以共沉澱法製備藍藻蛋白/羥基磷灰石複合薄膜評估Saos-2 cells骨傳導性之探討

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研究生：	陳彥志 Yen-chih Chen
論文名稱：	以共沉澱法製備藍藻蛋白/羥基磷灰石複合薄膜評估Saos-2 cells骨傳導性之探討 Assessments of osteoconductivity of Saos-2 cells on the cyanophycin/hydroxyapatite composite films prepared by coprecipitation
指導教授：	曾文祺 Wen-Chi Tseng
口試委員:	方翠筠 Tsuei-Yun Fang 林析右 Shi-Yow Lin 鄭如忠 Professor Ru-Jong Jeng
學位類別：	碩士 Master
系所名稱：	工程學院 - 化學工程系 Department of Chemical Engineering
論文出版年：	2012
畢業學年度：	100
語文別：	中文
論文頁數：	62
中文關鍵詞：	藍藻蛋白、羥基磷灰石、共沉澱法
外文關鍵詞：	cyanophycin, hydroxyapatite, coprecipitation
相關次數：	點閱：217 下載：3
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上一筆

藍藻蛋白是一種天然性的高分子，經由基因重組方式所產得的藍藻蛋白其結構為aspartic acid、arginine及lysine等三種胺基酸所組成。目前藍藻蛋白並未商業化量產，還沒有明確的應用實例。期望藉由藍藻蛋白具有良好的生物相容性，拓展藍藻蛋白在生醫材料的應用上。
由於一般的高分子材料少有骨傳導性，對於提升骨細胞的增值分化能力有限，故本研究以化學共沉澱法生成羥基磷灰石並混入藍藻蛋白製成薄膜，並利用骨肉瘤骨生成細胞 (sarcoma osteogenic, Saos-2)所具有骨母細胞的特性於藍藻蛋白/羥基磷灰石複合薄膜上培養。
實驗中以MTT法測試藍藻蛋白/羥基磷灰石複合薄膜對其Saos-2細胞之毒性。藉由鹼性磷酸酶會將透明4-Nitrophenyl phosphate水解成黃色的p-Nitrophenol，以紫外光及可見光光譜儀量測其鹼性磷酸酶含量。以XRD及FTIR確定其結構與成分，並經接觸角測量儀量測其材料親疏水性，並以SEM觀察材料表面微觀結構及Saos-2細胞貼附型態。
實驗結果顯示混入的羥基磷灰石其疏水性明顯增高；在SEM觀察材料表面型態，其藍藻蛋白/羥基磷灰石薄膜表面呈粗糙不帄。藍藍藻蛋白/羥基磷灰石薄膜經48小時的細胞培養未發現其毒性並有助於細胞生長；培養72小時測量其骨肉瘤骨生成細胞鹼性磷酸酶含量，比藍藻蛋白薄膜提升6~8倍。
總結以上，將羥基磷灰石混入到藍藻蛋白，有助於提高Saos-2細胞的增值與分化。

Cyanophycin (CGP) is a natural polymer. The structure of recombinant CGP consists of a poly-aspartic acid backbone with arginine and lysine as side chains. Due to the biocompatibility of cyanophycin, it can be used in biomaterial applications.
Hydroxyapatite (HAP) is an important biomaterial used in hard bone tissue substitute because of its bioactive and excellent osteoconductivity. In this study, cyanophycin/ hydroxyapatite composites film was prepared by coprecipitation method to enhance the osteoconductivity of cyanophycin.
The physical and chemical property of cyanophycin/hydroxyapatite films were analyzed by scanning electron microscopy, Fourier transform- ed infrared spectroscopy, X-ray diffraction and contact angle. The cell viability was testd by using MTT assay and the cell morphology was observed with SEM. The alkaline phosphatase (ALP) activity was measured by using enzymatic colorimetry.
The experimential results showed that the hydroxyapatite mixed with hydrophobic significantly higher. The cyanophycin/hydroxyapatite films exhibited rounghness when the surface morphology was observed by SEM. The cell viability showed minimal toxicity when Saos-2 cells on the CGP/HAP flims for 48 hr；the Saos-2 cells on CGP/HAP film also had improved ALP activity .
In conclusion, CGP/HAP film can guide bone cell differentiation. It has a great potential of biomaterials applications for bone tissue engineering.

