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研究生:安小龍
研究生(外文):Hsiao-Lung An
論文名稱:高葡萄糖對大鼠骨髓間葉幹細胞之硬骨分化路徑之影響探討
論文名稱(外文):The Effect of High Glucose on the Osteogenic Pathway of Mesenchymal Stem Cells Derived from Rat Bone Marrow
指導教授:李文婷李文婷引用關係
指導教授(外文):Wen-Tyng Li
學位類別:碩士
校院名稱:中原大學
系所名稱:生物醫學工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:124
中文關鍵詞:典型 Wnt 訊號活性氧物種骨質生成高血糖高葡萄糖骨髓間葉幹細胞
外文關鍵詞:osteogenesiscanonical Wnt signalingreactive oxygen speciesmesenchymal stem cellhigh glucosehyperglycemia
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先前研究顯示,糖尿病會抑制大鼠骨髓間葉幹細胞 (Mesenchymal stem cells, MSCs)的生長能力及硬骨分化能力,並促進其細胞凋亡及脂肪分化能力。因此,本研究目的在進一步探討高葡萄糖環境對大鼠骨髓MSCs硬骨分化能力及路徑。首先以四氧嘧啶破壞大鼠胰臟 β 細胞誘發其產生第一型糖尿病,比較正常以及糖尿病鼠的骨髓 MSCs 培養在5.5 mM、20 mM 及 33.3 mM 葡萄糖濃度下的細胞存活率、胞內 ATP含量以及活性氧物種 (Reactive oxygen species, ROS)含量的差異。並檢測硬骨分化轉錄因子 Runx2 基因的表現量、早期硬骨分化標記-鹼性磷酸酶 (Alkaline phosphatase, ALP) 的基因表現及蛋白質活性,以及晚期硬骨分化指標-鈣沉積量。另外,也量測高糖環境對硬骨分化時典型 Wnt 訊號的表現。結果發現,高糖環境會降低骨髓 MSCs 增生特性及細胞內 ATP 含量,糖尿病骨髓 MSCs 於體外培養的增生速度低於正常組骨髓 MSCs。高糖環境會增加細胞內 ROS 的含量,導致細胞凋亡比例增加,糖尿病骨髓 MSCs 的 ROS 含量高於正常組骨髓 MSCs,細胞凋亡的現象也明顯高於正常組骨髓 MSCs。高糖環境會抑制骨髓 MSCs 硬骨分化時 ALP 活性及鈣沉積量,ALP、Runx2 基因的表現也會受到抑制,糖尿病骨髓 MSCs表現的硬骨分化標記明顯低於正常組骨髓 MSCs。高糖環境會抑制骨髓 MSCs 硬骨分化時 β-catenin 的表現,而糖尿病骨髓 MSCs 的 β-catenin 的表現明顯低於正常組骨髓 MSCs。綜上所述,糖尿病可透過長期高血糖引起高氧化壓力而造成細胞內典型 Wnt/β-catenin 訊號異常低下,進而抑制骨髓 MSCs 硬骨分化的能力。未來仍需進一步使用拮抗技術以驗證典型 Wnt 訊號對於骨髓 MSCs 於高糖高氧化壓力下硬骨分化能力的影響。

Previous studies show that diabetes inhibits the growth ability of and osteogenic differentiation of mesenchymal stem cells (MSCs) derived from rat bone marrow. It also promotes apoptotic and adipogenic differentiation of MSCs. Therefore, the aim of this study was to investigate the effect of high glucose on osteogenesis of rat bone marrow MSCs and related signaling pathway. First of all, type I diabetes rat model with pancreas β cell dysfunction was established by peritoneal injection