臺灣博碩士論文加值系統

骨質疏鬆症主要症狀為骨質流失、骨密度下降，導致骨折發生。本研究目的在評估骨髓間葉幹細胞(mesenchymal stem cells, MSCs) 受dexamethasone (Dex) 、雙磷酸鹽類Alendronate (Ale) 或樹枝狀高分子poly (amidoamine) (Pamam) 搭載Ale (Paman-Ale) 等藥物作用，混合氫氧基磷灰石(Hydroxyapatite, HA) ，包覆於mPEG-PLGA溫感性水膠中；在二維、三維培養環境下進行硬骨分化之影響；並探討MSCs、誘導分化藥物及水膠在糖皮質類固醇所誘發之骨質疏鬆症小鼠的治療成效。硬骨分化實驗結果發現以100 nM Dex、100 nM Ale及10 nM Pamam-Ale能提升MSCs之鹼性磷酸酶(Alkaline phosphatase, ALP)活性及ALP、第一型膠原蛋白基因表現，其中以Pamam-Ale效果最佳；添加HA或包覆水膠後，則以Ale提升ALP表現量最佳，Dex促進鈣沉積為高。將分化藥物、MSCs、HA和水膠製備成複合性材料，注入骨質疏鬆症小鼠股骨內，透過骨密度及骨組織切片染色，發現治療組與控制組的皮質骨骨密度並無統計上顯著差異，但治療組海綿骨骨密度增加；由組織染色可觀察到HA 分佈於皮質骨內且能減少海綿骨空隙。本研究結果證明MSCs 、HA 、Ale 和水膠組成之複合性材料具骨誘導分化及修復骨缺損能力。

The major symptoms of osteoporosis are bone loss and decreasing bone mineral density, which eventually lead to bone fracture. The specific aims of the study were to evaluate the effect on osteogenesis of mesenchymal stem cells (MSCs) in two or three-dimensional culture environment with the supplements of dexamethasone (Dex), bisphosphonate drug Alendronate (Ale) or dendrimer poly(amidoamine) (Pamam) loaded with Alendronate (Pamam-Ale), and combined with hydroxyapatite (HA) and mPEG-PLGA thermoreversible hydrogel. The therapeutic effect in osteoporosis mice induced b glucocorticoid by using the combination treatment of MSCs, osteogenic inducing drugs and hydrogel was also observed. The results from osteogenic induction showed that 100 nM Dex,100 nM Ale and 10 nM Pamam-Ale could enhance alkaline phosphatase (ALP) activity and ALP and type I collagen gene expression. Highest level of ALP activity was found in the group of Pamam-Ale. When in combining with HA or hydrogel, highest level of ALP activity was found in the group of Ale and highest level of calcium deposition was seen in the group of Dex. The results from the femurs of osteoporosis mice injected with the combination of osteogenic inducing drugs, MSCs, HA and hydrogel found that no significant difference in the bone mineral densities of cortex bones between control and treatment groups. But the bone mineral densities of spongy bones from the treatment groups were higher than those of control group. Tissue sections showed that HA was distributed in the cortex bone and the porosity decreased in the spongy bone. In summary, the combination of osteogenic inducing drugs, MSCs, HA and hydrogel has the potential in osteoinduction and may be applied in bone defect repair.