中文摘要..................................................................... I
Abstract .................................................................. III
誌謝..........................................................................V
目錄.........................................................................VI
第一章 緒論............................................................................1
1.1前言...................................................................... 1
1.2研究動機.................................................................. 2
文獻回顧..................................................................... 3
2.1骨骼組織簡介............................................................................3
2.1.1骨骼組成及構造........................................................................... 3
2.1.2 骨骼之生長過程............................................................................5
2.1.3 骨修復材料............................................................................7
2.2 羥基磷灰石........................................................................... 9
2.2.1 羥基磷灰石簡介........................................................................... 9
2.2.2 多孔性的羥基磷灰石.................................................... 12
2.2.3 羥基磷灰石/有機高分子複合材料 .........................................13
2.2.4製備羥基磷灰石方法......................................................16
2.3藍藻蛋白..................................................................17
2.3.1藍藻蛋白簡介........................................................... 17
2.3.2 藍藻蛋白合成...........................................................18
2.3.3 藍藻蛋白性質.......................................................... 18
2.3.4 藍藻蛋白之生產........................................................ 19
第三章 實驗................................................................. 21
3.1 實驗材料.................................................................21
3.1.1 菌株.................................................................. 21
3.1.2 細胞株................................................................ 21
3.1.3 細胞培養基............................................................ 21
3.1.4 抗生素.................................................................21
3.2 實驗藥品................................................................ 21
3.3 藥品配置................................................................ 23
3.3 實驗器材................................................................ 26
3.4 實驗設備................................................................ 27
3.5 實驗流程.................................................................28
3.5.1 藍藻蛋白生產...........................................................29
3.5.1.2 培養大腸桿菌於瓊脂板上...............................................29
3.5.1.3 培養大腸桿菌於2 mL LB培養基 ........................................ 29
3.5.1.4 培養大腸桿菌於60 mL LB培養基 ....................................... 29
3.5.1.5 培養大腸桿菌於3L發酵槽...............................................30
3.5.1.6 藍藻蛋白純化.........................................................30
3.5.2培養骨肉瘤骨生成細胞Saos-2 .............................................32
3.5.2.1解凍Saos-2細胞 .......................................................32
3.5.2.2繼代培養Saos-2........................................................32
3.5.3 CGP/HAP 複合材料薄膜 ..................................................33
3.5.3.1 製備soluble CGP/HAP 複合材料薄膜 ....................................33
3.5.3.2 0.5％ (v/v)戊二醛交聯CGP/HAP 複合材料薄膜 ............ ..............35
3.5.3.3製備0.4％ (w/v) 藍藻蛋白薄膜 ........................................ 36
3.6 X光繞射 (X-ray diffraction)分析 CGP/HAP ................................ 36
3.7傅立葉轉換紅外線光譜儀 (FTIR) 分析CGP/HAP ................................36
3.8 以MTT呈色法測試Saos-2 cell在 CGP/HAP薄膜上之細胞毒性.....................36
3.9 Saos-2 cell在 CGP/HAP薄膜上之鹼性磷酸酶定量測試 ........................ 38
3.10蛋白質定量分析 (Bradford protein-bindding assay) ........................40
3.11 以掃描式電子顯微鏡 (SEM) 觀察CGP/HAP材料表面微觀結構 ...................41
3.12 以掃描式電子顯微鏡 (SEM) 觀察Saos-2 cell在CGP/HAP材料表面上的貼附型態.. 41
3.13接觸角量測.............................................................. 42
第四章 結果與討論........................................................... 44
4.1鑑定材料成分及成膜結果................................................... 44
4.1.1以X-ray diffraction 鑑定結果 ...........................................44
4.1.2 以傅立葉轉換紅外光譜儀.................................................45
4.1.3 以肉眼觀察藍藻蛋白/羥基磷灰 ...........................................45
4.2分析藍藻蛋白/羥基磷灰石複合材料特性 ......................................46
4.2.1 水接觸角量測.......................................................... 46
4.2.2 以掃描式電子顯微鏡觀察CGP/HAP薄膜表面形態 ............................ 46
4.3骨肉瘤骨生成細胞於藍藻蛋白/羥基磷灰石材料上生長及分化 ....................47
4.3.1骨肉瘤骨生成細胞於CGP/HAP複合材料上之毒性測試 ..........................47
4.3.2骨肉瘤骨生成細胞於CGP/HAP複合材料貼附情況 ..............................47
4.3.3骨肉瘤骨生成細胞於CGP/HAP複合材料上之鹼性磷酸酶測試.....................47
第五章結論...................................................................49
參考文獻.................................................................... 50
表 2.1 有機骨基質成分及各項功能...............................................4
表 2.2 各類型的骨細胞及功能...................................................5
表 2.3 羥基磷灰石基本物理化學性質............................................11
表 3.1 製備CGP/HAP複合薄膜藥品用量...........................................34
圖2.1骨之生長示意圖...........................................................6
圖2.2 骨細胞於骨替代材料上生長新骨之示意圖....................................9
圖2.3羥基磷灰石的晶體結構....................................................11
圖2.4藍綠藻菌中藍藻蛋白結構..................................................17
圖2.5使用基因重組菌株，其藍藻蛋白結構........................................20
圖3.1 實驗流程圖.............................................................28
圖3.2反應裝置圖..............................................................34
圖3.3戊二醛與lysine交聯反應機制..............................................35
圖3.4 MTT與粒腺體反應過程....................................................37
圖3.5 ALP assay 反應機.......................................................39
圖3.6 介面力與接觸角關係圖...................................................42
圖6.1 於25℃反應條件下之X-ray繞射圖譜........................................54
圖6.2 不同溫度下HAP之X-ray 繞射圖譜..........................................54
圖6.3 於37℃反應條件下CGP/HAP複合物之X-ray 繞射圖譜..........................55
圖6.4 以傅立葉紅外線光譜儀分析CGP/HAP之官能基................................56
圖6.5 CGP/HAP 薄膜戊二醛交聯前後比較圖.......................................57
圖6.6 接觸角測量.............................................................58
圖6.7 CGP/HAP 薄膜表面形態...................................................59
圖6.8 培養Saos-2 48小時於不同比例CGP/HAP薄膜上之存活率比較圖.................60
圖6.9 Saos-2細胞於不同比例CGP/HAP薄膜之貼附情況..............................61
圖6.10 Saos-2 在72小時於不同比例CGP/HAP薄膜之鹼性磷酸酶測....................62

                                

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