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研究生:林臻
研究生(外文):Chen Lin
論文名稱:幾丁聚醣支架混摻去細胞腦基質對神經幹細胞分化之影響
論文名稱(外文):Effects of Chitosan-Based Scaffolds Incorporating Rat Decellularized Brain Extracellular Matrix on Differentiation of Neural Stem Cells
指導教授:楊台鴻
指導教授(外文):Tai-Horng Young
口試委員:婁培人李亦宸洪智煌
口試委員(外文):Pei-Jen LouYi-Chen LiChih-Huang Hung
口試日期:2019-07-24
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫學工程學研究所
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:2019
畢業學年度:108
語文別:英文
論文頁數:46
中文關鍵詞:幾丁聚醣三維支架神經幹細胞分化去細胞化大腦細胞外基質
外文關鍵詞:chitosan3-D scaffoldNSC differentiationdecellularizationbrain ECM
DOI:10.6342/NTU202000525
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神經幹細胞是具有自我更新以及分化能力的細胞,可以分化成神經細胞和神經膠質細胞的型態,其來源除了胚胎外,在成體次腦室區也發現神經幹細胞的存在,其再生與分化的能力,對損傷後便無法再修復的中樞身經系統而言儼然是盞明燈,以期利用在神經退化性疾病、脊髓受傷、腦部創傷等神經損傷疾病的治療上。
本研究先是利用含有鹼性成纖維細胞生長因子的培養基,將取自大鼠胚胎大腦皮質的神經幹細胞培養成球,進一步在二維幾丁聚醣塗佈或三維幾丁聚醣支架上培養。鑒於神經幹細胞的分化命運和周圍環境息息相關,為了刺激神經幹細胞趨向神經細胞的分化,我們假設「將幾丁聚醣混摻經去細胞化處理的大鼠腦」有助於達到這個目標,此假設乃建立在過去研究指出,腦中細胞外基質含有些許蛋白成分如膠原蛋白,以及大量糖胺聚糖,可提供影響細胞分化的生物化學刺激。
另外,本實驗培養神經幹細胞係使用一種取名為Medium B的培養基,配方在本研究室先前的研究中確立,以DMEM/F-12為基底添加特定濃度的纖連蛋白(fibronectin)及表皮生長因子(EGF),有助於提升神經幹細胞的爬行能力同時維持原本幹細胞之特性,研究結果顯示在二維培養如此,三維支架培養亦然。
從免疫螢光圖中,可以看到在培養3-7天的組別,不論是巢蛋白(nestin)、維管蛋白(MAP2及βIII-tubulin)、膠質纖維酸性蛋白(GFAP)都有所表現;故以西方點墨法對分化比例做進一步的比較,結果顯示在二維塗佈培養的環境下,相較於單純幾丁聚醣塗佈,混有去細胞化腦的組別MAP2蛋白表現確實有所提升。
然而從三維支架培養的免疫螢光結果,並無法看出MAP2蛋白的表現在混摻去細胞化腦後是否有所影響;進一步做爬行測試,將種滿神經幹細胞的支架覆蓋到Transwell上,於七天後將Transwell翻面,將從支架通過Transwell爬行至反面的細胞固定並進行染色、計數,結果同樣顯示無論是單純幾丁聚醣支架或混摻有去細胞化腦的幾丁聚醣支架組別,其分化結果並無差異。因此,本研究認為將幾丁聚醣支架確實可作為體外培養神經幹細胞的材料,同時在Medium B的培養環境下有助細胞進行遷移,但在三維環境下混摻去細胞化腦對MAP2蛋白表現提升的能力及混摻之比例仍有待進一步探究。
Neural stem cells (NSCs) can be obtained from various sources and have been proved to be capable of self-renewal and differentiation. With features of proliferation and differentiation, NSCs are considered to be a reservoir to replace the lost cells, making NSC-based therapy a potential treatment for neurodegenerative diseases, traumatic brain injuries or other neuro-related diseases.
