博碩士論文 963204058 詳細資訊




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姓名 何玲儀(Ling-Yi Ho)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 可見光調控神經細胞之基因表現及突觸生長
(Visible Light Regulates Gene Expression and Neurite Outgrowth of Nerve Cells)
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摘要(中) 可見光被視為一種感光性的治療藥劑,且能應用於組織與細胞上。然而,在基因表現層面上,尚未有研究觀察光對於培養在collagen-coated plates上的類神經細胞(PC12 cells)的影響。本研究將描述在不同波長的發光二極體LED光(i.e., 470 (藍光 LED), 525 (綠光 LED), 600 (黃光 LED), 及 630 (紅光 LED) nm)照射下,PC12 cells培養於含有Collagen Type Ι 塗佈之細胞培養皿上且在添加神經成長因子(NGF)的情況下之突觸生長。神經成長因子(NGF)在PC12 cells 上有兩個接受器(receptors),分別為Trk-A和p75NTR(NGFR)。本研究利用RTPCR的方法來分析在有照射或無照射特殊波長光下之神經細胞(PC12 cells)上的Trk-A和p75NTR的神經元基因層面的表現。結果發現,PC12 cells 在添加NGF下照射特殊波長LED光三十小時或十分鐘,p75 neurotrophin receptor (NGFR)的基因表現呈現負調控,其表現甚至低於無添加NGF且無照光情況下之Normal control組;而不管PC12 cells有無照射LED光,在有添加NGF情況下tyrosine kinase receptor (Trk-A)之基因表現呈現負調控情況。這樣的結果顯示,光的照射PC12 cells對於Trk-A之基因表現並無顯著的影響,而光的照射可以調控p75NTR的基因表現。使人聯想神經細胞如PC12 cells在照射光的影響下,在細胞上可能存在光接受器(light receptor)且在光照射後在基因表現上會有訊息的傳遞。
  突觸蛋白(Synaptophysin)為一種從突觸囊泡中釋出的神經傳送素,本論文將研究利用Synaptophysin免疫細胞化學方法觀察PC12 cells在照射不同波長光後之神經元的聯結進而分析光照對於PC12 cells之影響。
摘要(英) Visible light was regarded as phototherapeutic agents for tissue and cell application. However, the effect of light on nerve-like PC12 cells cultured on collagen-coated plates on the gene expression level has not yet investigated. Here I report the neurite outgrowth of PC12 cells on collagen-coated plates under light emitting diode (LED) irradiation at several wavelengths (i.e., 470 (Blue light LED), 525 (Green light LED), 600 (Yellow light LED), and 630 (Red light LED) nm). The biological effects of nerve growth factor (NGF) are mediated by two structurally unrelated receptors, i.e., Trk-A and p75NTR (NGFR). Neuronal gene expression (RNA of p75NTR and Trk-A) of nerve cells (PC12 cells) with and without irradiation of specific wavelength of light was analyzed from RT-PCR assay to investigate the regulation of neurite outgrowth by light in gene expression level. It was found that the gene expression of p75 neurotrophin receptor (NGFR) on PC12 cells was down-regulated under irradiation of any specific wavelength of LED light for 30 hrs or 10 min, while the expression of tyrosine kinase receptor (Trk-A) was down-regulated when cells were cultured with NGF addition no matter how the cells were irradiated with LED light or not. These results indicate that no significant effect of light on TrkA expression of PC12 cells, while visible light regulated the gene expression of p75NTR on PC12 cells. It is suggested that nerve cells such as PC12 cells have photo-receptors and signal transduction on gene expression is generated by light irradiation on the PC12 cells.
Synaptophysin (38 kDa) is a protein found as a neurotransmitter contained in small synaptic vesicles, located inside the presynaptic terminals. Neuronal connection on PC12 cells under irradiation of different wavelength of light from LED was also investigated from Synaptophysin-immunoreactivity method to find the effect of light on the formation of synapse connection between cells in this study.
