紅色螢光豬羊水幹細胞移植於巴金森氏症模式大鼠之治療潛能

Abstract

巴金森氏症 (Parkinson's disease) 為一漸進性的神經退化性疾病，其病因乃由於患者中腦黑質中的多巴胺神經元 (dopaminergic neuron) 死亡，無法製造足夠的多巴胺 (dopamine)，導致神經傳導出現問題，造成患者產生一些活動障礙，臨床上之症狀包括靜止性震顫 (resting tremor)、動作遲緩 (bradykinesia)、軀幹僵硬 (rigidity) 以及步態姿勢不穩定 (postural instability) 等；目前臨床上使用藥物治療和手術治療雖可於發病初期緩解其症狀，然隨病程之延長其療效將弱化，終將無法阻止病症之惡化。而近年研究指出，羊水幹細胞 (amniotic fluid stem cells, AFSCs) 具有分化為三胚層等不同組織細胞之能力，且表現多能性幹細胞之特異性分子標誌－Oct-4，經移植於體內後亦不會形成畸胎瘤，乃一新興且極具潛力之幹細胞來源。因此，探討以羊水幹細胞治療巴金森氏症之可行性並釐清其治療機轉，將有助於巴金森氏症新興療法之發展。 本研究使用來自於紅色螢光蛋白質轉基因豬 (DsRed transgenic pig) 的羊水幹細胞，進行異種移植至巴金森氏症模式大鼠中，以探討羊水幹細胞應用於治療巴金森氏症之可行性。首先，利用音蝟因子 (sonic hedgehog, SHH)、第八纖維母細胞生長因子 (fibroblast growth factor, FGF8)、鹼性成纖維細胞生長因子 (basic fibroblast growth factor, bFGF) 及腦源性神經滋養因子 (brain-derived neurotrophic factor, BDNF) 等生長因子之添加於紅色螢光豬羊水幹細胞的培養液中以進行多巴胺神經分化 (dopaminergic neuronal differentiation)，結果可觀察到細胞外觀之轉變，開始出現類似神經細胞之細長形態且會表現神經細胞的特異性標誌－β-III微管蛋白 (β-III tubulin)；直至誘導分化第12天時，可在誘導分化組中發現表現多巴胺神經元之特異性標誌－酪胺酸羥化酶 (tyrosine hydroxylase, TH) 的細胞，顯示紅色螢光豬羊水幹細胞已被成功誘導分化成為類多巴胺神經，此結果也暗示豬來源之羊水幹細胞應用於巴金森氏症新興療法開發之可行性。而後將紅色螢光豬羊水幹細胞移植至使用六-羥基多巴胺 (6-hydroxydopamine, 6-OHDA) 破壞單側內側前腦束 (medial forebrain bundle, MFB) 之巴金森氏症疾病模式大鼠腦中，以評估羊水幹細胞應用於巴金森氏症之治療效果。在進行六-羥基多巴胺破壞後兩週，經由免疫組織化學染色可見破壞側之黑質紋狀體徑路 (nigrostriatal pathway) 有明顯的多巴胺神經元缺失；續將紅色螢光豬羊水幹細胞移植至巴金森氏症模式大鼠腦中，並於移植後兩週經由阿樸嗎啡 (apomorphine) 誘發之旋轉行為評估其治療效果。結果顯示，接受細胞移植的組別之旋轉行為顯著地低於巴金森氏症模式大鼠控制組及接受磷酸鹽緩衝液 (phosphate buffered saline, PBS) 注射的對照組 (p < 0.01)；而至移植後四週，巴金森氏症模式大鼠之旋轉行為的圈數下降約40% 且極顯著地低於另兩個組別 (p < 0.001)。進一步觀察大鼠腦中多巴胺神經纖維之表現，結果顯示，接受紅色螢光豬羊水幹細胞移植的組別之多巴胺神經纖維密度顯著地高於未移植細胞的巴金森氏症模式大鼠控制組及接受PBS注射之對照組 (p < 0.001)。上述結果顯示，移植紅色螢光豬羊水幹細胞可顯著地改善巴金森氏症模式大鼠之旋轉行為缺陷並減緩其運動功能退化之進程。此外，欲釐清羊水幹細胞治療巴金森氏症之可能機轉，利用免疫螢光染色於移植後24小時、48小時、72小時及四週，追蹤腦組織切片中所移植進去的紅色螢光豬羊水幹細胞。結果在移植後24小時、48小時和72小時，皆能在移植處發現許多表現紅色螢光的細胞，然此些紅色螢光豬羊水幹細胞並未開始往神經系 (neural lineage) 進行分化；至移植後四週時，雖於移植處所找到的表現紅色螢光的細胞大都為細胞碎屑 (cellular debris)，但仍可於週邊找到極少存活的紅色螢光豬羊水幹細胞，且分化為表現酪胺酸羥化酶之多巴胺神經元。 綜合上述，紅色螢光豬來源之羊水幹細胞可於體外適當培養環境下經誘導分化為類多巴胺神經，若移植至體內，紅色螢光豬羊水幹細胞於移植後四週仍可存活於大鼠紋狀體中，並分化為表現酪胺酸羥化酶之多巴胺神經元，且能顯著地改善巴金森氏症模式大鼠之旋轉行為缺陷。是以羊水幹細胞乃一具有潛力之幹細胞來源應用於後續臨床上巴金森氏症細胞療法之開發。