with alloxan. Cell viability, intracellular ATP level, and reactive oxygen species (ROS) level were measured in bone marrow MSCs derived from normal or diabetic rats grown in 5.5 mM, 20 mM and 33.3 mM glucose. The gene expression of Runx2 which is a transcription factor of osteoblast differentiation, gene expression and protein activity of alkaline phosphatase (ALP) which is early osteogenic marker, calcium deposition which is late osteogenic marker were determined in MSCs incubated in osteogenic medium containing different concentration of glucose. In addition, the expression of canonical Wnt signaling was also determined in MSCs incubated in osteogenic medium containing high concentration of glucose. The results showed that proliferation and ATP content of MSCs were decreased in high concentration of glucose, and diabetic MSCs exhibited lower growth rate than that of normal MSCs. High glucose induced more ROS production and resulted in higher percentage of apoptic cells compared to those of cells grown in 5.5 mM glucose. Higher amount of ROS and apoptotic cells were observed in diabetic MSCs than in normal MSCs. Both normal and diabetic MSCs had lower ALP activity, and decreased ALP and Runx2 gene expression in high glucose condition. Calcium deposition was also decreased in high glucose condition. Higher inhibition on osteogenic markers were observed in diabetic MSCs than in normal MSCs. Expression of β-catenin was decreased in high glucose condition, and higher inhibition on β-catenin expression was observed in diabetic MSCs than in normal MSCs. In summary, diabetes could inhibit osteogenic potential of MSCs through down-regulation of canonical Wnt signaling by hyperglycemia induced high oxidative stress. In the future, more investigation using antagonist technology will prove whether canonical Wnt signaling plays an important role in osteogenesis of MSCs at hyperglycemia or high glucose state.

