請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47013完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳志成(Chih-Cheng Chen) | |
| dc.contributor.author | Yuan-Ren Cheng | en |
| dc.contributor.author | 鄭淵仁 | zh_TW |
| dc.date.accessioned | 2021-06-15T05:45:16Z | - |
| dc.date.available | 2010-08-20 | |
| dc.date.copyright | 2010-08-20 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-19 | |
| dc.identifier.citation | 1. Bargmann, C.I., Thomas, J.H. & Horvitz, H.R. Chemosensory cell function in the behavior and development of Caenorhabditis elegans. Cold Spring Harb Symp Quant Biol 55, 529-538 (1990).
2. Hart, A.C., Kass, J., Shapiro, J.E. & Kaplan, J.M. Distinct signaling pathways mediate touch and osmosensory responses in a polymodal sensory neuron. J Neurosci 19, 1952-1958 (1999). 3. Kaplan, J.M. & Horvitz, H.R. A dual mechanosensory and chemosensory neuron in Caenorhabditis elegans. Proc Natl Acad Sci U S A 90, 2227-2231 (1993). 4. Chalfie, M. Neurosensory mechanotransduction. Nat Rev Mol Cell Biol 10, 44-52 (2009). 5. Sukharev, S.I., Blount, P., Martinac, B., Blattner, F.R. & Kung, C. A large-conductance mechanosensitive channel in E. coli encoded by mscL alone. Nature 368, 265-268 (1994). 6. Sukharev, S.I., Martinac, B., Arshavsky, V.Y. & Kung, C. Two types of mechanosensitive channels in the Escherichia coli cell envelope: solubilization and functional reconstitution. Biophys J 65, 177-183 (1993). 7. Hong, K., Mano, I. & Driscoll, M. In vivo structure-function analyses of Caenorhabditis elegans MEC-4, a candidate mechanosensory ion channel subunit. J Neurosci 20, 2575-2588 (2000). 8. Schild, L., Schneeberger, E., Gautschi, I. & Firsov, D. Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation. J Gen Physiol 109, 15-26 (1997). 9. Kellenberger, S., Gautschi, I. & Schild, L. A single point mutation in the pore region of the epithelial Na+ channel changes ion selectivity by modifying molecular sieving. Proc Natl Acad Sci U S A 96, 4170-4175 (1999). 10. Ji, H.L., Bishop, L.R., Anderson, S.J., Fuller, C.M. & Benos, D.J. The role of Pre-H2 domains of alpha- and delta-epithelial Na+ channels in ion permeation, conductance, and amiloride sensitivity. J Biol Chem 279, 8428-8440 (2004). 11. Grunder, S., Jaeger, N.F., Gautschi, I., Schild, L. & Rossier, B.C. Identification of a highly conserved sequence at the N-terminus of the epithelial Na+ channel alpha subunit involved in gating. Pflugers Arch 438, 709-715 (1999). 12. Garcia-Anoveros, J. & Corey, D.P. Touch at the molecular level. Mechanosensation. Curr Biol 6, 541-543 (1996). 13. Royal, D.C., et al. Temperature-sensitive mutant of the Caenorhabditis elegans neurotoxic MEC-4(d) DEG/ENaC channel identifies a site required for trafficking or surface maintenance. J Biol Chem 280, 41976-41986 (2005). 14. Goodman, M.B., et al. MEC-2 regulates C. elegans DEG/ENaC channels needed for mechanosensation. Nature 415, 1039-1042 (2002). 15. Chalfie, M. & Sulston, J. Developmental genetics of the mechanosensory neurons of Caenorhabditis elegans. Dev Biol 82, 358-370 (1981). 16. Chalfie, M. & Thomson, J.N. Organization of neuronal microtubules in the nematode Caenorhabditis elegans. J Cell Biol 82, 278-289 (1979). 17. Vogel, B.E. & Hedgecock, E.M. Hemicentin, a conserved extracellular member of the immunoglobulin superfamily, organizes epithelial and other cell attachments into oriented line-shaped junctions. Development 128, 883-894 (2001). 18. Emtage, L., Gu, G., Hartwieg, E. & Chalfie, M. Extracellular proteins organize the mechanosensory channel complex in C. elegans touch receptor neurons. Neuron 44, 795-807 (2004). 19. Birder, L.A. & Perl, E.R. Cutaneous sensory receptors. J Clin Neurophysiol 11, 534-552 (1994). 20. Lewin, G.R. & Moshourab, R. Mechanosensation and pain. J Neurobiol 61, 30-44 (2004). 21. Burnstock, G. Purinergic mechanosensory transduction and visceral pain. Mol Pain 5, 69 (2009). 