目錄
摘要 I
Abstract II
致謝 III
目錄 IV
圖目錄 X
縮寫表 XIII
第一章 緒論 1
1.1 前言 1
1.2 理論基礎 3
1.2.1 氫氧基磷灰石 4
1.2.2 水膠 4
1.2.3 溫感性水膠 5
1.2.3.1 溫度敏感性 6
1.2.3.2 降解特性 7
1.2.4 雙磷酸鹽類 8
1.2.5 樹枝狀高分子 9
1.2.6 骨髓間葉幹細胞 11
1.3 文獻回顧 11
1.3.1 溫感性水膠添加氫氧基磷灰石 11
1.3.2 雙磷酸鹽類藥物對細胞之影響 13
1.3.3 支架搭載骨誘導分化材料於骨組織工程之應用 13
1.4 研究目的與架構 15
第二章 材料與方法 18
2.1 材料合成 18
2.1.1 mPEG-PLGA Diblock Copolymer 之合成 18
2.1.2 氫氧基磷灰石合成方法 19
2.2 材料分析 20
2.2.1 場發射掃描式電子顯微鏡 20
2.2.2 X-ray 繞射儀 20
2.2.3 流變儀測試 20
2.2.4 傅立葉紅外線轉換光譜 21
2.3 藥品配製 21
2.3.1 非細胞培養所需藥品 21
2.3.1.1 麻醉藥 21
2.3.1.2 抗凝血劑 22
2.3.1.3 紅血球裂解液 22
2.3.1.4 蘇木紫-伊紅染劑 22
2.3.2 細胞培養所需藥品 23
2.3.2.1 磷酸鹽緩衝溶液 23
2.3.2.2 間葉幹細胞長期培養基 23
2.3.2.3 胰蛋白酶 24
2.3.2.4 台盼藍染劑 24
2.4 骨質疏鬆鼠模式 25
2.4.1 實驗動物來源 25
2.4.2 骨質疏鬆小鼠誘發 25
2.5 細胞培養與細胞操作技術 26
2.5.1 間葉幹細胞分離 26
2.5.2 細胞繼代 27
2.5.3 細胞計數 28
2.6 大鼠間葉幹細胞生長分析 28
2.6.1 MTT 分析試劑配製 28
2.6.2 MTT 分析方法 28
2.7 蛋白質定量分析 29
2.8 骨髓間葉幹細胞誘導分化 30
2.8.1 硬骨分化培養基配製 30
2.8.2 硬骨分化實驗流程 31
2.8.2.1 無水膠組別實驗設計 31
2.8.2.2 含水膠組別實驗設計 31
2.8.3 鹼性磷酸酶活性測定 32
2.8.4 鹼性磷酸酶染色分析 33
2.8.5 鈣沉澱測定 34
2.9 反轉錄聚合酶連鎖反應 35
2.9.1 萃取及純化Total RNA 36
2.9.2 反轉錄作用 37
2.9.3 聚合酶鏈鎖反應 37
2.9.4 DNA 瓊膠電泳 39
2.9.5 電泳影像分析 39
2.10 水膠及藥物移植治療 40
2.11 動物術後照顧 41
2.12 組織切片染色 41
2.12.1 取出骨頭及切片前處理 41
2.12.2 組織切片 42
2.12.3 切片染色 42
2.12.4 微計算機斷層掃描技術 43
2.13 統計分析 43
第三章 結果 44
3.1 氫氧基磷灰石材料分析 44
3.1.1 SEM 觀察粉末材料表面結構 44
3.1.2 HA 之鈣/磷比量測 45
3.1.3 X-ray 繞射儀觀察HA 粉末晶格結構 46
3.1.4 傅利葉轉換紅外線光譜儀 48
3.2 溫感性水膠mPEG-PLGA 材料分析 49
3.2.1 溶膠-凝膠相轉變 49
3.2.2 mPEG-PLGA 溫感性水膠FTIR 分析 49
3.2.3 mPEG-PLGA 溫感性水膠1H-NMR圖譜鑑定 50
3.2.4 mPEG-PLGA 水膠流變性質分析 51
3.3 分化藥物測試分析 53
3.3.1 HA 細胞毒性測試 53
3.3.2 Pamam-Ale 之細胞毒性測試 54
3.3.3 Pamam-Ale 之MSCs 硬骨分化濃度測試 54
3.3.4 藥物對於MSCs 硬骨分化最佳濃度評估 55
3.4 二維與三維培養環境對MSCs 硬骨分化能力影響 56
3.4.1 二維培養環境對MSCs 早期硬骨分化之影響 56
3.4.1.1 ALP 染色分析 56
3.4.1.2 ALP 活性分析 59
3.4.2 二維環境對MSCs 晚期硬骨分化之影響 62
3.4.3 二維培養環境對MSCs 硬骨分化基因表現之影響 64
3.4.4 三維培養環境對MSCs 早期硬骨分化之影響 65
3.4.4.1 ALP 染色分析 65
3.4.4.2 ALP 活性分析 66
3.4.5 水膠包覆細胞於三維培養環境下晚期硬骨分化之影響 67
3.4.5.1 鈣沉積染色分析 67
3.4.5.2 鈣沉積定量分析 68
3.5 微電腦斷層造影觀察骨修復 69
3.6 組織切片觀察骨修復 72
第四章 討論 74
4.1 材料特性分析 74
4.1.1 HA 粉末製備 74
4.1.2 mPEG-PLGA 相轉變 74
4.2 分化藥物對MSCs 細胞於二維培養環境下分化能力之影響 75
4.3 分化藥物對MSCs 於三維培養環境下分化能力之影響 77
4.4 微電腦斷層造影觀察骨修復 77
4.5 組織切片觀察骨修復 78
第五章 結論與未來工作 79
參考文獻 80
附錄 90
A. 儀器表 90
B. 藥品表 92
C. 附圖 94