摘要 I
Abstract II
致謝 IV
縮寫表 V
目錄 X
圖索引 XIV
表索引 XV
第一章 緒論 1
1.1 前言 1
1.2 理論基礎 2
1.2.1 高血糖 2
1.2.2 活性氧物種 4
1.2.3 幹細胞 5
1.3 研究目的與動機 7
1.4 研究架構 8
第二章 文獻回顧 9
2.1 高血糖及高濃度葡萄糖對間葉幹細胞的影響 9
2.2 Wnt 訊號分子路徑 11
2.3. 調控間葉幹細胞硬骨分化分子機制 12
第三章 材料與方法 14
3.1 糖尿病大鼠模式建立 14
3.1.1 藥物誘發糖尿病 14
3.1.2 血糖測量 14
3.2 藥品配製 16
3.2.1 非細胞培養所需藥品配製 16
3.2.1.1 麻醉藥配製 16
3.2.1.2 紅血球裂解液配製 16
3.2.1.3 抗凝血劑配製 17
3.2.2 細胞培養所需藥品配製 17
3.2.2.1 1X 磷酸鹽緩衝溶液 17
3.2.2.2 間葉幹細胞長期培養基 18
3.2.2.3 胰蛋白酶配製 18
3.2.2.4 台盼藍染劑配製 19
3.3 骨髓間葉幹細胞分離 19
3.4 細胞無菌培養 21
3.4.1 細胞繼代 21
3.4.2 細胞計數 22
3.4.3 細胞凍存 22
3.4.4 細胞解凍 23
3.5 高濃度葡萄糖對骨髓間葉幹細胞生長之影響分析 23
3.5.1 細胞存活率分析 23
3.5.1.1 MTT 細胞存活率分析法原理 23
3.5.1.2 MTT 試劑配製 24
3.5.1.3 骨髓間葉幹細胞 MTT 分析實驗流程 24
3.5.2 細胞內 ATP 含量分析 25
3.5.2.1 細胞內 ATP 分析法原理 25
3.5.2.2 ATP 分析試劑配製 25
3.5.2.3 骨髓間葉幹細胞 ATP 分析實驗流程 25
3.6 高濃度葡萄糖對骨髓間葉幹細胞凋亡之影響分析 26
3.6.1 細胞內活性氧物種產生量分析 26
3.6.1.1 活性氧物種含量分析法原理 26
3.6.1.2 H2DCFDA 螢光探針活性氧物種分析試劑配製 26
3.6.1.3骨髓間葉幹細胞活性氧物種分析實驗流程 26
3.6.2 Hoechst 33258 螢光染色分析 27
3.6.2.1 細胞凋亡螢光染色定性分析原理 27
3.6.2.2 Hoechst-33258 螢光染劑配置 27
3.6.2.3骨髓間葉幹細胞 Hoechst-33258 螢光染色凋亡定性實驗流程 27
3.6.3 流式細胞儀分析 28
3.6.3.1 流式細胞儀分析原理 28
3.6.3.2 流式細胞儀分析細胞凋亡實驗流程 28
3.7 蛋白質定量 30
3.7.1 蛋白質定量分析原理 30
3.7.2 細胞蛋白質 BCA 定量分析實驗流程 30
3.8 高濃度葡萄糖對骨髓間葉幹細胞硬骨分化之影響分析 32
3.8.1 骨髓間葉幹細胞硬骨分化能力分析 32
3.8.2 骨髓間葉幹細胞誘導硬骨分化培養基配製 32
3.8.3骨髓間葉幹細胞硬骨分化鹼性磷酸酶染色定性分析 33
3.8.3.1 鹼性磷酸酶染色原理 33
3.8.3.2 鹼性磷酸酶染色流程 34
3.8.4 骨髓間葉幹細胞硬骨分化鹼性磷酸酶定量分析 34
3.8.4.1 鹼性磷酸酶活性分析原理 34
3.8.4.2 鹼性磷酸酶活性分析流程 35
3.8.5 骨髓間葉幹細胞硬骨分化鈣沉積分析 35
3.8.5.1 鈣沉積分析原理 35
3.8.5.2 鈣沉積分析流程 36
3.9 骨髓間葉幹細胞之免疫螢光染色 36
3.9.1 免疫螢光染色原理 36
3.9.2 免疫螢光染色染色流程 38
3.10 骨髓間葉幹細胞基因表現分析 39
3.10.1 反轉錄聚合酶鏈鎖反應原理 39
3.10.2 總 RNA 的萃取及純化 39
3.10.3 總 RNA 定量 40
3.10.4 反轉錄作用 41
3.10.5 聚合酶鏈鎖反應 42
3.10.6 DNA 瓊脂膠體電泳 44
3.10.7 電泳影像分析 45
3.11 西方點墨法 46
3.11.1 西方點墨法分析原理 46
3.11.2 細胞蛋白質萃取 46
3.11.3 蛋白質電泳 48
3.11.4 蛋白質轉漬 51
3.11.5 抗體反應 52
3.11.6 化學冷光呈色法 54
3.11.7 去除抗體 55
3.12 統計分析 55
第四章 結果 56
