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研究生:蔣宗恆
研究生(外文):Chung-Heng Chiang
論文名稱:低氧和生長因子誘發樹突生長對神經細胞貼附能力的影響
論文名稱(外文):The changes of cell adhesion force during neural dendrite outgrowth induced by hypoxia and growth factors
指導教授:蘇芳慶蘇芳慶引用關係
指導教授(外文):Fong-Chin Su
學位類別:碩士
校院名稱:國立成功大學
系所名稱:醫學工程研究所碩博士班
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:73
中文關鍵詞:細胞刮取神經生長因子氯化鈷大白鼠腎上腺髓質嗜鉻細胞瘤細胞株
外文關鍵詞:PC12 cellsNGFCoCl2cytodetach
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神經元的貼附和突觸生長在大腦發展中扮演關鍵的角色。然而神經元的生物力學和突觸之間的關係仍未被深入探討。突觸生長可藉由神經生長因子或是化學模擬低氧調控對PC12細胞株誘發進而模擬活體內的神經元發展過程。在本研究中,我們針對細胞型態的改變和貼附性質的關係探討。藉由細胞刮取來量化不同突觸誘發模型中貼附性質的改變。實驗中選擇以缺氧模擬藥劑(CoCl2)和神經生長因子(NGF)作用24小時來對PC12細胞株進行分化。
結果顯示CoCl2和NGF都會造成細胞型態上顯著的差異, 包括細胞貼附面積、突觸生長和真圓度的改變;兩種分化模型唯一的型態差異在於NGF分化下細胞貼附面積顯著大於以CoCl2分化。在細胞力學表現上,兩者皆可顯著提高細胞最大貼附力和外力做功;在去除細胞尺度因素之後,單位面積下的最大貼附力只在NGF模型中有顯著上升; 分析分化後的細胞極性對刮取的影響發現在兩組分化模型中, 刮取方向平行細胞主軸所需施加的外力和功皆高於正交方向; 計算相對的細胞勁度發現兩組分化模型有顯著差異且細胞勁度的量測與方向無關。螢光染色發現增加低氧誘導因子(hypoxia inducible factor-1α ,HIF-1α)表現, 進一步確定CoCl2分化模型中細胞進行低氧的調控。
細胞的外型和結構相關,實驗結果發現類似的分化形態仍然存在不同的力學性質。目前的細胞模型無法描述攤平且不規則外型的細胞貼附性質,藉由對於分化細胞進行極性的探討使我們對於刮取機構的特性有更深入的了解,且在未來能有更廣泛的應用。
The cell attachment and neurite outgrowth are critical in neuron development. However, the relationships between cellular biomechanical property and neural outgrowth are still poor understanding. The neurite outgrowth can be induced by growth factor or chemical reagents in PC12 cells to imitate the neuron development in vitro. In present study, we are interested in the relationship between the changes of cell morphology and mechanical remodeling of cell adhesiveness. We used cytodetachment to quantitatively measure the maximal cell adhesion force of different induction treatments. The inducing model was achieved by 24 hr treatment of hypoxia mimic chemical reagent (CoCl2) or nerve growth factor (NGF) on PC12 cells.
Our results showed that both the CoCl2 and NGF treatments caused significantly morphological changes including cell area, neurite ourtgrowth and cell roundness; the only difference was that the cell area of NGF group was significantly greater than that of CoCl2. Comparing the mechanical behavior, we found that both of the treatments increase maximal adhesion force and total work; after dividing the force to the cell area, the normalized force significantly increased in NGF group. The cell orientation will effect maximal adhesion force and work after cell differentiation: comparing to the orthogonal direction, detaching in the direction parallel to cell orientation needed higher force and work. The NGF treatment significantly increased relative cell stiffness while it was cell orientation-independent. The immunofluorescent data showed that CoCl2 treatment caused a significant increase of HIF-1α protein and this confirmed that the cell was under hypoxia adaptation.
The cell shape is related to the internal cell structure, our result showed that the similar cell shapes may not correspond to the same mechanical properties. Presently, the cell adhesion model can’t depict the cells with irregular shapes. By analyzing the effects of cell orientations, we further understood the characteristic of cytodetachment and facilitated more applications in the future.
中文摘要 IV
ABSTRACT V
CONTENTS VI
LIST OF TABLES IX
LIST OF FIGURES X
CHAPTER 1 : INTRODUCTION 1
1.1 Neurons 2
1.1.1 Neural development in vivo 2
1.1.2 Neurite outgrowth and growth cone 3
1.2 PC12 differentiated models 3
1.2.1 Cell lineage 4
1.2.2 Hypoxia induced neurite outgrowth model 5
1.2.3 NGF induced model 8
1.3 Cell adhesion regulate neurite outgrowth 8
1.4 Cell Adhesion measurement 10
1.4.1 Purpose of this study 10
1.4.2 Hypothesis 10
CHAPTER 2 MATERIALS AND METHODS 11
2.1 Experimental models 11
2.1.1 Cell culture 11
2.1.2 Containers and solutions 12
2.1.3 Samples preparation 13
2.2 Experimental equipment 14
2.2.1 Microscope Working Station 14
2.2.2 Cytodetach Devices 16
2.2.3 Environment Control system 18
2.2.4 Cytodetachment procedure 19
2.3 Immunofluorescenct stain 20
2.4 The grading of cell differentiation and adhesiveness 21
2.4.1 Cell area 21
2.4.2 The measurement of cell roundness and cell orientation 22
2.4.3 Neurite outgrowth ratio 24
2.4.4 Adhesion force and work 24
2.4.5 Quantification of immunofluorescence 25
CHAPTER 3 RESULTS 28
3.1 Both CoCl2 and NGF cause morphological changes after 24hrs treatments 28
3.2 CoCl2 and NGF groups are significant different from Control group in area, roundness and neurite ratio. 39
3.3 Roundness is in proportion to area within CoCl2 and NGF treatment groups 40
3.4 Adhesion force increased in CoCl2 and NGF groups 41
3.5 Relationships between mechanics and morphology 42
3.5.1 Correlation between maximal adhesion force and cell area 42
3.5.2 Correlations between normalized maximal adhesion force and morphological characteristics 43
3.6 Both treatments increase total work 44
3.6.1 Maximal adhesion force is proportion to work, especially deformation work 45
3.7 NGF increased the cell stiffness 47
3.8 Only CoCl2 increase HIF-1α expression 48
3.9 Analyzing polarized cells in each group: Detaching CoCl2 and NGF induced PC12 cells in the directions perpendicular or parallel to the cell major axis 51
3.9.1 The maximal adhesion force and normalized force were lower in 0 degree group 51
3.9.2 Total work was lower if measured in the direction perpendicular to the major axis 53
3.9.3 Slope is the same despite different detaching directions 55
CHAPTER 4 DISCUSSION 57
4.1 Great variations in CoCl2 and NGF groups 57
4.2 Both treatments differentiated PC12 cells 57
4.3 The effects of the treatments on maximal adhesion force 57
4.4 The correlation between cell area and maximal adhesion force 58
4.5 The correlation between normalized maximal adhesion force and cell morphology 60
4.6 The correlation between maximal adhesion force and work 60
4.7 The change of cell stiffness 61
4.8 The cell orientation effect maximal adhesion force 64
4.9 Force-displacement figure 65
CHAPTER 5 CONCLUSION 68
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