圖目錄
圖1-1溫感性水膠之化學結構式 7
圖1-2酯鍵於中性與酸性水溶液之水解反應 7
圖1-3焦磷酸與雙磷酸鹽類化學結構 9
圖1-4研究架構 17
圖2-1 mPEG-PLGA 合成化學反應示意圖 18
圖2-2水膠複合物治療骨質疏鬆鼠流程 41
圖3-1 HA 粉末之SEM 影像 44
圖3-2 HA 粉末之EDX 分析 45
圖3-3氫氧基磷灰石之XRD 繞射圖譜 47
圖3-4氫氧基磷灰石之FTIR 光譜圖 48
圖3-5 mPEG-PLGA 水膠相轉變過程 49
圖3-6 mPEG-PLGA 水膠之FTIR 光譜圖 50
圖3-7 mPEG-PLGA 水膠之1H-NMR 頻譜圖 51
圖3-8 20 wt % mPEG-PLGA 水膠流變性質分析 52
圖3-9 MSCs 培養於不同濃度HA 之細胞毒性分析 53
圖3-10 MSCs 培養於不同濃度Pamam-Ale 之細胞毒性分析 54
圖3-11 MSCs 培養於不同濃度Pamam-Ale 之硬骨分化能力分析 55
圖3-12 MSCs 培養於不同濃度之分化藥物的ALP 活性分析 56
圖3-13不同分化藥物對MSCs 培養於二維環境下之ALP 染色分析 58
圖3-14不同分化藥物對MSCs 培養於二維環境下之鈣沉積染色分析 61
圖3-15經不同分化藥物對MSCs 培養於二維環境下之鈣沉積染色分析 63
圖3-16二維培養環境對MSCs 硬骨分化基因表現之影響 65
圖3-17不同分化藥物對MSCs 培養於三維環境下之ALP 染色分析 66
圖3-18不同分化藥物對MSCs 培養三維環境下之ALP 活性分析 67
圖3-19不同分化藥物對MSCs 培養於三維環境下之鈣沉積染色分析 68
圖3-20 不同分化藥物對MSCs 培養於三維環境下之鈣沉積定量分析 69
圖3-21 mPEG-PLGA 複合物注入股骨之皮質骨變化 70
圖3-22 mPEG-PLGA 複合物注入股骨之μ-CT 影像分析 71
圖3-23 mPEG-PLGA 水膠複合物治療骨鬆鼠骨組織切片 73

表目錄
表 1-1 PLA、PGA 與PLGA 高分子降解速率之比較 8
表 2-1蛋白質標準液配製表 30
表 2-2 PCR 反應溶液 37
表 2-3 RT-PCR 使用之引子核酸序列及反應參數 38
表 3-1 HA 之EDX 量測Ca/P 比 46

附錄
A.儀器表 91
B.藥品表 92
C.附圖 94

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