22. Burnstock, G. Purinergic receptors and pain. Curr Pharm Des 15, 1717-1735 (2009). 23. Cho, H., et al. A novel mechanosensitive channel identified in sensory neurons. Eur J Neurosci 23, 2543-2550 (2006). 24. Alessandri-Haber, N., et al. Hypotonicity induces TRPV4-mediated nociception in rat. Neuron 39, 497-511 (2003). 25. Hu, J., Chiang, L.Y., Koch, M. & Lewin, G.R. Evidence for a protein tether involved in somatic touch. EMBO J 29, 855-867. 26. Hu, J. & Lewin, G.R. Mechanosensitive currents in the neurites of cultured mouse sensory neurones. J Physiol 577, 815-828 (2006). 27. McCarter, G.C. & Levine, J.D. Ionic basis of a mechanotransduction current in adult rat dorsal root ganglion neurons. Mol Pain 2, 28 (2006). 28. McCarter, G.C., Reichling, D.B. & Levine, J.D. Mechanical transduction by rat dorsal root ganglion neurons in vitro. Neurosci Lett 273, 179-182 (1999). 29. Rugiero, F., Drew, L.J. & Wood, J.N. Kinetic properties of mechanically activated currents in spinal sensory neurons. J Physiol 588, 301-314. 30. Lin, Y.W., Cheng, C.M., Leduc, P.R. & Chen, C.C. Understanding sensory nerve mechanotransduction through localized elastomeric matrix control. PLoS One 4, e4293 (2009). 31. Cheng, C.M., et al. Probing localized neural mechanotransduction through surface-modified elastomeric matrices and electrophysiology. Nat Protoc 5, 714-724. 32. Leiss, M., Beckmann, K., Giros, A., Costell, M. & Fassler, R. The role of integrin binding sites in fibronectin matrix assembly in vivo. Curr Opin Cell Biol 20, 502-507 (2008). 33. Koerber, H.R. & Mendell, L.M. Functional specialization of central projections from identified primary afferent fibers. J Neurophysiol 60, 1597-1614 (1988). 34. Lawson, S.N. Peptides and cutaneous polymodal nociceptor neurones. Prog Brain Res 113, 369-385 (1996). 35. Stucky, C.L. & Lewin, G.R. Isolectin B(4)-positive and -negative nociceptors are functionally distinct. J Neurosci 19, 6497-6505 (1999). 36. Traub, R.J. & Mendell, L.M. The spinal projection of individual identified A-delta- and C-fibers. J Neurophysiol 59, 41-55 (1988). 37. Akopian, A.N., Chen, C.C., Ding, Y., Cesare, P. & Wood, J.N. A new member of the acid-sensing ion channel family. Neuroreport 11, 2217-2222 (2000). 38. Chen, C.C., England, S., Akopian, A.N. & Wood, J.N. A sensory neuron-specific, proton-gated ion channel. Proc Natl Acad Sci U S A 95, 10240-10245 (1998). 39. Sakai, H., Lingueglia, E., Champigny, G., Mattei, M.G. & Lazdunski, M. Cloning and functional expression of a novel degenerin-like Na+ channel gene in mammals. J Physiol 519 Pt 2, 323-333 (1999). 40. Waldmann, R. & Lazdunski, M. H(+)-gated cation channels: neuronal acid sensors in the NaC/DEG family of ion channels. Curr Opin Neurobiol 8, 418-424 (1998). 41. Lu, Y., et al. The ion channel ASIC2 is required for baroreceptor and autonomic control of the circulation. Neuron 64, 885-897 (2009). 42. Zhang, S., et al. MEC-2 is recruited to the putative mechanosensory complex in C. elegans touch receptor neurons through its stomatin-like domain. Curr Biol 14, 1888-1896 (2004). 43. Hunt, C.C. & Ottoson, D. Receptor potential and impulse activity in isolated mammalian spindles. J Physiol 230, 49P-50P (1973). 44. Loewenstein, W.R. & Skalak, R. Mechanical transmission in a Pacinian corpuscle. An analysis and a theory. J Physiol 182, 346-378 (1966). 45. Park, J.W., Vahidi, B., Taylor, A.M., Rhee, S.W. & Jeon, N.L. Microfluidic culture platform for neuroscience research. Nat Protoc 1, 2128-2136 (2006). 46. von Philipsborn, A.C., et al. Microcontact printing of axon guidance molecules for generation of graded patterns. Nat Protoc 1, 1322-1328 (2006). 47. Molliver, D.C., et al. ASIC3, an acid-sensing ion channel, is expressed in metaboreceptive sensory neurons. Mol Pain 1, 35 (2005). 48. Lin, Y.W., et al. Identification and characterization of a subset of mouse sensory neurons that express acid-sensing ion channel 3. Neuroscience 151, 544-557 (2008). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47013 | - |
