- 學位論文
特定單脈衝電磁場刺激誘導大鼠骨髓幹細胞分化至造骨細胞及蝕骨細胞之探討
Biological Effects of Specific PEMF Stimulation on Rat Bone Marrow Stem Cells to Induce Differentiation of Osteoblast and Osteoclast
摘要
隨著組織工程的研究發展，目前已知幹細胞具有多功能分化及自我更新的能力，且證實造骨細胞與蝕骨細胞分別是由間葉幹細胞與造血幹細胞分化而來；其中蝕骨細胞可由血源性幹細胞中之單核球細胞聚集、融合而成。故本研究欲利用脈衝式電磁場來刺激骨髓間葉幹細胞的增生或分化，並利用具有螢光穩定性之水溶性硒化鎘/硫化鋅（CdSe/ZnS）奈米量子點分別標定大鼠骨髓間葉幹細胞及單核球細胞，以觀測量子點於細胞融合分化後之蝕骨細胞中的分布情形。 本研究之實驗設計包括：一、建立大鼠骨髓間葉幹細胞之體外培養模式，並觀察細胞誘導分化情形。二、於三種磁場強度（1.3 G、2.4 G與3.2 G）下，將骨髓間葉幹細胞進行每天2小時的單脈衝電磁場刺激；並於第0、3、6、9、12與14天測定細胞生長力，及於第14天觀察細胞分化情形。三、進一步選擇兩種磁場強度（1.3 G與1.9 G）進行每天1小時的刺激，以期誘導骨髓間葉幹細胞分化成造骨細胞；並於第0、3、7、10和14天測定細胞生長力，及進行骨細胞特定之指標分析。四、將量子點分別與大鼠骨髓間葉幹細胞和單核球細胞進行培養；並以共軛焦顯微鏡觀察於分化成之蝕骨細胞中量子點的分布情形，同時進行蝕骨細胞的定量及定性分析。 本研究成功的純化及培養大鼠骨髓間葉幹細胞，並將其誘導分化為造骨細胞、脂肪細胞與軟骨細胞。而造骨細胞於誘導後第7天有鹼性磷酸酵素表現，第14天有骨礦化小節表現；脂肪細胞於誘導後第7天可於細胞質中發現脂肪油小滴；軟骨細胞於分化後第14天有GAGs表現。再者，於1.3 G與1.9 G之單脈衝電磁場刺激下，可達到促進細胞增生的效果，但長期於2.4 G與3.2 G之單脈衝電磁場刺激下，則會抑制細胞的生長速率。於添加造骨細胞誘導分化劑下，可發現1.9 G之單脈衝電磁場刺激可促進造骨細胞分化，反之於未添加分化劑時，電磁場刺激並不會改變細胞型態及分化能力。此外，於量子點添加培養一天後，可觀察到量子點聚集式分布於細胞質與細胞核周圍，但培養至第5天後，細胞內量子點開始減少；不過於10天的培養過程中，量子點並未改變細胞分化機制。由此可知，細胞是以胞噬機制攝入量子點。 於此，可發現透過單脈衝電磁場刺激能促進骨髓間葉幹細胞增生，但無法直接誘發造骨細胞的分化，然於誘導分化過程中，其有加速分化的效果。並且於此已成功建立利用奈米量子點標定細胞之架構，然量子點之毒性問題仍有待解決。於此，期望本研究可提供細胞工程學家一新視野，並作為骨組織相關研究之應用參考。
並列摘要
The core concept of bone tissue engineering is to accelerate bone repair and regeneration by guiding cells into scaffolds to form new bone tissues. Cells, scaffolds, and growth factors are the essential elements in tissue engineering. Besides, pulsed electromagnetic fields (PEMF) system has been well developed for many years and applied to bioreactors for bone tissue engineering in enhancing osteoblastic proliferation successfully in our laboratory. Stem cell, which is multipotent and self-renewal, is divided into mesenchymal stem cell (MSC) and hematopoietic stem cell (HSC) generally. Osteoclast is a multinucleated giant cell and osteoclastic formation is under the process of aggregation and fusion of monocytes, derived from HSCs. Osteoblasts can be derived from MSCs to provide source for bone tissue engineering. The amount of MSCs is low in vivo, therefore, we tried to applied PEMF to enhance the proliferation and differentiation of MSCs in vitro. Semiconductor quantum dot (QD) nanocrystal, synthesized by CdSe/ZnS is water-soluble and fluorescence stable for long-term imaging and labeling of rat bone marrow MSCs and monocytes to observe the differentiation of osteoclasts in vitro. Part I of this research was to establish rat bone marrow MSCs culture model in vitro, induce differentiation and perform qualitative analysis of differentiated cell types. Part II was to evaluate the effect of PEMF on MSCs proliferation. PEMFs (7.5 Hz) with three magnetic intensities, 1.3, 2.4, and 3.2 G, were applied to MSCs with three seeding cell densities, 50, 500, and 1000 cells/cm2. Cell viability was determined by MTT assay at day 0, 3, 6, 9, 12, and 14. The differentiation of MSCs was assessed by alkaline phosphatase (ALP) stain, von Kossa stain, alcian blue stain and oil red-O stain after 14-day PEMF exposure. Part III was to examine the effect of PEMF on osteoblastic differentiation from MSCs. MSCs were also exposed to 14-day PEMF stimulation of 1 hour at each day, and cell viability was determined by MTT assay at day 0, 3, 7, 10, and 14 day. In experiment of Part IV fluorescent, QDs were used to label rat bone marrow MSCs and monocytes for 10 days and the cytotoxicity in different cells as well as the osteoclastic formation derived from monocytes were evaluated. Cell viabilities of MSCs and osteoclasts were also determined by MTT assay, the characteristics and the number of osteoclasts derived from monocytes was examined by TRAP stain, and the distributions and the photostability of QDs were observed and reported by confocal microscope. The results showed that bone marrow MSCs could be isolated, expanded, and induced differentiated into osteoblasts, adipocytes, and chondrocytes in vitro. Besides, ALP and bone mineralization nodules expressions were observed at day 7 and 14, respectively, after osteoblastic differentiation. Fat droplets were present within cytoplasm at day 7 after adipotic differentiation. GAGs also expressed at day 14 in chondrocytes. The proliferation of MSCs was promoted by PEMF stimulation with 1.3 and 1.9 G, but the inhibitory effect was observed under the exposure of PEMF stimulation with 2.4 and 3.2 G. The morphology and the multipotent potential of MSCs did not change after PEMF stimulation. Moreover, PEMF of 1.9 G had the significantly synergistic effect with appropriate supplements in MSCs on facilitating osteoblastic differentiation. In the study of QDs labeling, QDs appeared the outside surroundings of nuclei after coculture with MSCs, and the level of QDs decreased after day 5 and 10 of coculture. After QDs cocultured with monocytes for 10 days, they did not affect osteoclastic differentiation. It indicated that endocytosis might occur for QDs to enter the cells. It was proved that single PEMF stimulation with specific parameters had the influence on enhancing the proliferation of MSCs, but not inducing the differentiation of osteoblasts from MSCs directly. It was interesting that PEMF had synergistic and facilitated effect on osteoblastic differentiation of MSCs in the presence of osteoblastic differentiation supplements. We also successfully used QDs to label MSCs and target osteoclasts. QDs may become a powerful and potential tool for clinical diagnosis afterwards. We successfully applied PEMF stimulation to facilitate the proliferation of MSCs, and we also successfully used fluorescent QDs to label MSCs and osteoclasts. It would be better if the cytotoxicity of QDs is decreased. It would supply an insight for cell engineering as well as a novel application for bone tissue engineering.