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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蘇剛毅 | |
| dc.contributor.author | Chia-Hua Chou | en |
| dc.contributor.author | 周佳樺 | zh_TW |
| dc.date.accessioned | 2021-06-08T04:01:44Z | - |
| dc.date.copyright | 2018-09-04 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-06 | |
| dc.identifier.citation | 1. Liu, B.P. and S.M. Strittmatter, Semaphorin-mediated axonal guidance via Rho-related G proteins. Curr Opin Cell Biol, 2001. 13(5): p. 619-26.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22086 | - |
| dc.description.abstract | CRMP-1 (collapsin response mediator protein 1)屬於CRMP家族的成員之一，CRMP家族成員有CRMP1-5，其表現量皆具有高度專一性，他們是一群表現在細胞質中的磷蛋白質 (phosphoprotein)，並且高度表現於神經系統中。過去研究顯示CRMP-1會參與Sema 3A的訊息傳遞路徑而導致生長點潰縮 (growth cone collapse)，而缺乏CRMP-1也會影響小鼠的神經發育，進一步使小鼠的空間學習與記憶能力下降。另外，過去研究也發現，CRMP-1扮演著抑制癌細胞侵犯及轉移的角色，並且在非小細胞肺癌的病人中，CRMP-1表現量與存活率具有正相關。然而，有另一個CRMP-1的亞型被稱作LCRMP-1 (long-form CRMP-1)，LCRMP-1與CRMP-1不同之處為其N端的第一外顯子 (exon 1)以及分子量。而LCRMP-1在非小細胞肺癌中卻是扮演促進癌症侵犯及轉移的角色。但是至今我們對於LCRMP-1在生理上的功能卻仍不清楚。因此，我們利用基因剔除技術將小鼠的Lcrmp-1基因剔除，並且想利用此動物模型進一部探討LCRMP-1在生理上所扮演的角色。我們成功建立了Lcrmp-1基因剔除小鼠，並且確定剔除鼠可以在胚胎發育中發育成型，而且在出生後不會死亡。另外也確認基因剔除的小鼠沒有表現LCRMP-1蛋白。在成功建立基因剔除小鼠模型的同時，我們利用野生型小鼠來檢驗LCRMP-1及Short-form CRMP-1在正常小鼠體內的表現量分佈。我們發現LCRMP-1在小鼠的腦部中表現量最高，而在其他的器官中表現量都極低，而在LCRMP-1表現量次高的小鼠睪丸中，LCRMP-1相較於Short-form CRMP-1表現高出許多。這可能顯示LCRMP-1在小鼠腦部及睪丸扮演著重要的角色。另外我們將不同時期的野生型小鼠胚胎分離出來，並且觀察LCRMP-1以及Short-form CRMP-1在各個時期胚胎及器官中的表現量，結果顯示，LCRMP-1與Short-form CRMP-1的表現量變化具有相同的趨勢。他們皆是在晚期胚胎的腦中具有大量的表現，在出生前後達到最高表現，而在出生之後表現量就會降低。而在其他胚胎器官中，LCRMP-1及Short-form CRMP-1的表現量都極低。此外，在Lcrmp-1剔除鼠的組織病理學分析中，我們發現剔除鼠的腦部組織與野生型小鼠比較之下並無異常，然而卻發現剔除鼠的睪丸組織中有高比例出現生精小管中的精子生成異常情形。腦部神經細胞標記分佈情形的分析中，我們發現剔除鼠在數個神經標記的表現皆與野生型小鼠一致。而癲癇誘發實驗與酒精誘發腦部受損的實驗中皆無法引起LCRMP-1蛋白表現量的改變。我們再確認了Lcrmp-1剔除鼠中有高比例的生精小管精子生成異常後，也發現這些異常的小鼠的確無法產生具有正常形態及活動力的精細胞，這些異常的精細胞皆呈現圓形並取缺乏精子的尾巴。而且在統計Lcrmp-1剔除小鼠生育情況後，我們發現確實有部分基因剔除鼠具有不孕的情形。我們進一步檢驗剔除鼠的睪丸切片，發現生精小管中缺乏具有SCP3表現的初級精母細胞。也就是說，Lcrmp-1基因剔除小鼠會有高比例的精子生成異常可能是由於缺乏精母細胞的減數分裂所造成的。 | zh_TW |
| dc.description.abstract | CRMP-1 (collapsin response mediator protein 1) is one of the families of cytosolic phosphoproteins expressed exclusively in the nervous system. It is thought to be a part of the signal transduction pathway implicated in growth cone collapse during neural development. CRMP-1 has been reported to act as a tumor invasion and metastasis suppressor in non-small cell lung cancer (NSCLC). One of the CRMP-1 transcript variants, called long-form CRMP-1 (LCRMP-1) has recently been reported as a cancer invasion enhancer in NSCLC. They are different in the molecular weight and N-terminal exon 1. However, there is no one investigating the physiological function of LCRMP-1 in vivo. Here, we developed the Lcrmp-1 specific knockout mouse model to investigate the physiological function of LCRMP-1. We profiled LCRMP-1 mRNA expression level in major organs, and found that LCRMP-1 express much higher in several brain parts than other organs. In addition, the expression of LCRMP-1 in testis is second to brain, and it is much higher than short-form CRMP-1 expression in testis. Then, we isolated embryos and their organs form different embryonic stages and postnatal stages, and profiled the LCRMP-1 and short-form CRMP-1 expression. The results showed that LCRMP-1 expression was higher in the brain of late embryonic stage and the early postnatal stage. We have established the Lcrmp-1 knockout mouse model and confirmed its genotype in both gene and protein level. Histopathology examination in Lcrmp-1-/- mice showed normal in brain but loss of germ cell generation in testis. Therefore, we aimed to focus on brain and testis to explore the physiological function of LCRMP-1. We found there was no difference in brain neuronal markers expression between wild type and knockout mice. Furthermore, the seizure induction and ethanol uptake stress did not change the expression of LCRMP-1 in wild type mice. In the examination of testis sections and sperm smears, we found there were high percentage of knockout mice existing the abnormality of germ cell production and lack of motile spermatozoa. In addition, some of knockout mice were infertile in our observation. The SCP3 stain in the testis section implied the loss of primary spermatocytes might be associated with the deficient spermatogenesis and mice infertility. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T04:01:44Z (GMT). No. of bitstreams: 1 ntu-107-R05424001-1.pdf: 12684225 bytes, checksum: 41d3a31f4a0793777cb51a996352a076 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 口試委員會審定書 I
