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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王致恬 | |
dc.contributor.author | Yen-Ju Chen | en |
dc.contributor.author | 陳彦儒 | zh_TW |
dc.date.accessioned | 2021-06-17T07:04:31Z | - |
dc.date.available | 2024-07-31 | |
dc.date.copyright | 2019-07-31 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-07-29 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72723 | - |
dc.description.abstract | 帕金森氏症 (Parkinson’s Disease)為第二常見之神經退化性疾病,臨床症狀包括運動遲緩、靜止時震顫,以及肢體僵直。帕金森氏症主要的運動症狀源於位 在中腦黑質(substantia nigra)的多巴胺神經元 (dopamine neuron)死亡。已知黑 質中的多巴胺神經元的軸突末梢投射至位在基底核的紋狀體(striatum);當多巴胺 神經元於軸突末梢釋放神經傳導物質多巴胺時,紋狀體的神經元可依據不同類型 的多巴胺受器(dopamine receptor)接收這些來自黑質的訊號,進而給予調控運動 功能的上游指令。因此在帕金森氏症中,黑質的多巴胺神經元大量死亡,導致紋 狀體失去充足的多巴胺,進而造成運動症狀出現。此外,過去研究指出,不論是 遺傳性或偶發性的帕金森氏症,都牽涉路易氏體(Lewy bodies)在腦組織中的形 成。路易氏體是蛋白質在細胞中的堆積,研究發現其主要成分為胺基酸第 129 位 點之絲胺酸(Ser129)磷酸化的 α-突觸核蛋白 (α-Synuclein)堆積所致,且這些 異常堆疊的蛋白可在中樞神經系統中傳播,並特定地造成黑質多巴胺神經元死亡。 而在遺傳的研究也發現 α-突觸核蛋白的表現量(copy numbers)增加或是點突變都 可能導致帕金森氏症。然而現今關於 α-突觸核蛋白堆疊並造成多巴胺神經元死亡 的特定原因尚不清楚。因此在本篇研究中,我們期望在成年公鼠上建立一個可研 究帕金森氏症病程的模型。藉由立體定位儀手術及活體電穿孔技術,我們將帶有 HA 標記的外源基因轉染至成年公鼠的黑質中。轉染的 DNA 種類包含人類野生型 α-突觸核蛋白(模擬表現量增加)以及人類 α-突觸核蛋白 A53T 點突變。藉由免疫 螢光染色,我們發現 HA 以及第 129 位點之絲胺酸(Ser129)磷酸化的 α-突觸核蛋 白訊號出現在被轉染的黑質以及未被轉染的異側黑質,並且由黑質所投射的紋狀 體區域中也偵測到訊號;此外,將鼠腦分成六區進行西方墨點法,外源 α-突觸核蛋白和磷酸化之 α-突觸核蛋白的訊號分佈在數個區塊,這些結果表示外源表現之 α-突觸核蛋白在大鼠腦中可誘導產生堆積並傳播。接著,為了評估過量表現野生型 或突變型 α-突觸核蛋白後對運動功能的影響,我們藉由行為實驗分析大鼠步態, 分析的參數包括後肢腳掌角度(foot angle)、後肢步距(step length),以及後肢步 幅(stride length)。在術後 4 至 6 個月時,表現過量人類野生型 α-突觸核蛋白以及 人類 α-突觸核蛋白 A53T 點突變之大鼠後肢腳掌角度增加,表示大鼠維持動作平 衡的能力受到損傷。最後,由於多巴胺神經元大量死亡是帕金森氏症的病徵之一; 藉由免疫螢光染色,我們發現受術後的老鼠在 1 至 6 個月中,多巴胺神經元尚未 顯著減少,表示我們在大鼠模型中所觀察到的現象仍處在帕金森氏症的早期病程。 綜合上述,我們認為以活體電穿孔技術表現人類致病 α-突觸核蛋白基因於成年公 鼠,可使 α-突觸核蛋白產生堆積後於腦中傳播,並且在行為上出現早期病徵,藉 此可以做為研究如何延緩帕金森氏症的模型。 | zh_TW |
dc.description.abstract | Parkinson’s disease (PD) is the second most common neurodegenerative disease, clinically characterized by bradykinesia, resting tremor, and rigidity. The etiology of PD is attributed to the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc), further decreasing the release of dopamine (DA) through the nigrostriatal pathway in the striatum, where dopamine binds to different subtypes of dopamine receptors, producing various responses (activation/inhibition) for the fine control of locomotor activity. Loss of these dopamine neurons in substantia nigra pars compacta results in insufficient levels of dopamine, thus breaking the balance of neurotransmission in the striatum. Specifically, the hallmark in both familial and sporadic PD is the presence of intra-neuronal proteinaceous inclusions, termed “Lewy Bodies”, mainly composed by phosphorylated alpha-Synuclein (α-Syn), which can be distributed across the whole brain during PD progression. However, how α-Syn aggregates propagate across brain or other systems remains unclear. In this study, we aim to establish a rat model to mimic PD during adulthood. With the technique of in vivo electroporation, we successfully transfected the pathological forms of human α-Syn (hα-Syn and hα-Syn-A53T) in the rat SNc. The results from immunostaining and immunoblotting showed that ectopic α-Syn and phosphorylated α-Syn (α-Syn-pS129) appeared in both sites of SNc, CPu, and other brain regions that were far away from the injected site, suggesting the ability of pathological α-Syn to propagate and aggregate within the CNS. In order to assess locomotor activities upon PD progression, we conducted the gait pattern analysis and found that foot angle was increased at 4-6 months post transfection of one-side SNc with hα-Syn or hα-Syn-A53T, indicating that the impaired ability in maintaining balance caused by these pathogenic α-Syn forms. Moreover, the numbers of DA neurons within SNc remained unchanged by transfection of these pathological α-Syn forms, implying that this model reflects an early stage of PD progression. Overall, this in vivo electroporation technique can be used as a gene delivery method to introduce the human pathogenic α-Syn in adult rats, serving as a suitable model in searching therapeutic methods for delaying the early-stage PDs. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T07:04:31Z (GMT). No. of bitstreams: 1 ntu-108-R06B43015-1.pdf: 58904122 bytes, checksum: 4f2ee59c08c1bf1d8432046270ddda12 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 口試委員會審定書 i
