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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 錢宗良 | zh_TW |
| dc.contributor.advisor | Chung-Liang Chien | en |
| dc.contributor.author | 龔加鳳 | zh_TW |
| dc.contributor.author | Chia-Feng Kung | en |
| dc.date.accessioned | 2023-03-27T17:02:45Z | - |
| dc.date.available | 2023-11-09 | - |
| dc.date.copyright | 2023-03-20 | - |
| dc.date.issued | 2022 | - |
| dc.date.submitted | 2022-12-23 | - |
| dc.identifier.citation | Brisson BK and Barton ER (2013). New modulators for IGF-I activity within IGF-I processing products. Front Endocrinol (Lausanne), 4: 42.
Chen SJ, Tsai JC, Lin TY, Chang CK, Tseng TH and Chien CL (2012). Brain-derived neurotrophic factor-transfected and nontransfected 3T3 fibroblasts enhance migratory neuroblasts and functional restoration in mice with intracerebral hemorrhage. J Neuropathol Exp Neurol, 71: 1123-1136. Chen W, He B, Tong W, Zeng J and Zheng P (2019). Astrocytic insulin-like growth factor-1 protects neurons against excitotoxicity. Front Cell Neurosci, 13: 298. Chien CL, Liu TC, Ho CL and Lu KS (2005). Overexpression of neuronal intermediate filament protein alpha-internexin in PC12 cells. J Neurosci Res, 80: 693-706. Choi JE, Lee SS, Sunde DA, Huizar I, Haugk KL, Thannickal VJ, Vittal R, Plymate SR and Schnapp LM (2009). Insulin-like growth factor-I receptor blockade improves outcome in mouse model of lung injury. Am J Respir Crit Care Med, 179: 212-219. Chou PC, Tsai YC, Chen SJ, Tsai LK and Chien CL (2019). Intracerebral transplantation of erythropoietin-producing fibroblasts facilitates neurogenesis and functional recovery in an ischemic stroke model. Brain Behav, 9: e01274. Costales J and Kolevzon A (2016). The therapeutic potential of insulin-like growth factor-1 in central nervous system disorders. Neurosci Biobehav Rev, 63: 207-222. D'Ercole AJ, Applewhite GT and Underwood LE (1980). Evidence that somatomedin is synthesized by multiple tissues in the fetus. Dev Biol, 75: 315-328. DeTure MA and Dickson DW (2019). The neuropathological diagnosis of Alzheimer's disease. Mol Neurodegener, 14: 32. Digicaylioglu M, Garden G, Timberlake S, Fletcher L and Lipton SA (2004). Acute neuroprotective synergy of erythropoietin and insulin-like growth factor I. Proc Natl Acad Sci U S A, 101: 9855-9860. Didonna A and Opal P (2019). The role of neurofilament aggregation in neurodegeneration: lessons from rare inherited neurological disorders. Mol. Neurodegener. , 14. Doré S, Kar S and Quirion R (1997). Insulin-like growth factor I protects and rescues hippocampal neurons against beta-amyloid- and human amylin-induced toxicity. Proc Natl Acad Sci U S A, 94: 4772-4777. Fernandez AM and Torres-Aleman I (2012). The many faces of insulin-like peptide signalling in the brain. Nat Rev Neurosci, 13: 225-239. Greene LA and Tischler AS (1976). Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A, 73: 2424-2428. Hamaguchi Y, Fujimoto M, Matsushita T, Hasegawa M, Takehara K and Sato S (2008). Elevated serum insulin-like growth factor (IGF-1) and IGF binding protein-3 levels in patients with systemic sclerosis: possible role in development of fibrosis. J Rheumatol, 35: 2363-2371. Hung CF, Rohani MG, Lee SS, Chen P and Schnapp LM (2013). Role of IGF-1 pathway in lung fibroblast activation. Respir Res, 14: 102. Kasprzak A and Szaflarski W (2020). Role