4.1 高濃度葡萄糖對骨髓間葉幹細胞之生長影響 56
4.1.1 細胞存活率分析 56
4.1.2 細胞內 ATP 含量 60
4.2 高濃度葡萄糖對骨髓間葉幹細胞之凋亡影響 63
4.2.1 細胞內活性氧物種含量 63
4.2.2 細胞染色質濃縮 65
4.2.3 Annexin V/PI 雙染流式細胞儀分析 67
4.3高濃度葡萄糖對骨髓間葉幹細胞分化能力之影響 70
4.3.1早期硬骨分化標記-鹼性磷酸酶活性表現量 70
4.3.2 晚期硬骨分化標記-鈣沉積分析 73
4.3.3 硬骨分化基因表現的影響 76
4.4 高葡萄糖對骨髓間葉幹細胞 Wnt/β-catenin 路徑之影響 79
4.4.1 高葡萄糖對硬骨分化時 GSK-3β 及 pGSK-3β 表現的影響 79
4.4.2 高葡萄糖對骨髓間葉幹細胞硬骨分化時 β-catenin 表現的影響 85
第五章 討論 89
5.1 高血糖與高濃度葡萄糖對骨髓間葉幹細胞之影響 89
5.2高血糖與高濃度葡萄糖對骨髓間葉幹細胞之硬骨分化能力影響 91
第六章 結論與未來展望 95
參考文獻 96
附錄表 104
A. 藥品表 104
B. 儀器表 108



圖索引
圖 1.1 高葡萄糖對大鼠骨髓間葉幹細胞硬骨分化影響與路徑之研究架構 8
圖 3.1 正常與糖尿病鼠血糖值 15
圖 3.2 骨髓間葉幹細胞之細胞群落 21
圖 3.3 ATP 分析之螢光素酶化學反應原理 25
圖 4.1 高濃度葡萄糖對骨髓間葉幹細胞之存活率影響 58
圖 4.2 正常組與糖尿病組之骨髓間葉幹細胞生長速率 59
圖 4.3 高濃度葡萄糖對骨髓間葉幹細胞內之 ATP 含量影響 62
圖 4.4 高濃度葡萄糖對骨髓間葉幹細胞內產生活性氧物種之影響 64
圖 4.5 高濃度葡萄糖對骨髓間葉幹細胞之染色質濃縮影響 66
圖 4.6 高濃度葡萄糖對骨髓間葉幹細胞之凋亡與壞死影響 69
圖 4.7高葡萄糖對骨髓間葉幹細胞之鹼性磷酸酶影響 71
圖 4.8 高葡萄糖對骨髓間葉幹細胞之 ALP 活性影響 72
圖 4.9 高葡萄糖對骨髓間葉幹細胞之鈣沉積染色 74
圖 4.10 高葡萄糖對骨髓間葉幹細胞之鈣沉積定量表現影響 75
圖 4.11 高葡萄糖對骨髓間葉幹細胞硬骨分化基因表現量的影響 78
圖 4.12 高葡萄糖對骨髓間葉幹細胞硬骨分化時 GSK-3β 基因與蛋白質表現量的影響 81
圖 4.13 高葡萄糖對骨髓間葉幹細胞硬骨分化之 GSK-3β 免疫螢光染色 82
圖 4.14 高葡萄糖對骨髓間葉幹細胞硬骨分化時 pGSK-3β 蛋白質表現量影響 83
圖 4.15 高葡萄糖對骨髓間葉幹細胞硬骨分化之 pGSK-3β 免疫螢光染色 84
圖 4.16 高葡萄糖對骨髓間葉幹細胞硬骨分化時 β-catenin 基因與蛋白質表現量的影響 87
圖 4.17 高葡萄糖對骨髓間葉幹細胞硬骨分化之 β-catenin 免疫螢光染色 88



表索引
表 3.1 5X binding buffer 配方表 30
表 3.2 蛋白質標準曲線製作表 31
表 3.3 硬骨分化誘導因子配方表 33
表 3.4 免疫螢光染色阻斷液配方表 37
表 3.5 免疫螢光染色抗體種類及稀釋倍率 37
表 3.6 RT-PCR 使用之引子核酸序列及反應參數 43
表 3.7 6 X Loading dye 配方表 45
表 3.8 Nonidet-P 40 (NP-40) 細胞裂解液配方表 47
表 3.9 4X Sample buffer 配方表 47
表 3.10 4X 焦集膠體溶液配方表 49
表 3.11 4X 分離膠體溶液配方表 49
表 3.12 SDS-PAGE 膠體配方表 50
表 3.13 1X Running buffer 配方表 50
表 3.14 Towbin transfer buffer with SDS 配方表 51
表 3.15 Ponceau S 染劑配方表 52
表 3.16 5 % 阻斷液配方表 52
表 3.17 一級抗體稀釋比例表 53
表 3.18 Tris-buffered saline with Tween-20 (TBST) 溶液配方表 53
表 3.19 二級抗體稀釋比例表 53
表 3.20 Stripping buffer 配方表 55

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