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra that leads to dopamine level decreases in the brain. Its clinical symptoms include resting tremor, rigidity, bradykinesia and postural instability. However, present pharmacological and surgical therapies can only postpone the emergence of motor symptoms, none have been able to reverse the degeneration of dopaminergic neurons. Recent studies have shown that amniotic fluid stem cells (AFSCs) are capable of differentiating into cell types from all three embryonic germ layers and expressing pluripotent stem cell marker, Oct-4. Most importantly, AFSCs won't induce teratoma formation in vivo when applied on clinical transplantation. Therefore, to investigate the therapeutic potential and possible mechanisms of AFSCs in PD is beneficial to developing a new and more effective way for treating PD. In this study, we used the AFSCs isolated from transgenic DsRed pigs for xenotransplantation in parkinsonian rats. First, we found that DsRed porcine AFSCs (pAFSCs) could differentiate into dopaminergic neuron-like cells in vitro. After induction with dopaminergic neuronal differentiation medium which was composed of sonic hedgehog, fibroblast growth factor 8, basic fibroblast growth factor, and brain-derived neurotrophic factor, the cells developed a neuronal morphology expressing the neuronal marker β-III tubulin. Additionally, the differentiated DsRed pAFSCs could express dopaminergic neuronal specific marker tyrosine hydroxylase (TH) 12 days after induction. These results implied the feasibility of using pAFSCs as a cell source for developing therapies of PD. Next, Sprague Dawley rats unilaterally lesioned by 6-hydroxydopamine (6-OHDA) in the medial forebrain bundle were used as the parkinsonian model animal to evaluate the therapeutic effect of AFSCs in PD. After 2 weeks of 6-OHDA lesioning, the immunoreactivity of dopaminergic neuronal marker-TH reduced in the nigrostriatal pathway compared with the sham lesioned control group. Following DsRed pAFSCs transplantation into striatum, apomorphine-induced rotations were significantly less than the PD model control and sham grafted control group that only received PBS injection 2 weeks after transplantation (p < 0.01). Moreover, the rotations number of the DsRed pAFSCs transplanted group were reduced by about 40% and showed very significantly lower than the other two groups 4 weeks after transplantation (p < 0.001). TH positive dopaminergic neurons and fibers could be observed in the graft side by immunohistochemistry of the brain sections and the density of ipsilateral TH positive fibers were significantly increased after 4 weeks following transplantation of DsRed pAFSCs (p < 0.001). These results suggested that transplantation of DsRed pAFSCs could significantly alleviate the asymmetric rotational behavior of the PD rats and prevent further deterioration. Furthermore, we tracked the grafted cells by immunofluorescence staining of the brain sections at 24, 48, 72 hours and 4 weeks after cell transplantation in order to figure out the possible therapeutic mechanism. We found that the transplanted DsRed pAFSCs stayed around the graft site in striatum but had not yet started to differentiate into neural lineage at 24, 48 and 72 hours after transplantation. Until 4 weeks after transplantation, the xenografted cells could survive and differentiate into dopaminergic neurons though most of the grafts become cellular debris that left in the graft site. In conclusion, the results show that DsRed pAFSCs could differentiate into dopaminergic neuron-like cells in vitro under the specific culture condition, and when transplanted in vivo, DsRed pAFSCs could not only survive and differentiate into dopaminergic neurons but also significantly promote the functional behavioral recovery of the PD rats 4 weeks after transplantation. Hence, AFSCs might be a promising cell source for clinical cell therapy development of Parkinson's disease.