Assay of mitochondrial ROS level in GLA-null

CMs 98Figure 3-15 Assay of cell death in GLA-null CMs 99Figure 4-1. Characterization of an FD patient with the IVS4+919G>A mutation 119Figure 4-2 The generation of FD IVS4+919G>A patient-specific iPSCs 120Figure 4-3 The generation of FD IVS4+919G>A patient-specific iPSCs 121Figure 4-4

CRISPR/Cas9 Corrected FD GLA IVS4+919G>A mutated iPSCs 122Figure 4-5 Quantification of corrected and mutant cell number by ddPCR detection assay 123Figure 4-6 Screening the clones was transfected CRISPR/Cas9 corrected clones 124Figure 4-7 Confirmation of selected CRISPR/Cas9 mediated clone

s genotype 125Figure 4-8 Validation of GLA protein and enzyme activity in CRISPR/Cas9 mediated clones 126Figure 4-9 Characterization of the ECs morphology from isogenic ctrl and FD-iPSCs 127Figure 4-10 Characterization of the pluripotency from isogenic ctrl and FD-iPSCs 128Figure 4-11 Ch

aracterization of the ECs differentiated from isogenic ctrl and FD-iPSCs 129Figure 4-12 Characterization of the ECs differentiated ECs from isogenic ctrl and FD-iPSCs during shear stress 130Figure 4-13 Validation of GLA genotype in isogenic ctrl and FD iPSCs differentiated ECs 131Figure 4-1

4 Attenuations of GLA-deficient induced Gb3 accumulation in isogenic iPSCs-ECs by TEM assay 132Figure 4-15 Attenuations of GLA-deficient induced Gb3 accumulation in isogenic iPSCs-ECs by immunofluorescence 133Figure 4-16 Reversal of autophagic impairment in isogenic iPSCs-ECs 134Figure 4-17

Reversal of the expression of regulated autophagic protein expression in isogenic iPSCs-ECs 135Figure 4-18 Reversal of the autophagic vesicle and ROS in isogenic iPSCs-ECs 136Figure 4-19 Differential expression analyses of isogenic ctrl and FD-ECs derived from iPSCs 137Figure 4-20 Cytokine

releases and inflammatory status in isogenic ctrl and FD-ECs 138Figure 4-21 Cytokine releases and inflammatory gene expression in isogenic ctrl and FD-ECs 139Figure 4-22 Monocytes adhesion in isogenic ctrl compared with FD-ECs 140Figure 4-23 Isogenic ctrl-ECs attenuate NF-kBan

d MAPK signaling pathway in comparison to FD-ECs 141Table 1. List of primers used to amplify the predicted off-target gene loci. 100Table 2. Sequences of the primers used to analyze stemness markers by RT-PCR 101Table 3. Antibodies used in the Result 2 102Table 4. Summary of Healthy Cont

rols and Patients’ cell lines Used in the study. 142Table 5. On-target Summary 143Table 6. Different SNV/InDels summary for Off-target 144Table 7. Sequences of the primers used for Probe, RT-PCR, and qPCR 145Table 8. Antibodies used in the Result 3 147