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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 謝松蒼 | |
| dc.contributor.author | Ming-Fong Chang | en |
| dc.contributor.author | 張銘峰 | zh_TW |
| dc.date.accessioned | 2021-06-08T03:12:00Z | - |
| dc.date.copyright | 2017-09-08 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-03-10 | |
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In vivo gene delivery to the lung using polyethylenimine and fractured polyamidoamine dendrimers. J Gene Med 2, 269-278, doi:10.1002/1521-2254(200007/08)2:4<269::AID-JGM112>3.0.CO;2-F (2000). 33 Zimmermann, M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16, 109-110 (1983). 34 Chiang, H. Y. et al. Reinnervation of muscular targets by nerve regeneration through guidance conduits. J Neuropathol Exp Neurol 64, 576-587 (2005). 35 Rubinson, D. A. et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 33, 401-406, doi:10.1038/ng1117 (2003). 36 Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263-267 (1996). 37 Dull, T. et al. A third-generation lentivirus vector with a conditional packaging system. J Virol 72, 8463-8471 (1998). 38 Blomer, U., Naldini, L., Verma, I. 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Comparison of adeno-associated viral vector serotypes for spinal cord and motor neuron gene delivery. Hum Gene Ther 22, 1129-1135, doi:10.1089/hum.2011.008 (2011). 53 Lonez, C., Vandenbranden, M. & Ruysschaert, J. M. Cationic lipids activate intracellular signaling pathways. Adv Drug Deliv Rev 64, 1749-1758, doi:10.1016/j.addr.2012.05.009 (2012). 54 Kabanov, A. V. Polymer genomics: an insight into pharmacology and toxicology of nanomedicines. Adv Drug Deliv Rev 58, 1597-1621, doi:10.1016/j.addr.2006.09.019 (2006). 55 Akhtar, S. Cationic nanosystems for the delivery of small interfering ribonucleic acid therapeutics: a focus on toxicogenomics. Expert Opin Drug Metab Toxicol 6, 1347-1362, doi:10.1517/17425255.2010.518611 (2010). 56 O'Mahony, A. M., Godinho, B. M., Cryan, J. F. & O'Driscoll, C. M. Non-viral nanosystems for gene and small interfering RNA delivery to the central nervous system: formulating the solution. J Pharm Sci 102, 3469-3484, doi:10.1002/jps.23672 (2013). 57 Smith, G. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/20950 | - |
| dc.description.abstract | 對背根神經節進行基因轉殖是可以被應用於治療周邊神經病變的一個有力的治療方式，然而過去的進行方式如背根神經節內注射或脊髓穿刺都存在有許多缺點，包括有容易造成神邊神經損傷或轉殖效益低落等缺點。所以在我們的研究中採用聚乙烯亞胺(PEI)混合可表現綠色螢光(GFP)之DNA植體的方式並透過脊神經注射來達成背根神經節之基因轉殖。在轉殖後一週，我們可發現在脊神經注射組有 82.8% ± 1.70% 的神經細胞試轉殖成功比對背根神經節內注射組別也有(81.3% ± 5.1%, p = 0.82)相較於脊髓穿刺組別有非常高之基因轉殖成功率。此外GFP 在背根神經節內的大神經細胞與小神經細胞的轉殖效益也是相同的。另外在安全性測試中，我們也證實透過脊神經注射不會引起神經損傷、發炎、行為改變等變化。我們的結果證明透過脊神注射聚乙烯亞胺(PEI)混合DNA植體來進行背根神經節的基因轉殖是一個安全且有效的方法。 | zh_TW |
| dc.description.abstract | Delivering gene constructs into the dorsal root ganglia (DRG) is a powerful but challenging therapeutic strategy for sensory disorders affecting the DRG and their peripheral processes. The current delivery methods of direct intra-DRG injection and intrathecal injection have several disadvantages, including potential injury to DRG neurons and low transfection efficiency, respectively. This study aimed to develop a spinal nerve injection strategy to deliver polyethylenimine mixed with plasmid (PEI/DNA polyplexes) containing green fluorescent protein (GFP). Using this spinal nerve injection approach, PEI/DNA polyplexes were delivered to DRG neurons without nerve injury. Within one week of the delivery, GFP expression was detected in 82.8% ± 1.70% of DRG neurons, comparable to the levels obtained by intra-DRG injection (81.3% ± 5.1%, p = 0.82) but much higher than those obtained by intrathecal injection. The degree of GFP expression by neurofilament(+) and peripherin(+) DRG neurons was similar. The safety of this approach was documented by the absence of injury marker expression, including activation transcription factor 3 and ionized calcium binding adaptor molecule 1 for neurons and glia, respectively, as well as the absence of behavioral changes. These results demonstrated the efficacy and safety of delivering PEI/DNA polyplexes to DRG neurons via spinal nerve injection. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T03:12:00Z (GMT). No. of bitstreams: 1 ntu-106-D96446002-1.pdf: 5646914 bytes, checksum: 4b483c85e72036c9ff66102990c6bb37 (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | Introduction…………………………………………………1