致謝 ii 中文摘要 iii Abstract v Contents vii Introduction 1 1. Parkinson’s disease 1 1-1. Prevalence of PD 1 1-2. Etiology of PD 1 1-3. Pathophysiology of PD 2 2. Alpha-Synuclein (α-Syn) 3 2-1. Structure and function of α-Syn 3 2-2. Toxicity of aggregated α-Syn 4 2-3. Propagation of α-Syn 5 3. Previous animal models for the study of PD 6 3-1. Neurotoxin-induced PD models 6 3-2. Transgenic PD models 8 3-3. The α-Syn preformed fibril-induced PD models 8 4. Assessment of gait patterns 9 5. Purpose of the study 10 Materials and Methods 12 2.1 Animals 12 2.2 Plasmid construction and subcloning 12 2.3 Stereotaxic surgery 16 2.4 Heart perfusion and brain fixation 17 2.5 Cryostat section 19 2.6 Immunofluorescence staining 19 2.7 Preparation of brain tissue 20 2.8 Immunoblotting 21 2.9 Behavioral assessment 23 2.10 Gait analysis 23 2.11 Statistics 24 Results 25 3.1 The immunoreactivity of iGluSnFR indicates the success of the in vivo electroporation for adult rat transfection. 25 3.2 In vivo electroporation allows overexpression of hα-Syn and hα-Syn-A53T in the adult rat SNc. 25 3.3 Immunoreactivity of syn211 is detectable within the injected SNc after several months post transfection. 27 3.4 Overexpressing hα-Syn or hα-Syn-A53T induces the aggregation of α-Syn in the rat SNc at 5-7 days post transfection. 28 3.5 Immunoreactivity of pS129 was remarkably increased after 2 months post transfection. 29 3.6 Aggregated α-Syn can be detected in the striatum. 30 3.7 Signals of ectopic α-Syn appeared in the caudate putamen (CPu). 31 3.8 Ectopic α-Syn may propagate across brain after transfection. 32 3.9 The aggregates of α-Syn may propagate (detected by the signals of pS129) across the brain after transfection with hα-Syn-A53T. 33 3.10 The numbers of dopamine neurons within SNc remained unchanged. 33 3.11 Gait pattern analysis 34 3.11.1 Foot Angle 35 3.11.2 Step Length 37 3.11.3 Stride Length 38 3.12 Changes in body after transfection 40 Discussion 41 4.1 Potential for α-Syn to propagate in the CNS. 42 4.2 Size of α-Syn and pS129 in immunoblotting. 43 4.3 The TH immunoreactivity of DA neurons remained similar after overexpressing the pathological forms of α-Syn. 44 4.4 The hα-Syn-A53T groups displays a stronger effect on step/stride length. 45 4.5 Pros and cons of our PD rat model and future directions. 46 Conclusion 48 References: 49 Figure 1. Hallmarks of Parkinson’s disease. 55 Figure 2. The nigrostriatal pathway in the rat. 56 Figure 3. Alpha-Synuclein (α-Syn) plays a critical role in PD progression. 57 Figure 4. Plasmid DNA construction. 59 Figure 5. Stereotaxic surgery and in vivo electroporation. 60 Figure 6. Schematic flowchart for the experimental protocol. 61 Figure 7. The diagram of the gait pattern analysis system for rats. 62 Figure 8. In vivo electroporation successfully allows ectopic iGluSnFR expressed in the rat SNc. 63 Figure 9. In vivo electroporation successfully allows ectopic human α-Syn expressed in the rat SNc. 64 Figure 10. Signals of ectopic α-Syn appeared around the SNc but not in cortex and hippocampus. 65 Figure 11. hα-Syn or hα-Syn-A53T was overexpressed in SNc-DA neurons by in vivo electroporation. 67 Figure 12. α-Syn was slightly aggregated in the rat SNc at 5-7 days after transfection. 