致謝 III ABSTRACT IV 中文摘要 VI 1. INTRODUCTION 1 1.1 Neuron development and axon guidance 2 1.2 The role of CRMP family in neuron development 3 1.3 Crmp-1 gene organization 4 1.4 CRMP-1 physiological function 5 1.5 CRMP-1 and cancers 6 1.6 Rationale 7 1.7 The central nervous system 7 1.7.1 Cortex 8 1.7.2 Prefrontal cortex 8 1.7.3 Hippocampus 8 1.7.4 Amygdala 9 1.7.5 Cerebellum 9 1.8 Stress induced neuronal damage 9 1.9 Mice reproductive system 9 1.9.1 Sertoli cell 10 1.9.2 Spermatogonium 10 1.9.3 Spermatocyte 11 1.9.4 Spermatid 11 1.10 Spermatogenesis and spermiogenesis 12 1.11 Male infertility 13 2. SPECIFIC AIM 14 3. MATERIALS AND METHODS 16 3.1 Knockout mice model and animal maintenance 17 3.2 Genotyping and PCR 17 3.3 RNA extraction and quantitative RT-PCR analysis 18 3.4 Protein extraction and western blot analysis 19 3.5 Mouse embryo isolation 20 3.6 Mouse histopathology sample preparation 20 3.7 Pathology interpretation 21 3.8 Immunofluorescence assay 21 3.9 Seizure induction procedure 22 3.10 Ethanol treated procedure 22 3.11 Mouse sperm isolation and smear preparation 22 3.12 Immunohistochemistry staining 23 3.13 Statistical analysis 24 4. RESULTS 25 4.1 Generation of LCRMP-1 deficient mice 26 4.2 Designing the Lcrmp-1 genotyping primers and PCR conditions 26 4.3 LCRMP-1 deficiency does not cause embryonic lethality. 27 4.4 Confirming of the LCRMP-1 expression of different genotypes 27 4.5 Definition of LCRMP-1 expression profile 28 4.6 Histopathological examination of mouse organs. 30 4.7 Neuronal marker expression pattern in WT and KO mice 31 4.8 Seizure induction and ethanol treated did not alter brain LCRMP-1 expression in wild type mouse. 32 4.9 The knockout mice exhibit high percentage of germ cell degeneration. 33 4.10 Calculation of the fertility rate in the Lcrmp-1 knockout mice. 34 4.11 Loss of synaptonemal complex protein in Lcrmp-1 knockout mice 35 5. DISCUSSION 36 5.1 The knockout strategy of LCRMP-1 deficient mice 37 5.2 The strategy of PCR primer designing 37 5.3 The LCRMP-1 deficient mice can grow up and breed offspring 38 5.4 The genotype was confirmed by protein expression. 39 5.5 LCRMP-1 is highly expressed in mouse brain 40 5.6 Histopathological examination of mice organs 41 5.7 Brain neuronal markers expression is normal in knockout mice. 42 5.8 The stress inductions do not alter the LCRMP-1 expression in wild type mice. 42 5.9 Testis sections and sperm smear show abnormality in spermatogenesis. 43 5.10 Some of the knockout mice exist infertility phenomenon. 45 5.11 Synaptonemal complex formation is deficient in Lcrmp-1 knockout mice. 45 6. CONCLUSION AND FUTURE PERSPECTIVES 47 7. FIGURES 49 8. TABLES 72 9. REFERENCES 79 APPENDIX 92 | |
| dc.language.iso | en | |
| dc.subject | 生精小管異常 | zh_TW |
| dc.subject | 小鼠不孕症 | zh_TW |
| dc.subject | 神經標記表現 | zh_TW |
| dc.subject | Lcrmp-1基因剔除小鼠 | zh_TW |
| dc.subject | 無精症 | zh_TW |
| dc.subject | neuronal markers pattern | en |
| dc.subject | mice infertility | en |
| dc.subject | seminiferous tubules abnormality | en |
| dc.subject | LCRMP-1 knockout mice | en |
| dc.subject | azoospermia | en |
| dc.title | 以基因剔除策略探討LCRMP-1在神經與精子生成之生理功能 | zh_TW |
| dc.title | Characterization of LCRMP-1 Physiological Function in Neuron and Spermatogenesis by Gene Targeting Strategy | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林亮音,楊雅倩 | |
| dc.subject.keyword | Lcrmp-1基因剔除小鼠,神經標記表現,生精小管異常,無精症,小鼠不孕症, | zh_TW |
| dc.subject.keyword | LCRMP-1 knockout mice,neuronal markers pattern,seminiferous tubules abnormality,azoospermia,mice infertility, | en |
| dc.relation.page | 95 | |
| dc.identifier.doi | 10.6342/NTU201802420 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2018-08-07 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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