| dc.description.abstract | 在感覺神經系統的力學傳導中，延展力活化通道扮演了很重要的角色，早先的研究已經發現直接施力於神經細胞本體可以引發力學活化電流，但是這些研究都無法在近似生理環境以及分子的層級探討專一性的施力下會引發什麼樣的反應。因此我們團隊發展了一套可以探討在體外類似生理環境下偵測感覺神經系統力學傳導的方法。首先我們將小鼠的背根神經元分離並且培養在有細胞外間質塗層 (例如: 纖維網蛋白) 的聚二甲基矽氧烷 (PDMS) 彈性介質上，待其纖維生長；再施力於彈性介質上，藉由彈性介質的形變牽引神經纖維，引發延展力活化電流再以電生理之方式紀錄。另外，在膜片鉗的電生理紀錄中我們也仔細監測輸入阻抗是否因為力學刺激而造成紀錄誤差。藉此技術，我們發現了一群背根神經元細胞會對延展力反應並且表現延展力活化電流，經過一連串的藥理實驗後我們發現有一群細胞的張力活化電流對於Amiloride有反應會產生抑制效果，而另外一群細胞則不對Amiloride有任何反應。其後，我們更加深入的以單一細胞層級反轉錄多聚酶鏈鎖反應技術去探討這些細胞中對於Amiloride反應的酸敏感通道基因表現，發現部份酸敏感通道經常表現於這些對於Amiloride有反應的細胞中，這樣的結果可能表示小鼠的酸敏感通道可能在一部份背根神經節的感覺神經系統力學傳導中扮演著特殊的角色。 | zh_TW |
| dc.description.abstract | Stretch-activated channels play an important role in neuronsensory mechanotransduction. Previous studies have revealed that direct mechanical indentation on neuron soma and neurites or applying radial stretch on neural cells can produce stretch-activated currents in dissociated sensory neurons. However, none of these methods have concerned the mechanical specificity in micro-environment in stretching neurons. We have recently developed a novel platform to probe the localized neurosensory mechanotransduciton in a physiologically relevant condition. We grew neurite-bearing dorsal root ganglion (DRG) neurons on elastic matrix (PDMS) anchored with specific extracellular matrix (ECM) such as fibronectin. While applying indentation onto the elastic-matrix, we can deliver a mechanical stimulation to stretch single neurite via the ECM tethering molecules. To obtain a consistent stretch response, we placed the indentation pipette ~10μm away from the neurite, and 100μm away from the cell soma, then programmed the movement of micromanipulator, and monitored reproducibility of the indentation with flurosphere movement. We used whole-cell patch clamp recording to investigate subtypes of stretch-activated currents delivered by ECM tethering neurites in dissociated dorsal root ganglion neurons. Furthermore, by a series of pharmacological examination, we found an amiloride sensitive and an amiloride insensitive stretch-activated current expressed in different neurons. In addition, I performed the single cell RT-PCR to screening the ASIC family (a mammalian DEG/ENaC channels), and found the ASIC1b and ASIC3 expression in the amiloride sensitive neurons. These results suggest that members of DEG/ENaC/ASIC family may participate in the neurosensory mechanotransduction in a subset of dorsal root ganglion neurons. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T05:45:16Z (GMT). No. of bitstreams: 1 ntu-99-R97b41037-1.pdf: 7016721 bytes, checksum: 0ad0dd373ad95a492de78c2d247f2752 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 口試委員審定書……………......................................................................................ii
Acknoledgement……………........................................................................................iii 摘要……………..........................................................................................................iv Abstract………………………………………………………………………….…v~vi Contents…………………………………………………………………………...vii~x List of figures…………………………………………………………………...vii~viii Figure 1. experimental setting and protocol……………………………………...45~46 Figure 2. The dorsal root ganglion primary culture on PDMS…………………..47~48 Figure 3. Flurosphere displacement during indentation……………………….…49~50 Figure 4. Representative stretch-activated response ……………………….……51~52 Figure 5. Representative stretch-activated response with TTX presents in the bathing solution………………………………………………………………………..….53~54 Figure 6. Sequence of stretch-activated current trace in presented of amiloride…………………………………………………………………………55~56 Figure 7. Cell size distribution and AP duration of recorded neurons………….57~58 Figure 8. Result of single cell RT-PCR…………………………………………59~60 List of tables…………………………………………………………………………viii Table 1. Sequence and melting tempature of used primer…………………….