| 摘要: | 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甚至多巴胺細胞等，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，故極需要尋找另一種間質幹細胞之來源，然而臍帶組織可培養出很多代之間質幹細胞，並且本團隊利用一種新的培養方法內含人類臍帶血清，分離出一種具多分化能力(multipotent potential)帶IGF1R+之間質幹細胞，因此本研究欲探討此間質幹細胞生成之分子機轉，並利用此間質幹細胞來治療腦中風及巴金森動物模式，可想而知的是對於治療神經系統退化性疾病應可提共一種神經細胞復原及再生的好方法之一。第一年研究結果第一年將建立人類IGF1R+臍帶間質幹細胞體外培養模式，所須之工作包括收集臍帶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2% 之臍帶血清及EGF 及FGF 等物質，待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(Figure 1)及帶有SSEA-4, SSEA-3, IGF1R,Oct-4 等之自我更新(self-renewal) 之標誌， 同時作分盤觀察其生長速率及進行Differentiation stage，此時培養基中加入含B27, SHH, NGF, FGF8, retinoic acid 及β-ME等物質，脂肪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有三種胚層之細胞，90/12/21 修訂包括ㄧ些特別之細胞標誌或染色如oil red O stain, Alizni red S stain，Alcian blue, insulin,SMA, MAP-2, Neu-N Nestin 及GFAP 等，所以藉此體外培養模式的確可以證實IGF1R+臍帶間質幹細胞經處理後會分化成各種細胞。第二年研究結果將已建立之人類IGF1R+臍帶間質幹細胞體外培養模式之後，利用此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功能(body swing testand locomotor activity)之恢復情況，期間以MRI 及MRS 觀察neuronal activity 恢復之情形(Figure 2)，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IGF1R+臍帶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。第三年研究結果將已建立之人類IGF1R+臍帶間質幹細胞體外培養模式之後，利用此間質幹細胞在體外誘導分化成多巴胺細胞(Tyrosin hydroxylase-TH) ，再以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 andNeu-N)及以Confocal microscopy 之技術檢視IGF1R+臍帶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 ofmesenchymal stem cells (MSCs), the use of bone marrow-derived cells is not alwaysacceptable due to the high risk of viral infection and the significant decreases in cellnumber and proliferation/differentiation capacity with age. Thus, the search forpossible alternative MSCs sources remains a valid issue. In this study, we haveisolated MSCs from the umbilical cord, the matrix of umbilical, to obtain clonallyexpanded MSCs (hUCMSCs) that are of multilineage differentiation potential. ThesehUCMSCs have been propagated in culture for more than 40 population doublings.The immunophenotype of these clonally expanded cells is consistent with thatreported for bone marrow MSCs. Under appropriate induction conditions, these cellscan differentiate into adipocyte, chondrocyte and osteogenic lineages in vitro. Inaddition, these cells were also able to differentiate into neuroglial-like cells underappropriate induction conditions. Furthermore, rats receiving intracerebral hUCMSCstransplantation showed significantly improved neurological dysfunction followingcerebral ischemia than vehicle-treated control rats. Intracerebrally administeredhUCMSCs enter brain, survive, migrate, and improve functional recovery after stroke.Transplanted hUCMSCs migrated towards the ischemic boundary zone anddifferentiated into glial cells (GFAP+), neuron (Nestin+, MAP-2+ and Neu-N+,CXCR4, Doublecortin) and vascular endothelial cells (vWF+) to enhance neuroplasticeffect in ischemic brain. Cortical neuronal activity as evaluated by Proton MRspectroscopy (1H-MRS) was also much increased on transplantation group incomparison to control. In addition, significantly increased modulation of neurotrophicfactor expression in the ischemic hemisphere was also found in the hUCMSCstransplantation group. The cells from umbilical cord could be regarded as a richsource of primitive cells characterized by mesenchymal stem cells that are readilyexpanded in culture with many generations. We conclude that hUCMSCs might beused as a more primitive source of MSCs with extended doublings for experimentaland therapeutic applications. |
