萊伯氏遺傳視神經病變 (LHON) 是首個被發現由粒線體 DNA (mtDNA)點突變引起的疾病，該疾病普遍於青少年時期發病，且好發於男性。該疾病會造成無痛的急性單眼或雙眼的視力下降並伴隨著中心視野缺損與變色能力下降。突變的粒線體DNA會導致呼吸鏈複合物1(Respiratory Complex 1)功能受損，造成活性氧 (ROS)過度上升，進而使得視神經內的視網膜神經節細胞 (RGC) 凋亡。檢查LHON患者的眼底血管呈現了水腫與曲折的外觀，這是LHON的特徵。在一些報導中指出少數LHON患者中會伴隨一些其他症狀，例如肌張力障礙、心臟功能障礙等。至今為止LHON 與血管之間的調控機制與關係尚未明朗。人類臍靜脈內皮細胞 (HUVEC) 是血管生成研究中常用的內皮細胞類型。我們利用魚藤酮 (Rotenone)建立了一個體外模型樣本來模擬人類誘導型多能幹細胞 (hIPSC)所生成的LHON的疾病模型，並且使用HUVEC建立了LHON患者眼外的血管體外模型。我們進行了血管新生實驗與細胞陣列因子實驗。與對照組相比，經過魚藤酮模擬的LHON疾病模型所生成的血管新生能力較對照組來的低。進一步進行血管新生陣列因子實驗 (Human Angiogenesis Array)發現一些與血管新生相關的細胞因子表達下降。總體而言我們的研究對於LHON疾病與血管間的關係有著不少的貢獻，這對於未來的治療策略中提出了另一種方向。

Leber's Hereditary Optic Neuropathy (LHON) is the first disease found to be caused by point mutations in mitochondrial DNA (mtDNA). The disease usually occurs in adolescence and predominantly affects males. Symptoms include painless acute loss of vision, central visual field loss, and decreased ability to distinguish colors. A mutation in mitochondrial DNA can impair the function of respiratory complex 1, leading to an increase in reactive oxygen species (ROS). This, in turn, results in apoptosis in retinal ganglion cells (RGCs) in the optic nerve. Examination of fundus blood vessels in LHON patients has shown dilation and tortuosity of optic nerve head blood vessels, which are characteristic features of LHON. Additionally, a small number of LHON patients have been reported to experience other symptoms such as dystonia or cardiac dysfunction. The relationship between LHON and blood vessels remains unclear. Human umbilical vein endothelial cells (HUVECs) are commonly used in angiogenesis studies as a representative endothelial cell type. To simulate the LHON disease model using human induced pluripotent stem cells (IPSCs), we employed rotenone and HUVECs to establish an in vitro model of blood vessels outside the eye for LHON patients. We conducted a tube formation assay and utilized a human angiogenesis array. Compared to the control group, the rotenone-induced LHON disease model exhibited a lower ability to induce angiogenesis. Further studies using the human angiogenesis array revealed a decrease in the expression of certain cytokines associated with angiogenesis. In conclusion, our research has made significant contributions to understanding the correlation between LHON disease and blood vessels, suggesting a new direction for future treatment strategies.

Abstract I
中文摘要 II
Abbreviation III
誌謝 IV
Table of Contents VI
Chapter 1: Introductio 1
1.1 The blood supply in eyes 2
1.2 Leber’s hereditary optic neuropathy 2
1.3 Vascular features in patients with LHON 3
1.4 The extraocular symptoms in LHON patients 3
1.5 Mitochondrial Apoptosis 4
1.6 Modeling LHON using IPSC-derived retinal organoids 4
1.7 Modeling LHON by Rotenone-Induced Mitochondrial dysfunction 5
1.8 Human umbilical vein endothelial cells 6
1.9 Motivation and Specific Aims 6
Chapter 2: Materials and Methods 8
2.1 Organize the method section by categories generation of hIPSC 9
2.2 Alkaline phosphatase staining 9
2.3 Immunofluorescence staining 10
2.4 Differentiation of IPSCs into RGCs 11
2.5 Reverse transcription polymerase chain reaction (RT-PCR) 12
2.6 Maintenance of Human umbilical vein endothelial cell (HUVEC) cell line 12
2.7 Cell Viability/Cytotoxicity Assay 12
2.8 Mitochondrial superoxide assay 13
2.9 Mitochondrial membrane potential assay 13
2.10 Annexin V staining 13
2.11 Conditioned medium preparation 14
2.12 Tube Formation Assays 14
2.13 Human Angiogenesis Array and Human Cytokine Array 15
2.14 Statistics Analysis 15
Chapter 3: Results 16
3.1 Generation of the PBMC-Derived iPSC 17
3.2 Differentiation of iPSC into RGCs 18
3.3 Rotenone induces RGC degeneration as the LHON patient's disease model. 19
3.4 Cytotoxicity effect of Rotenone on HUVECs 20
3.5 Assessment of the angiogenic capacity of HUVECs under different conditioned mediums. 21
3.6 Analysis of the Secretomes from Normal or Rotenone-treated RGCs 23
Chapter 4: Discussion 25
Chapter 5: Figures 31
Chapter 6: References 59

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