…61~62 Chapter 1 Introduction………………………………………………………...…..1~14 1.1 Mechanical sensation……………………………………………………...…..2~3 1.2 Mechanical sensitive complex………………………………………….….....3~7 1.2.1 Stretch-activated channels MEC-4 and MEC-10…………………………...4~5 1.2.2 Membrane associated protein MEC-2 and MEC-6……………………………5 1.2.3 The cytoskeleton protein MEC-7 and MEC-12…………………………….5~6 1.2.4 The extracellular protein MEC-1 and MEC-5…………………………………6 1.2.5 Others proteins involved in mechanical sensitive complex…………………...7 1.3 Study of neuronal sensory mechanical transduction…………………………7~11 1.3.1 Cutaneous mechanoreceptors in primary afferent dorsal root ganglion neurons………………………………………………………………….…….8 1.3.2 Mechanical activated conductance in primary afferent dorsal root ganglion neurons……………………………………………………………………9~10 1.3.3 Potential blocker of mammalian stretch-activated channels……………..10~11 1.4 ECM-tethering force delivery platform for analyzing the stretch-activated channels………………………………………………………………………11~12 Chapter 2 Materials and Methods ……………………………………………….13~22 2.1 ECM-tethered types of stretch-activated current……………………….……14~19 2.1.1 PDMS preparation and fibronectin coating…………………………….…14~15 2.1.2 Dorsal root ganglia neurons primary culture………………………………15~16 2.1.3 Electrophysiology recording………………………………………………...…16 2.1.4 Mechanical stimulation ………………………………………………………..17 2.1.5 Pharmacological test…………………………………………………………...17 2.2 Identify cell types by single cell RT-PCR…………………………………...18~21 2.2.1 Harvesting cell for Single cell RT-PCR……………………………………18~19 2.2.2 Single Cell RNA purification…………………………………………………..19 2.2.3 Reverse transcription………………………………………………….……19~20 2.2.4 Single cell nested PCR…………………………………………………..…20~21 Chapter 3 Results………………………………………………………………...22~31 3.1 Establish an improved platform for studying ECM-tethering stretch-activated channels………………………………………………………………………………23 3.1.1 The effect of fibronectin coating on neuron seeding and neurite outgrowth on PDMS……………………………………………………………………………...…24 3.1.2 Ideal cell densities for high efficiency of recording……………………………25 3.1.3 A stationary mechanical stimulus in a better control of time resolution…………………………………………………………………………25~26 3.2 Stretch-activated current…………………………………………………..…26~29 3.2.1 A subset of the stretch-activated current was amiloride sensitive…………28~29 3.3 Distribution of the stretch-activated current in mechanoreceptors and nociceptors……………………………………………………………………….29~30 3.4 Analyzing the molecular aspect of different types of stretch-activated current……………………………………………………………………………30~31 3.5 Summary…………………………………………………………………………31 Chapter 4 Disscussion…………………………………………………………..……32 4.1 A close conversation between experimental design and real world experience……………………………………………………………………..…33~35 4.1.1 The advantage of fibronectin coated PDMS…………………………………...34 4.1.2 The limitation of this technique………………………………………………..35 4.2 The ECM-tethering stretch-activated current………………………………..35~37 Reference……………………………………………………………………...…38~44 | |
| dc.language.iso | zh-TW | |
| dc.title | 探討感覺神經元中力學傳導經細胞外間質牽引引發之延展力活化電流 | zh_TW |
| dc.title | To investigate ECM-tethering Stretch-activated Currents in Neurosensory Mechanotransduction | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 閔明源(Ming-Yuan Min),嚴震東(Chen-Tung Yen),林以文(Yi-Wen Lin) | |
| dc.subject.keyword | 力學傳導,延展力活化電流,纖維網蛋白,背根神經元,Amiloride,酸敏感通道, | zh_TW |
| dc.subject.keyword | Mechanotransduction,Stretch activate current,Fibronectin,Dorsal root ganglion,Amiloride,ASICs, | en |
| dc.relation.page | 62 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-19 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 動物學研究所 | zh_TW |
| 顯示於系所單位： | 動物學研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-99-1.pdf 目前未授權公開取用 | 6.85 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。