Chapter 2. Material and Methods…………………………………3 Axotomy by transection of the sciatic nerve……………………………3 Polymer conjugate synthesis……………………………………………3 Intrathecal injection………………………………………………………4 Lumbar L5 intra-DRG injection…………………………………………4 Spinal nerve injection of PEI/DNA polyplexes……………………4 Spinal nerve crush……………………………………………………………5 Animal behavioral tests………………………………………………………5 Neurophysiological studies…………………………………………………6 Tissue preparation and quantitative multiple-labeling immunofluorescence in DRG neurons…………………………………………………………6 Nerve pathology…………………………………………………………8 Immunohistochemistry in dermal sheets and quantitation of dermal nerve fibers………………………………………………………………………8 Western Blot……………………………………………………………8 Experimental design and statistical analysis……………………9 Chapter 3. Results……………………………………………………………10 Evaluation of delivering gene constructs into DRG: dye injection………10 Demonstration of green fluorescent protein (GFP) expression in the dorsal root ganglia (DRG) after spinal nerve injection……………………………10 Assessment of glial cell activation in the dorsal root ganglia (DRG) after intrathecal, spinal nerve and intra-DRG injection………………………11 Quantitation of gene expression patterns in DRG neurons………………11 Expression patterns of GFP in DRG neurons after spinal nerve injection and intra-DRG injection………………………………………………………12 Safety assessment of gene delivery though spinal nerve injection: multi-modality evaluation………………………………………………12 Expression of activating transcription factor 3 (ATF3) after spinal nerve injection……………………………………………………………………15 Assessment of glial cells and macrophage activation in the spinal nerve after spinal nerve injection……………………………………………………15 Assessment of astrocyte and microglia activation in the spinal cord after spinal nerve injection……………………………………………………15 Integrity of DRG neurons and their processes after spinal nerve injection…16 Chapter 4. Discussion…………………………………………………………18 Technical considerations of spinal nerve injections: comparison with other approaches…………………………………………………………18 Quantifying DRG neurons of different phenotypes…………………19 Safety of delivering gene constructs to the DRG via spinal nerve injection..20 References …………………………………………………………………22 Figures and Figure legends………………………………………………………………………27 Figure 1. Introducing dye into dorsal root ganglia (DRG) neurons via different methods…………………………………………………………………27 Figure 2. Expression of green fluorescent protein (GFP) in the dorsal root ganglia (DRG) via spinal nerve injection……………………………………29 Figure 3. Expression of green fluorescent protein (GFP) and ionized calcium binding adaptor molecule 1 (Iba1) in the dorsal root ganglia (DRG) following different injection approaches……………………………………………….31 Figure 4. Quantitation of dorsal root ganglia neurons……………………….33 Figure 5. Expression of green fluorescent protein (GFP) in the dorsal root ganglia (DRG) after gene constructs were delivered via spinal nerve injection and intra-DRG injection…………………………………………………….35 Figure 6. Change in hindlimb posture, behavioral tests, and nerve conduction studies after spinal nerve injection……………………………………….…37 Figure 7. Expression pattern of phosphorylated neurofilament and macrophage as injury markers in dorsal root ganglia (DRG) after spinal nerve injection……………………………………………………………………40 Figure 8. Patterns of neurofilament expression and macrophage infiltration as injury markers in the dorsal root ganglia (DRG) after spinal nerve injection……………………………………………………………………42 Figure 9. Expression of activating transcription factor 3 (ATF3) and green fluorescent protein (GFP) in the dorsal root ganglia (DRG) and spinal cord after spinal nerve injection (injection group) in comparison with sciatic nerve transection (axotomy group), spinal nerve crush (crush group), and intra-DRG injection (intra-DRG injection group)……………………………………44 Figure 10. Expression of ionized calcium binding adaptor molecule 1 (Iba1) and green fluorescent protein (GFP) in the spinal nerve after spinal nerve injection (injection group) compared to sciatic nerve transection (axotomy group) ), spinal nerve crush (crush group) and intra-DRG injection (intra-DRG injection group)……………………………………………………………………46 Figure 11. Expression of glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1) in the spinal cord after spinal nerve injection (spinal nerve injection group) in comparison with sciatic nerve transection (axotomy group), spinal nerve crush (crush group), and intra-DRG injection (intra-DRG injection)…………………………………………48 Figure 12. Pathology of the sciatic nerve and nerve terminals in the skin after spinal nerve injection…………………………………………………………50 Table 1. Table of all antibodies………………………………………………………52 | |
| dc.language.iso | en | |
| dc.subject | 基因轉殖 | zh_TW |
| dc.subject | 背根神經節 | zh_TW |
| dc.subject | transfection | en |
| dc.subject | Dorsal root ganglion | en |
| dc.title | 透過脊神經注射達成非病毒載體之大鼠背根神經節基因轉殖 | zh_TW |
| dc.title | Effective gene expression in the rat dorsal root ganglia with a non-viral vector delivered via spinal nerve injection | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 吳君泰,潘俊良,曾拓榮,江皓郁,呂俊宏 | |
| dc.subject.keyword | 背根神經節,基因轉殖, | zh_TW |
| dc.subject.keyword | Dorsal root ganglion,transfection, | en |
| dc.relation.page | 52 | |
| dc.identifier.doi | 10.6342/NTU201700684 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2017-03-10 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 解剖學暨細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 解剖學暨細胞生物學科所 | |
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