of Alternatively Spliced Messenger RNA (mRNA) Isoforms of the insulin-like growth factor 1 (IGF1) in selected human tumors. Int J Mol Sci, 21. Kim I, Kim CH, Yim YS and Ahn YS (2008). Autocrine function of erythropoietin in IGF-1-induced erythropoietin biosynthesis. Neuroreport, 19: 1699-1703. Lee WC, Chen YY, Kan D and Chien CL (2012). A neuronal death model: overexpression of neuronal intermediate filament protein peripherin in PC12 cells. J. Biomed. Sci., 19: 8. Li YC, Chen SJ and Chien CL (2015). Erythropoietin produced by genetic‐modified NIH/3T3 fibroblasts enhances the survival of degenerating neurons. Brain and Behavior, 5. Lunn JS, Sakowski SA, Hur J and Feldman EL (2011). Stem cell technology for neurodegenerative diseases. Ann Neurol, 70: 353-361. Mascotti F, Cáceres A, Pfenninger KH and Quiroga S (1997). Expression and distribution of IGF-1 receptors containing a beta-subunit variant (betagc) in developing neurons. J Neurosci, 17: 1447-1459. Nieto-Estevez V, Defterali C and Vicario-Abejon C (2016). IGF-I: a key growth factor that regulates neurogenesis and synaptogenesis from embryonic to adult stages of the brain. Front Neurosci, 10: 52. Oberbauer AM (2013). The regulation of IGF-1 gene transcription and splicing during development and aging. Frontiers in Endocrinology, 4. Okada N, Kawakita T, Mishima K, Saito I, Miyashita H, Yoshida S, Shimmura S and Tsubota K (2011). Clusterin promotes corneal epithelial cell growth through upregulation of hepatocyte growth factor by mesenchymal cells in vitro. Invest Ophthalmol Vis Sci, 52: 2905-2910. Pan W and Kastin AJ (2000). Interactions of IGF-1 with the blood-brain barrier in vivo and in situ. Neuroendocrinology, 72: 171-178. Park WJ, You SH, Choi HA, Chu YJ and Kim GJ (2015). Over-expression of recombinant proteins with N-terminal His-tag via subcellular uneven distribution in Escherichia coli. Acta Biochim Biophys Sin (Shanghai), 47: 488-495. Philippou A, Maridaki M, Pneumaticos S and Koutsilieris M (2014). The complexity of the IGF1 gene splicing, posttranslational modification and bioactivity. Mol Med, 20: 202-214. Poreba E and Durzynska J (2020). Nuclear localization and actions of the insulin-like growth factor 1 (IGF-1) system components: transcriptional regulation and DNA damage response. Mutat Res Rev Mutat Res, 784: 108307. Puche JE and Castilla-Cortázar I (2012). Human conditions of insulin-like growth factor-I (IGF-I) deficiency. J. Transl. Med, 10: 224. Rotwein P (2017). Diversification of the insulin-like growth factor 1 gene in mammals. PLoS One, 12: e0189642. Sivandzade F and Cucullo L (2021). Regenerative stem cell therapy for neurodegenerative diseases: an overview. Int. J. Mol. Sci., 22. Slavin BR, Sarhane KA, von Guionneau N, Hanwright PJ, Qiu C, Mao H-Q, Höke A and Tuffaha SH (2021). Insulin-like growth factor-1: a promising therapeutic target for peripheral nerve injury. Front. bioeng. biotechnol., 9. Subirós N, Del Barco DG and Coro-Antich RM (2012). Erythropoietin: still on the neuroprotection road. Ther Adv Neurol Disord, 5: 161-173. Takeda N, Manabe I, Uchino Y, Eguchi K, Matsumoto S, Nishimura S, Shindo T, Sano M, Otsu K, Snider P, Conway SJ and Nagai R (2010). Cardiac fibroblasts are essential for the adaptive response of the murine heart to pressure overload. J Clin Invest, 120: 254-265. Takeuchi T, Wang L, Mori S, Nakagawa K, Yoshikura H and Kanda T (2008). Characterization of mouse 3T3-swiss albino cells available in Japan: necessity of quality control when used as feeders. Jpn J Infect Dis, 61: 9-12. Todaro GJ and Green H (1963). Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol, 17: 299-313. Xu K and Rubin H (1990). Cell transformation as aberrant differentiation: Environmentslly dependent spontaneous transformation of NIH 3T3 cells. Cell Res., 1: 197-206. Yoneyama Y, Lanzerstorfer P, Niwa H, Umehara T, Shibano T, Yokoyama S, Chida K, Weghuber J, Hakuno F and Takahashi S-I (2018). IRS-1 acts as an endocytic regulator of IGF-I receptor to facilitate sustained IGF signaling. eLife, 7:e32893.. Yuan A, Rao MV, Sasaki T, Chen Y, Kumar A, Veeranna, Liem RK, Eyer J, Peterson AC, Julien JP and Nixon RA (2006). Alpha-internexin is structurally and functionally associated with the neurofilament triplet proteins in the mature CNS. J Neurosci, 26: 10006-10019. Yuan LJ, Wang XW, Wang HT, Zhang M, Sun JW and Chen WF (2019). G protein-coupled estrogen receptor is involved in the neuroprotective effect of IGF-1 against MPTP/MPP (+)-induced dopaminergic neuronal injury. J Steroid Biochem Mol Biol, 192: 105384. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/86803 | - |
| dc.description.abstract | 類胰島素生長因子 (Insulin-like growth factor-1, IGF-1) 是一種多胜肽激素，在人體中有許多組織會生成製造。IGF-1被認為和發育、生長、分化和神經保護作用有關。IGF-1能夠通過血腦障壁 (Blood-brain barrier)，而因為這項特徵使得IGF-1被認為是一種具有潛力治療神經疾病的因子。另一方面來說，有許多類型的細胞像是纖維母細胞具有生成及釋放IGF-1。在我們實驗室先前的研究，轉染入腦衍生神經滋養因子 (brain-derived neurotrophic factor, BDNF) 或紅血球生成素 (Erythropoietin, EPO) 的NIH/3T3纖維母細胞株皆可以過量表達其轉染入的蛋白。而根據研究IGF-1具有的神經保護效果，本研究首先將嘗試建立過量表達IGF-1的NIH/3T3纖維母細胞株。
在本論文中，我們有兩個主要目的：(一) 建立可以過量表達IGF-1的纖維母細胞株；(二) 測試此過量表達IGF-1細胞株所釋放的IGF-1可能的生理功能。兩種分別帶有N端組氨酸標籤以及C端組氨酸標籤的IGF-1質體轉染入NIH/3T3纖維母細胞株。轉染的細胞經過濕黴素 (hygromycin) 篩選後，我們建立了兩株可以過量表達IGF-1的纖維母細胞株 (IGF1-3T3)。我們利用了逆轉錄聚合酶鏈式反應 (RT-PCR)、逆轉錄即時聚合酶鏈式反應 (RT-QPCR)、細胞免疫染色、西方墨點法 (Western-blot assay) 以及酶聯免疫吸附試驗 (ELISA) 等實驗方法對IGF1-3T3細胞株所釋放的IGF-1進行定性及定量分析。帶有組氨酸標籤的IGF-1 mRNA可經由RT-QPCR證實在細胞內表現量明顯增加。而在IGF1-3T3細胞株的培養液中也經由ELISA證實IGF-1分泌量亦明顯的增加。 為了測試IGF1-3T3釋放的IGF-1生物活性，我們利用IGF1-3T3的培養液和神經退化疾病細胞模式的細胞株，PC12-INT-EGFP。在利用神經生長因子 (Nerve growth factor, NGF) 誘導PC12-INT-EGFP細胞分化的第六天，給予含有IGF1-3T3培養液的條件培養基。經過培養48小時後，我們觀察到在含有IGF-1的條件培養基的組別中，可觀察到PC12-INT-EGFP細胞突起具有較高強度的中間絲蛋白綠色螢光，實驗顯示IGF-1具有影響神經細胞之生理功能。 本研究結果成功建立了兩株可以過量表達IGF-1的3T3纖維母細胞株，分別是N-His IGF1-3T3以及C-His IGF1-3T3。兩株細胞株經由實驗證實均可以釋放大量的IGF-1。因此，本研究所建立過量表達IGF-1的纖維母細胞株，將可以做為未來在研究神經疾病動物模式上一個可行測試的生物材料。 | zh_TW |
| dc.description.abstract | Insulin-like growth factor-1 (IGF-1) is a polypeptide hormone which is produced by multiple tissues in human. IGF-1 is considered to be related to development, growth, differentiation and neuroprotection. According to the characteristic of IGF-1 which is able to across the blood-brain barrier and have trophic effect on neuron, IGF-1 is regarded as a promising candidate to treat the neurological diseases. On the other hand, IGF-1 is suggested to be secreted from multiple cell types such as fibroblasts. In our laboratory, NIH/3T3 cells transfected with brain-derived neurotrophic factor (BDNF) or erythropoietin (EPO) could overexpress the protein respectively. Based on the neuroprotective effect of IGF-1 and the potential therapeutic effects of cell therapy on neurological diseases, we would first like to establish an IGF1-overexpressed 3T3 cell lines.
