摘要:近年來,腦中風是一種發生率相當高的疾病,腦中風對於生命及行為功能都造成極高的死亡率及威脅。一般臨床上第一線治療方式是給於tissue plasminogen activator (tPA),促使栓塊的裂解,恢復血流。但是tPA 除了有投與時間上的限制外,且癒後過程中可能會伴隨出血性轉化(hemorrhagic transformation)的發生,進而加劇中風病程的發展。至今,臨床上對於腦中風的相關治療上,一直都苦無良好的治療方法。近年來,針對中風病人相關性的研究發現,中風病人的腦部內若具有較好的血管脈絡,具有較高的存活率,除此之外,癒後結果也具為良好。由此結果可知,血管新生(angiogenesis)對於在中風性傷害的治療上,扮演了極為重要的角色。Angiogenesis的過程中包含許多的因子與細胞共同參與完成的,這其中主要包含內皮細胞(endothelial cell)及平滑肌細胞(smooth muscle cell),這兩者在angiogenesis 過程中是缺一不可的,藉由兩者細胞間的相互作用,功能性血管才能順利發展形成。在此過程中,angiopoients-1 (Ang-1)及Tie-2 之間的相互作用,有助於維持脈管的形成、成熟及結構的穩定。近年來,陸續有報告指出,可經由幹/前驅細胞的植入,誘發血管新生來達到治療的效果。常見的幹/前驅細胞是源自骨髓及周邊血液,但是兩者都具有細胞數量不足的問題。經研究結果顯示,Wharton’s jelly (WJ)中分離出的Mesenchymal stem cells (WJ-MSCs)具有極高分化與增生的潛質,而且用於植入性治療中不具有免疫排斥的作用。所以本研究目的是想利用WJ-MSCs 的分化潛質,檢測藉著加入不同的分化物質共同培養,是否能夠分化為內皮前驅細胞(endothelial progenitor cells; WJ-EPCs)及平滑肌前驅細胞(smooth muscle progenitor cells; WJ-SMPCs)(合稱脈管前驅細胞vascular progenitor cells; WJ-VPCs)以供腦中風植入治療之用。首先將這些經分化處理的細胞進行細胞定性的分析,接下來利用脈管生成能力檢測,相較於WJ-EPCs 與WJ-SMPCs 的單獨培養,觀察WJ-VPCs 在共同培養下是否有助於脈管的形成,並進一步檢測Ang-1/Tie-2 在此所扮演的角色,觀察WJ-VPCs 是否藉由Ang-1/Tie-2 的作用,提高脈管生成及穩定度。接下來,確認在WJ-VPCs 的共同培養下,是否有助於大腦血管細胞增生及提供神經細胞滋養作用,保護神經細胞,免於因缺氧性傷害,而走向死亡一途之作用。體外實驗的方法為,將WJ-VPCs 的培養液與初級培養所得的大腦血管細胞或神經細胞置於缺氧及正常環境下共同培養,收集上述共同培養的細胞及培養液,針對細胞凋亡及細胞增生相關因子,分別利用西方墨點法、免疫螢光染色及細胞凋亡試劑(TUNEL kit)進行分析。除此之外,利用酵素免疫分析法及細胞激素微陣分析法進行分析,針對促血管新生、促神經滋養因子及抗發炎因子進行檢測。藉此釐清是由何種細胞激素在此參與調控。在確認過上述實驗結果後,利用老鼠的中大腦動脈進行栓塞(middle cerebral artery occlusion; MCAO)手術,以此作為缺血性中風的動物模式,在經MCAO 後,將各組細胞(WJ-EPCs、WJ-SMPCs、WJ-VPCs 及WJ-VPCs-siRNA 細胞激素)植入,首先觀察植入細胞是否能夠受到受損區域的誘使而移行聚集於受損病兆處,執行修補作用;而另一方面,利用行為檢測、影像分析儀器及組織切片染色,確認植入之前驅細胞是否具有修補損傷之神經組織,維持組織活性,減緩發炎反應、誘發血管新生,進而增加神經細胞的存活率與促進新生的作用,藉由上述結果的分析來觀察WJ-VPCs 的治療效用。在確認WJ-VPCs 之治療效能後,藉由WJ-VPCs-siRNA 細胞激素的植入組,進一步釐清此細胞激素於WJ-VPCs 治療腦中風傷害中,所扮演的功能角色與調控機制。希冀藉由這些實驗的分析,WJ-VPCs 能夠開啟並提供缺血性中風,另一種嶄新及有效的細胞性療法。
Abstract: Stroke is one of the major causes of human morbidity and mortality in the world. Possiblestrategy for treatment stroke in clinical is thrombolytic therapy by using the tissue plasminogen activator (tPA) to restore the blood flow. However, the clinical use of the tPA has presented several problems, including the given time was liminted within a short time and hemorrhagic transformation could be induced to exacerbate devastating effects of stroke. Currently, there are still no effective theraputies. It is widely considered that stroke patients with the high denisty of cerebral vessels have better progress and survival rate. A growing number of studies show that angiogenesis and neurogenesis are coupled processes. Thus, angiogenesis plays an important role in the stroke therapy. Angiogenesis is a complex multistep process that involves various growth factors and different cell types, such as endothelial and smooth muscle cells. It is well knowed that a