| 摘要: | 目前針對治療神經系統退化性疾病還是沒有非常有效之方法，例如急性腦中風目前是十大死亡原中之第二位，然而腦中風之主要原因為腦中不同部位之血管栓塞或硬化阻塞造成進而產生突發性肢體偏癱，失語，手腳麻木等不同程度之神經功能喪失，目前之治療方式在急性期施以抗凝血劑，抗血小板凝集劑，甚至血栓溶解劑之藥物治療，其他之慢性神經系統退化性疾病如巴金森病及杭丁頓舞蹈症，則以復健及其他預防性之保守療法為主，疾病之預後不佳，多半為行動不便須家人照料，造成社會及國家資源之耗損。腦中風造成神經細胞快速死亡，而死亡之神經細胞及血管肉皮細胞無法再生及修補因此神經功能便喪失，因此，近年來有許多學者研究在中風之動物身上植入外來之幹細胞（Stem cell），例如有骨髓中胚層幹細胞，臍帶血幹細胞甚至是胚胎幹細胞等，每位學者都發現不僅能讓中風鼠之神經功能部份回復；且植入之幹細胞可分化成神經細胞及血管內皮細胞，此種治療方式對未來腦中風及神經退化性疾病帶來一線曙光。在許多文獻中都證實在幹細胞可在體內或體外分化為成熟之細胞例如肌肉細胞、肝臟細胞及神經細胞等，因此在骨髓幹細胞及臍帶血幹細胞慢慢被大多數人注意且被大量的運用於治療相關疾病之際，慢慢發現因老年人之骨髓較不易培養出幹細胞，而臍帶血之幹細胞數量過少，故極需要尋找另一間質幹細胞之來源，然而胎盤組織可培養出很多代之間質幹細胞，因此可想而知的是對於治療神經系統退化性疾病應可提共一使神經細胞復原及再生的好方法之一。本研究將分三年進行。第一年之工作 第一年將建立人類胎盤間質幹細胞體外培養模式，所須之工作包括收集胎盤組織分離血管並剪碎處理後置入細胞培養皿中加以培養，在進行 Proliferation stage 時培養基中加入含EGF 及FBS等物質，待胎盤間質幹細胞之數量到達一定程度時，進行FACS 分析以確定為間質幹細胞，同時作分盤及進行 Differentiation stage，此時培養基中加入含retinoic acid 及β-ME 等物質，胎盤間質幹細胞經分化處理後，我們首先觀查其細胞外形之變化，之後再利用免疫細胞染色之方法來觀查這些經分化過後之幹細胞是否表現一些具神經細胞之物質，包括 MAP-2, Neu-N, Nestin 及GFAP 等，所以藉此體外培養模式的確可以證實胎盤間質幹細胞經處理後會分化成神經細胞。第二年之工作 將已建立之成人胎盤間質幹細胞體外培養模式之後，利用胎盤間質幹細胞立體定位植入腦中風鼠腦中(中風七天後)，此後觀察老鼠每天之運動及感覺功能之恢復情況，期間以MRI及MRS觀察neuronal activity 恢復之情形，並於一個月後將實驗鼠之腦部取出，經切片染色觀察腦梗塞之大小，並以免疫化學染色之技術 (TUNEL, MAP-2 and Neu-N)在顯微鏡下觀察腦梗塞周圍神經細胞受保護之情行及以Confocal microscopy 之技術檢視胎盤間質幹細胞之腦內分化之情形，藉以與對照組比較是否有統計上之意義。第三年之工作 將已建立之成人胎盤間質幹細胞體外培養模式之後，利用胎盤間質幹細胞立體定位植入巴金森鼠腦中(6-OHDA lesioned 三週後)，此後觀察老鼠每天之運動及感覺功能之恢復情況(rotameter)，期間以MRI 及MRS 觀察neuronal activity 恢復之情形，並於三個月後將實驗鼠之腦部取出，經切片染色觀察並以免疫化學染色之技術(TH, MAP-2 and Neu-N)及以Confocal microscopy 之技術檢視胎盤間質幹細胞之腦內分化之情形，藉以與對照組比較是否有統計上之意義。
Despite the fact that bone marrow represents the main available source of mesenchymal stem cells (MSCs), the use of bone marrow-derived cells is not always acceptable due to the high risk of viral infection and the significant decreases in cell number and proliferation/differentiation capacity with age. Thus, the search for possible alternative MSCs sources remains a valid issue. In this study, we have isolated MSCs from the human placenta, to obtain clonally expanded MSCs (PMSCs) that are of multilineage differentiation potential. These PMSCs have been propagated in culture for more than 40 population doublings. The immunophenotype of these clonally expanded cells is consistent with that reported for PMSCs. Under appropriate induction conditions, these cells can differentiate into adipocyte, chondrocyte and osteogenic lineages in vitro. In addition, these cells were also able to differentiate into neuroglial-like cells under appropriate induction conditions. Furthermore, rats receiving intracerebral PMSCs transplantation showed significantly improved neurological dysfunction following cerebral ischemia than vehicle-treated control rats. Intracerebrally administered PMSCs enter brain, survive, migrate, and improve functional recovery after stroke. Transplanted PMSCs migrated towards the ischemic boundary zone and differentiated into glial cells (GFAP+), neuron (Nestin+, MAP-2+ and Neu-N+, CXCR4, Doublecortin) and vascular endothelial cells (vWF+) to enhance neuroplastic effect in ischemic brain. Cortical neuronal activity as evaluated by Proton MR spectroscopy (1H-MRS) was also much increased on transplantation group in comparison to control. In addition, significantly increasedmodulation of neurotrophic factor expression in the ischemic hemisphere was also found in the PMSCs transplantation group. The cells from adipose tissue could be regarded as a rich source of primitive cells characterized by mesenchymal stem cells that are readily expanded in culture with many generations. We conclude that PMSCs might be used as a more primitive source of MSCs with extended doublings for experimental and therapeutic applications. |