68 Figure 13. Signals of pSer129 can be barely detected within SNc from the rat transfected with HA. 69 Figure 14. α-Syn formed aggregates in the SNc after overexpressing hα-Syn. 70 Figure 15. α-Syn formed aggregates in the SNc after overexpressing hα-Syn-A53T. 71 Figure 16. Overexpressing hα-Syn or hα-Syn-A53T induced the high pS129 immunoreactivity (α-Syn aggregation) in the both sides of SNc. 72 Figure 17. The pS129 immunoreactivity for α-Syn was detectable in both sides of CPu after transfection. 74 Figure 18. Ectopic α-Syn spread to the ipsilateral CPu after expressing hα-Syn in the SNc at 6 days post transfection. 75 Figure 19. Ectopic α-Syn spread to the ipsilateral CPu after expressing hα-Syn-A53T in the SNc at 112 days post transfection. 76 Figure 20. The immunoreactivity of syn211 was shown in the CPu at 4-5 months post transfection. 78 Figure 21. Ectopic α-Syn can be detected in remote regions of the brain after transfection of SNc with hα-Syn-A53T. 80 Figure 22. The syn211 immunoreactivity showed the specificity for detecting the human form of α-Syn. 81 Figure 23. α-Syn-pS129 can be detected in the brains of the rats transfected with hα-Syn-A53T. 82 Figure 24. The numbers of dopamine neurons within SNc did not significantly decrease after transfection with HA. 83 Figure 25. The numbers of dopamine neurons within both sides of SNc remained unchanged after transfection of hα-Syn. 84 Figure 26. The numbers of dopamine neurons within both sides of SNc remained unchanged after transfection of hα-Syn-A53T. 85 Figure 27. Foot angle was changed after transfection of hα-Syn or hα-Syn-A53T. 87 Figure 28. The left hindlimb had the more obvious effect compared to the right hindlimb after transfection. 88 Figure 29. Step length was changed after in vivo electroporation. 90 Figure 30. Step length was decreased in the rats transfected with hα-Syn-A53T in the SNc. 92 Figure 31. Alteration of stride length after in vivo transfection. 94 Figure 32. Stride length was decreased in the rats transfected with hα-Syn-A53T in the SNc. 96 Figure 33. Body weight was changed at 4-6 months after in vivo transfection. 97 Figure 34. Establishing a rat model for PD with in vivo electroporation. 98 Table 1. Primers for PCR cloning 99 Table 2. Antibodies for immunofluorescence staining 100 Table 3. Antibodies for western blot 101 Appendix Figure 1. Signals of pS129 were present in the SNc and hippocampus of the rat transfected with hα-Syn-A53T at 61 days post transfection. 103 Appendix Figure 2. Signals of pS129 were present in the SNc, CPu, and hippocampus of the rat transfected with hα-Syn-A53T at 112 days post transfection. 105 Appendix Figure 3. Signals of pS129 were present in the SNc, CPu, and hippocampus of the rat transfected with hα-Syn at 179 days post transfection. 107 Appendix Figure 4. Signals of pS129 can barely detected within the SNc, CPu, and hippocampus of the rat transfected with HA at 40 days post transfection. 109 Appendix Figure 5. Signals of pS129 can barely detected within the SNc, CPu, and hippocampus of the rat transfected with HA at 159 days post transfection. 111 Society of Neuroscience (SfN) 2018 Abstract and Poster 112 Institute of Molecular and Cellular Biology (IMCB) 2019 Poster 115 | |
dc.language.iso | en | |
dc.title | 建立一種新型成年大鼠模型以研究帕金森病的進程 | zh_TW |
dc.title | Establishing a new adult rat model to study the progression of Parkinson’s disease | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 胡孟君,徐立中,盧主欽 | |
dc.subject.keyword | α-突觸核蛋白,帕金森氏症,步態分析,黑質紋狀體迴路,活體電穿孔, | zh_TW |
dc.subject.keyword | α-Synuclein,Parkinson’s disease,gait pattern analysis,nigrostriatal pathway,in vivo electroporation, | en |
dc.relation.page | 115 | |
dc.identifier.doi | 10.6342/NTU201902096 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-07-29 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
顯示於系所單位: | 分子與細胞生物學研究所 |
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