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| 題名: | 年齡對脂肪幹細胞之骨誘導分化影響 |
| 作者: | 曾富宏 |
| 貢獻者: | 生醫材料暨工程研究所 |
| 日期: | 2010 |
| 上傳時間: | 2010-10-20 11:14:26 (UTC+8) |
| 摘要: | 本研究目的，是探討年齡對脂肪間葉幹細胞(adipose-derive stem cells, ADSCs)之骨生成作用影響。基於先前實驗室建立之血小板濃厚液(platelet rich plasma)促使骨前驅細胞其增生及骨分化能力，再移植至去卵巢早老化老鼠(OVX-SAMP8)中，進行骨質疏鬆之預防與治療研究，此結果已發表至Journal of Nuclear Medicine國際期刊上。故本實驗，鑒於先前建立細胞移植模式外，將深入探討不同年齡ADSC於體外及骨鬆老鼠內之骨誘導分化影響。首先第一部分體外實驗，於早老化老鼠腹腔中，分別取得年輕族群（1月齡）與老年族群（10月齡）之脂肪幹細胞進行體外定性實驗，並試驗出最適生長環境，以利維持不同年齡之幹細胞活性，實驗結果證實年輕族群之脂肪幹細胞其增生速率(proliferation rate)、自我複製(self-renewal)能力及細胞骨誘導分化表現，皆優於老年族群之脂肪幹細胞。第二部分動物實驗，將延續體外試驗，進而應用於骨鬆動物中，探討不同年齡的脂肪幹細胞來源於體內環境中，是否影響其骨生成作用。骨質密度結果發現，此治療模式可修復骨質流失現象並優於老年族群脂肪幹細胞之骨修復能力。該兩大部分實驗之建立，有助於現今細胞治療中，提供不同年齡幹細胞給予受試者之臨床前發展。
The aim of this thesis is to study the effects of aging on the ability of bone formation in ADSCs. Previously, we have established a novel therapeutic approach based on transplantation with progenitor cells (NIH3T3), in order to prevent osteoporosis and prolong the lifespan of the ovariectomized-senescence-accelerated mice (OVX-SAMP8). We decided to further our previous study by determining the aging effect on osteogenic differentiation potential of ADSCs. ADSCs were isolated from adipose tissues which obtained from female SAMP8 mice with different ages for in vitro study. First, we used flow cytometry to characterize the isolated cells by examining the expression of surface markers such as CD45, CD34, Sca-1, CD44 and CD105. Second, we compared the aging effects on the growth kinetics and differentiation potential of ADSCs between young (one month old) and old (ten month old) from female SAMP8 mice, and found there was a significant difference in both the proliferation rate and osteo-differentiation potential. Differentiated ADSCs were transplanted into the bone marrow in osteoporotic mice (OVX-SAMP8). Therefore, transplantion of differentiated ADSC cells was shown effective in restoring bone mineral density from the right/left knees and femurs at 3 months and the differentiated young group (ADSC-1M) showed significantly higher bone regeneration in OVX-SAMP8 mice, comparing to the differentiated old group (ADSC-10M). In conclusion, these findings provided important insights on emerging cell-based therapeutic strategies, especially in the treatment of various bone disorders including osteoporosis. |
| 關聯: | 49頁 |
| 描述: | 致謝 I
中文摘要 II
英文摘要 III
第一章 緒論 1
第二章 理論基礎 7
2.1 脂肪幹細胞 7
2.2 骨骼重朔作用 8
2.3 骨質疏鬆症 9
2.4 骨質疏鬆症動物模式建立 10
2.5 雙能譜骨質密度儀 10
第三章 材料與方法 12
3.1 實驗材料 12
3.1.1 藥品與試劑: 12
3.1.2 主要儀器: 13
3.1.3 細胞與動物: 13
3.2 實驗方法 14
3.2.1 骨質疏鬆症動物模式之建立 14
3.2.1.1 實驗動物 14
3.2.1.2 早老化雌性鼠的卵巢切除手術(Ovariectomy，OVX) 14
3.2.2 體外試驗(in vitro): 比較ADSC-1M和ADSC-10M的骨誘導分化 影響 15
3.2.2.1 脂肪幹細胞的取得與培養 15
3.2.2.2 流式細胞技術 16
3.2.2.3 細胞染色法 (cytochemistry assay) 16
3.2.2.4 配置誘導分化的培養基 17
3.2.2.5 mRNA萃取(total mRNA extration) 17
3.2.2.6 反轉錄作用(reverse transcription, RT) 18
3.2.2.7 聚合酶連鎖反應(Polymerase Chain Reaction, PCR) 18
3.2.2.8 洋菜膠配法(Agarose gel preparation) 19
3.2.2.9 去卵巢早老化小鼠(SAMP8-OVX)的鑽骨手術 19
3.2.3 功能性評估(function index) 19
3.2.3.1 骨礦物質密度測定(bone mineral density, BMD) 19
第四章 結果 21
4.1 實驗設計： 22
4.2 利用早老化老鼠(SAMP8)建立脂肪幹細胞(ADSC)之分離、培養 22
4.3 從分離的貼附細胞中分析脂肪幹細胞(ADSC)的表面抗原 23
4.4 探討分離細胞的分化能力 23
4.5 在活體外環境下培養不同年齡取得的脂肪幹細胞比較老化上的差異 24
4.6 比較兩組幹細胞年輕(younger donor)及老年(order donor)的增生速率 24
4.7 比較兩組幹細胞年輕(younger donor)及老年(order donor) 幹細胞的自我更新複製的能力 25
4.8 在體外環境下利用基因表現探討年齡影響幹細胞的骨分化能力 25
4.9 在體外環境下利用Alizarin red染色表現探討年齡影響幹細胞的骨分化能力 26
4.10 在體內環境下(in vivo)利用BMD探討年齡影響脂肪幹細胞的骨誘導分化 26
第五章 討論 27
第六章 結論 32
第七章 參考文獻 33
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