||Biomechanical and Morphological Effects of Paclitaxel on PC-12 Cells
||Department of Mechanical Engineering
本研究利用PC-12類神經細胞施加不同濃度之化療藥物紫杉醇，模擬人體內神經細胞受其作用之形態與生物力學變化。首先利用倒立式螢光顯微鏡量測細胞之形貌參數、微管分佈量化指標，並利用原子力顯微鏡掃描形貌及壓印以估測其力學性質。結果顯示紫杉醇會破壞 PC-12 細胞之動態不穩定性(dynamic instability)，造成微管在細胞核附近過度集中，其微管分散率隨著紫杉醇濃度上升而逐漸上升。實驗組之楊氏模數普遍高於控制組，且其細胞核區和轉換區的楊氏模數有明顯差異，表示其微結構已經改變，且有可能破壞微管傳輸神經傳導物質之功能。
Chemotherapy-induced peripheral neuropathy (CIPN) is by far the top reason for patients to drop out of chemotherapy. 50% of patients suffer from CIPN after treated with paclitaxel, a cytotoxicity drug which targets microtubules of cells and kills it by disrupting the cell cycle. Most of CIPN symptoms is related to dysfunction or damage of patients’ neurons. This study focus on effect of paclitaxel concentrations effect on neuron cells. PC-12 cells are used as the model of neuron cells in this study. The morphological effect is examined using immunofluorescence staining. Multi indexes, include an index describing the dispersion of microtubules near nucleus area called dispersion ratio (RD), are then examined by processing cell images using software ImageJ. The biomechanical effect is tested using an atomic force microscope (AFM). Cell topography was first scanned and then indentation experiments of different area (nucleus and transition area) were performed. Cell high was measured by topographies and local apparent Young’s modulus was estimated by fitting the force-indentation data to the bottom-effect-cone-correction (BECC) model. This study find that paclitaxel affects the dispersion of microtubules in cells. The dispersion ratio increase with the increase of concentration of paclitaxel. This result suggests that there are more microtubules assembled in nucleus area of cells affected by paclitaxel, rather than spread through the whole soma or axon. The Young’s modulus of both nucleus and transition area of paclitaxel effected cells are higher than control group except the transition area of 1 μM group. Also, as the concentration of paclitaxel increase, the Young’s modulus increase. Combine these two results, this study suggests that paclitaxel enhance assembly of microtubules in area near nucleus, leading to the Young’s modulus increase. And the disorder or incorrect distribute of microtubules can cause dysfunction of neurons, which may result in CIPN.
第一章 緒論 1
1.1. 癌症與化療引起之周邊神經病變 1
1.2. 太平洋紫杉醇 (Paclitaxel) 3
1.3. 癌症相關細胞力學研究 5
1.4. 研究動機與目的 7
1.5. 本文架構 7
第二章 方法與實驗 8
2.1. PC-12類神經細胞培養程序與突觸誘發 9
2.2. 紫杉醇製備與施加於PC-12細胞 11
2.3. 細胞螢光染色 12
2.4. PC-12類神經細胞形貌分析 15
2.5. 原子力顯微鏡掃描 19
2.6. 細胞機械性質估測 23
2.7. 樣本高度修正 27
2.8. 細胞壓痕實驗設計 28
2.9. 資料處理與統計分析 30
第三章 結果 31
3.1. PC-12類神經細胞形貌分析 31
3.2. 原子力顯微鏡量測分析 43
第四章 討論 52
4.1. 細胞形貌 52
4.2. 原子力顯微鏡掃描與壓痕實驗 55
4.3. 結論 58
4.4. 建議 60
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