靜水壓力增強胎盤幹細胞分化為成骨細胞之鈣沉積

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鄭志堅(ChIh-Chien Cheng) 查詢紙本館藏

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機械工程學系

論文名稱

靜水壓力增強胎盤幹細胞分化為成骨細胞之鈣沉積
(Hydrostatic Pressure Facilitates Calcium Deposition in Osteoblastic Differentiation of Placenta-Derived Multipotent Cells (PDMCs))

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摘要(中)

人體的骨骼形成起始於成骨細胞及骨髓間葉幹細胞(BMMSCs)，其會受到培養表面的影響。雖然骨組織在受到創傷後具有再生的能力，但修復的能力卻不一致，且可能產生不良的功能與結果。目前，有一些不同的方法來促進骨頭修復，如外固定或在骨折處加入無機鹽結晶來增進癒合。然而，若損傷區域太大或是骨質疏鬆患者，其破骨細胞的能力大於成骨細胞的能力，則修復能力將受到不同程度的限制。因此，在某些臨床的狀況裡，如骨質疏鬆，增加成骨細胞和前趨細胞的生醫材料工程或許是必要的改進，而在全世界人口老化趨勢下，骨質疏鬆已經是一個龐大的臨床問題。
組織工程是結合生命科學以及工程原理的一個新興學門。有越來越多的學者開始著手進行這方面的研究，其中探討機械物理因素對於細胞分化生長的研究近來也引起廣泛的注意。
近年來許多新的研究發現幹細胞具有高度分化的可塑性，而分化的主要決定條件之一在於幹細胞所處的微環境。目前的研究主要針對生化因子 (例如生長因子、細胞激素、藥物等等)對幹細胞分化的影響，但現在逐漸發現，組織工程材料或是物理因素對細胞也可能具有很重要的影響。
本研究從人類胎盤純化出多功能的細胞(PDMCs)，此細胞有能力分化成所有三個胚層的細胞表現型，其中包含成骨細胞。PDMCs的優勢是可方便容易取得大量的細胞，且不需藉由侵入性的方式就可以獲得細胞來源，與骨髓間質幹細胞有所不同。而且PDMCs 擁有免疫抑制的特性，在同種異體治療的應用上應該是可被高度考慮的。
所以，本文的特定目標為探討靜水壓力影響PDMCs成為並增進具有分化成為成骨性質的能力。我們覺得這些細胞可以是成骨細胞的前驅細胞之好的來源，本文的研究數據可能會有針對骨質疏鬆治療的關連性，以及在未來提供實際的臨床價值。

摘要(英)

Human bone derives from its cellular component, the osteoblast (OB) and its progenitor the bone marrow mesenchymal stem cell (BMMSC), could be affected by the surface and environment on which it was cultured. Although bone tissue is capable of regenerating after damage or injury, the quality of healing could vary and might result in poor function and outcome. Recently, a number of different methods to improve bone healing have been used, including inner/outer fixation and addition of inorganic crystals at the fracture site. However, if the injured surface/area is wide, or in the case of osteoporosis (OP) where bone resorption by osteoclasts (OCs) exceeds bone formation by OBs, the healing might still be compromised even with such augmentation to intrinsic capacity. Thus, the addition of OBs and progenitors to engineered biomaterials may be necessary to improve outcome in certain clinical conditions, including postmenopausal OP, which with the aging of the population is a clinical problem of epidemic proportions.
In the past few years, the field of adult stem cell research has been revolutionized with the discovery of plasticity. Key to determining the differentiation lineage is the microenvironment in which the stem cell resides in. While the majority of studies have focused on the effects of biochemical factors (i.e., growth factors, cytokines, drugs etc) on stem cell differentiation, it is now increasingly clear that tissue-engineered materials and physical factors might also exert important effects on cells.
In this study, we first isolated a population of multipotent cells from the human term placenta capable of differentiation into phenotypes of all three germ layers, including an osteoblastic phenotype. These placenta-derived multipotent cells (PDMCs) are abundant in numbers and can be obtained without the need of invasive procedures, unlike BMMSCs. Moreover, PDMCs possess significant immunosuppressive properties, which making these cells good candidates for therapeutic applications in an allogeneic setting.
The aim of our study is to discover how the osteoblastic differentiation capabilities of PDMCs are affected by using hydrostatic pressure. Our study showed that these cells provide an excellent source of OB progenitors and data from our study might have clinical therapeutic implications of OP.

關鍵字(中)

★ 靜水壓力
★ 胎盤幹細胞

關鍵字(英)

★ hydrostatic pressure
★ stem cell
★ osteoblastic differentiation

論文目次

CHAPTER 1. Introduction ….....................................................................................1
1.1. Research Motivation-Osteoporosis Clinical Overview.........................................1
1.2. Literature Survey of Stem cells ..………………………………………...........9
1.3. Research Purpose …………………..……………………………………….....13
1.4. Organization of Thesis ....……….…………………….…………………….....14
CHAPTER 2. Osteo-differentiation of Placenta-Derived Multipotent cells (PDMCs)…………………………………………………………………………..15
2.1 Isolation and ex vivo expansion of PDMCs.........................................................16
2.2. Phenotypic characterization of PDMCs .............................................................17
2.3. Osteoblastic differentiation ................................................................................18
2.3.1. Alkaline phosphatase activity assay .......................................................... 19
2.3.2. Alizarin red staining .................................................................................. 20
2.3.3. Quantitative reverse-transcriptase polymerase chain reaction...................... 21
2.4. Immunophenotype of Human Placenta-Derived Multipotent Cells .................. 22
2.5. Osteogenic Differentiation................................................................................. 24
2.5.1. Osteogenic Differentiation ........................................................................ 25
2.6. Characterization of Differentiated PDMCs .................................................... 28
2.7. Summary…………………………………...…………………..……………....31
CHAPTER 3. Hydrostatic Pressure Stimulation on PDMCs …………..…..........34
3.1. Isolation and ex vivo expansion of PDMCs ...................................................... 35
3.2. Statement for using of human materials .............................................................36
3.3. Osteoblastic differentiation of PDMCs ............................................................. 37
3.4. Hydrostatic pressure stimulation ....................................................................... 38
3.4.1. Alizarin red staining after Hydrostatic pressure stimulation………….….…42
3.4.2. Calcium and phosphate deposition assay ......................................................43
3.4.3. Total RNA extraction and quantitative reverse-transcriptase polymerase chain reaction ......................................................................................................... 44
3.5. Statistics analysis.................................................................................................45
3.6. Results …………..……………..……………………………............................46
3.6.1. Calcium deposition was enhanced by hydrostatic pressure stimulation ….. 47
3.6.2. Quantification of osteoblast differentiation gene expressions in PDMCs under hydrostatic pressure stimulation………..…………………….……... 50
3.6.3. Quantification of BMP family and BMP receptor gene expression in PDMCs under hydrostatic pressure stimulation ......................................................... 52
CHAPTER 4. Conclusions…………………….........................................................56
CHAPTER 5. Future work …………..……….........................................................61
REFERENCES ..........................................................................................................62

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指導教授

鍾志昂(Chih-An Chung)

審核日期

2015-7-13

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