In this study, NSCs were derived from rat embryonic cortex and were first cultured with medium added with basic fibroblast growth factor (bFGF) to form neurospheres. By means of lyophilization, chitosan-based scaffolds with good porosity could be constructed easily. In order to better trigger NSCs to differentiate into neurons, we hypothesized that incorporating chitosan scaffolds and decellularized rat brains could reach this goal. Previous studies have already shown that there are ECM proteins and glycosaminoglycans (GAGs) retained in decellularized rat brains, and we have ensured that this result remained after our decellularization process.
Instead of medium with 10% serum, a common medium for NSC culture, here NSCs were cultured with a formula called Medium B, which is DMEM/F-12 with certain concentration of epidermal growth factor (EGF) and fibronectin added. It has been proved that Medium B helps NSCs proliferate and migrate out of spheres but has no effect on differentiation. This case was proved to remain in three-dimensional culture.
From immunofluorescent images, markers for NSCs, glial cells and neurons could all be seen in both 2-D and 3-D culture systems. Western blotting served as a technique to further compare differentiation percentage. The western blotting results of 2-D culture suggested that NSCs in system with decellularized brains showed stronger expression of MAP2.
However, we further flipped NSC-seeded scaffolds onto Transwells and fixed the migrated cell from scaffolds to inserts for immunofluorescence analysis. The results showed that there was no difference in differentiation percentage between groups of Chi-scaffold and DBChi-scaffold. Thus, this study suggests that chitosan scaffold with Medium B is a suitable system for NSCs culture but further research is needed for the effect of decellularized brain on differentiation in 3-D culture.
口試委員審定書 ---------------------------------i
致謝 ---------------------------------ii
中文摘要 ---------------------------------iii
Abstract ---------------------------------v
Content ---------------------------------vii
List of Figures ---------------------------------x
List of Tables ---------------------------------xii
Chapter 1 Introduction ---------------------------------1
1.1 Cells: neural stem cell (NSC) ---------------------------------1
1.1.1 Sources of NSCs and its differentiation ability ---------------------------------1
1.1.2 Endogenous and exogenous neural stem cells for brain repair ------------3
1.2 Scaffolds: chitosan as a choice of biomaterial ---------------------------------5
1.3 Growth factors: extracellular matrix existing in the brain -------------7
Chapter 2 Materials and Methods ---------------------------------8
2.1 Decellularization of rat brains ---------------------------------8
2.1.1 Protocol of decellularization ---------------------------------8
2.1.2 Validation of decellularization and analysis of retained substances ---------8
2.2 Cell culture ---------------------------------10
2.2.1 Isolation and culture of neural stem cells ---------------------------------10
2.2.2 Preparation of culture media ---------------------------------12
2.2.3 Preparation of seeding substrates ---------------------------------13
2.2.3.1 Chitosan-coated surfaces ---------------------------------13
2.2.3.2 DBChi-coated surfaces ---------------------------------13
2.2.3.3 Chitosan-based scaffolds ---------------------------------15
2.3 Morphological characterization ---------------------------------16
2.4 Immunofluorescence analysis ---------------------------------16
2.5 Western blotting ---------------------------------18
2.6 Migration test ---------------------------------19
Chapter 3 Results ---------------------------------20
3.1 Decellularization analysis ---------------------------------20
3.2 Properties of NSCs for culture ---------------------------------20
3.3 Microscopic image of NSCs cultured on different substrates (2-D) -----------21
3.4 Protein expression of NSCs cultured on different substrates on day 3 (2-D) ---22
3.5 Scanning electron microscopic image of NSCs cultured on chitosan scaffolds ---23
3.6 Immunofluorescent image of NSCs cultured on chitosan-based scaffolds ---------24
3.7 Immunofluorescent image of NSCs migrated from scaffolds to inserts ---------25
Chapter4 Discussion ---------------------------------26
Chapter 5 Conclusion ---------------------------------29
Figures ---------------------------------30
References ---------------------------------44
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