tight cooperation between endothelial and smooth muscle cells is critical for the development of functional neovessels. In particular, angiopoients-1 (Ang-1) and their receptor Tie-2 have been shown to play an important role in communication between endothelial cells and smooth muscle cells, and then regulate the vessel formation, maturation and stability. Accumulated studies showed that cell-based therapy is a promoising strategy to induce angiogenesis in stroke patients. But the number of stem/progenitor cells from the bone marrow and peripheral blood is limited. Mesenchymal stem cells (MSCs) isolated from Wharton’s jelly (WJ) have multilineage differentiation and proliferative potential and little immune response. The goal was to isolate cells from WJ and differentiate into endothelial progenitor cells (WJ-EPCs) and smooth muscle progenitor cells (WJ-SMPCs) and termed them as vascular progenitor cells (WJ-VPCs). After the cell characterized individually, we examine the vessel-forming ability of WJ-VPCs and analyze the role of Ang-1/Tie-2 signaling in the beneficial effect of WJ-VPCs coadminstration. And next, we examine the potential of WJ-VPCs to contribute to vascular regeneration and neuronprotection and to provide therapeutic benefit for stroke. In vitro study, the angiogenesis and neurotrophic function of WJ-VPCs on cerebral endothelial cells (CECs) and cortical neurons were determined. We use the conditioned medium of WJ-VPCs to treat the CECs and cortical neurons in the normal and hypoxia, and then collect the cell and cultured medium for western blot, immunofluroresent staining and TUNEL assay analysis. ELISA and cytokine array were used to examine whether the cytokines involve in the therapeutic mechanism of WJ-VPCs. In vivo study, we transplant the WJ-EPCs, WJ-SMPCs, WJ-VPCs and cytokine siRNA-transfected WJ-VPCs into mice after middle cerebral artery occlusion (MCAO). First, we examine the homing activity of transplanted cells. Second, by using the image analysis machine and immunohistochemistry results, we compare the blood flow, vascular density, neuronal survival, inflammation, infarct area and neurobehavioral outcomes between the different groups to examine the therapeutic effects of WJ-VPCs. In this study, we hypothesize that the coadminstration of WJ-VPCs may be more efficient than single-cell-based therapy in producing stable and functional vessels for ischemic stroke. Movever, compared to the WJ-VPCs-transplanted mice, the therapeutic effects of cytokine siRNA-transfected WJ-VPCs on MCAO-mice were be observed to elucidate the regulatory role of cytokine in ischemic storke. These studies suggest that a combination of WJ-VPCs could be a novel and practical cell-based therapy for ischemic stroke.