In this study, we aimed to (1) establish IGF-1 overexpressed NIH/3T3 fibroblast cell lines and (2) examine the potential functional effects of IGF-1 secreted from IGF1-3T3 stable clones. Two plasmid DNA, N-terminal polyhistidine-tagged IGF-1 and C-terminal polyhistidine-tagged IGF-1, were transfected into NIH/3T3 cells respectively. Two stable IGF-1 overexpressed NIH/3T3 fibroblast cell lines were established after selected by hygromycin. IGF-1 secreted from two IGF-1 overexpressed cell lines were examined by reverse-transcriptase PCR (RT-PCR), reverse-transcriptase quantitative real-time PCR (RT-QPCR), immunocytochemistry, Western-blot assay and enzyme-linked immunosorbent assay (ELISA). The polyhistidine tagged IGF-1 cDNA fragment from transfected cells was detected by PCR and higher mRNA level could be demonstrated by RT-QPCR. The significantly higher concentration of secreted IGF-1 was also detected in the culture supernatants of two IGF1-3T3 cell lines by ELISA analysis. Subsequently, we examined the bioactivity of secreted IGF-1 via culturing the cell model of neurodegeneration, PC12-INT-EGFP cell line with conditioned media. The conditioned media, which were comprised of the culture supernatant of IGF1-3T3 cells, was supplied to PC12-INT-EGFP cells on the sixth day after induced by nerve growth factor (NGF). After culturing with conditioned media for 48 hours, stronger green fluorescence intensity in the processes of NGF-induced PC12-INT-EGFP cells could be observed in the groups supplied with IGF-1. From these observations, it is suggested that IGF-1 might have functional effects in the PC12-INT-EGFP cell model. In summary, we established two IGF1-overexpressed 3T3 cell lines, N-His IGF1-3T3 and C-His IGF1-3T3, and the abundant secreted IGF-1 was well demonstrated. The results of functional assay also suggest that the secreted IGF-1 exert bioactivity. Therefore, the IGF1-overexpressed cell lines we established in this study might be offered as potential biomaterials for the study of animal models with neurological diseases. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-03-27T17:02:45Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2023-03-27T17:02:45Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
誌謝 ii 摘要 iii Abstract v Contents viii List of Figures ix List of Tables xi Abbreviation xii Chapter 1: Introduction 1 Chapter 2: Materials and Methods 6 Chapter 3: Results 19 Chapter 4: Discussion 28 Figures and Figure Legends 33 Tables 55 Supplementary Figures 58 References 76 | - |
| dc.language.iso | en | - |
| dc.title | 建立過度表達類胰島素生長因子的NIH/3T3纖維母細胞株以應用在未來之治療 | zh_TW |
| dc.title | Establishment of IGF-1 overexpressed NIH/3T3 fibroblast cell lines for the potential therapeutic application | en |
| dc.title.alternative | Establishment of IGF-1 overexpressed NIH/3T3 fibroblast cell lines for the potential therapeutic application | - |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-1 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 彭偉豪;廖孟琳 | zh_TW |
| dc.contributor.oralexamcommittee | Wei-Hao Peng;Meng-Lin Liao | en |
| dc.subject.keyword | 類胰島素生長因子,組氨酸標籤,NIH/3T3纖維母細胞株,過量表達,PC12細胞株, | zh_TW |
| dc.subject.keyword | IGF-1,polyhistidine tag,NIH/3T3 fibroblast cell line,overexpression,PC12 cell line, | en |
| dc.relation.page | 81 | - |
| dc.identifier.doi | 10.6342/NTU202210167 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2022-12-26 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 解剖學暨細胞生物學研究所 | - |
| dc.date.embargo-lift | 2027-11-25 | - |
| 顯示於系所單位： | 解剖學暨細胞生物學科所 | |
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