關鍵字(中) ★ PC12
★ 神經細胞
★ 突觸生長
★ RTPCR
關鍵字(英) ★ neurite outgrowth
★ nerve cells
★ PC12
★ RTPCR
論文目次 Chapter 1 Introduction ................................................................................................................ 1
1.1 Introduction .................................................................................................................. 1
1.1.1 Nerve cells and nerve-like cell line (pheochromocytoma, PC12 cells) ............. 1
1.1.2 Cell signaling ..................................................................................................... 3
1.1.2.1 Signaling molecules and their receptors ................................................. 3
1.1.2.2 Growth factors ........................................................................................ 3
1.1.3 The effect of NGF treatment on PC12 cells ...................................................... 3
1.1.3.1 Nerve growth factor and nerve growth factor receptor .......................... 3
1.1.3.2 Signaling mechanism of nerve growth factor ......................................... 4
1.1.3.3 Gene expression on PC12 cells after NGF addition ............................... 9
1.1.4 Polymerase chain reaction (PCR) ...................................................................... 9
1.1.4.1 Polymerase chain reaction (PCR) ........................................................... 9
1.1.4.2 Reverse transcription polymerase chain reaction (RTPCR) and
Quantitative real time polymerase chain reaction (qRT-PCR) ......................... 11
1.1.5 Nerve cell connection between cells ............................................................... 13
1.2 The effect of visible light on cells .............................................................................. 13
1.2.1 The effect of visible light : Phototherapy ........................................................ 13
1.2.2 The effect of visible light on nerve cells ......................................................... 14
Chapter 2 Materials and Methods ............................................................................................. 16
2.1 Chemicals ................................................................................................................... 16
2.2 Consumables ............................................................................................................... 19
2.3 Instruments ................................................................................................................. 20
2.4 Experimental ............................................................................................................... 21
2.4.1 Preparation of cell cultured medium and NGF solution .................................. 21
2.4.1.1 Cell cultured medium (1 L) .................................................................. 21
2.4.1.2 Preparation of NGF stock solution ....................................................... 21
2.4.2 Cell cultivation ................................................................................................ 21
2.4.3 Cell cultivated plates preparation .................................................................... 21
2.4.4 Cell differentiation ........................................................................................... 22
2.4.5 LED light irradiation ....................................................................................... 22
2.4.6 Phosphate buffer saline (PBS) preparation ...................................................... 22
2.4.7 Cell density measurement ................................................................................ 23
2.4.8 Isolation of total RNA ..................................................................................... 23
2.4.9 RTPCR assay ................................................................................................... 24
2.4.9.1 Primers Design ..................................................................................... 24
2.4.9.2 Single-strand cDNA synthesis .............................................................. 24
2.4.9.3 PCR steps .............................................................................................. 25
2.4.10 Immunocytochemistry methods .................................................................... 26
2.4.11 Statistical analysis .......................................................................................... 27
2.4.11.1 Definition of differentiation on PC12 cells ........................................ 27
2.4.11.2 Neurite outgrowth ratio....................................................................... 28
2.4.11.3 Differentiation ratio ............................................................................ 28
2.4.11.4 Gene expression ratio ......................................................................... 28
Chpter 3 Results and Discussion .............................................................................................. 30
3.1 Culture conditions of PC12 cells ................................................................................ 30
3.2 Morphology of PC12 cells under irradiation of LED light and mixed light .............. 32
3.3 Neurite outgrowth length on PC12 cells under irradiation of different wavelength
from LED lights ................................................................................................................ 45
3.4 Differentiation ratio on PC12 cells under irradiation of different wavelength from
LED lights ........................................................................................................................ 49
3.5 Relationship between neurite outgrowth length and differentiation ratio .................. 54
3.6 Gene expression on PC12 cells .................................................................................. 58
3.6.1 Gene expression of p75NTR on PC12 cells .................................................... 58
3.6.2 Gene expression of Trk-A on PC12 cells......................................................... 66
3.6.3 Gene expression of BDNF on PC12 cells ....................................................... 74
3.7 The effect of different wavelength of light on gene expression of PC12 cells ........... 81
3.7.1 Visible light affects the gene expression of p75NTR on PC12 cells ............... 81
Chapter 4 Conclusion ............................................................................................................... 93
Chapter 5 References ................................................................................................................ 95
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指導教授 樋口亞紺(Akon Higuchi) 審核日期 2009-7-6
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