利用人胚幹細胞建立一篩選具刺激多巴胺神經細胞分化潛力以改善帕金森氏症之植化素的體外模式

Abstract

本研究的實驗目的是以人類胚幹細胞株（human embryonic stem cell, hESC）H9細胞株建立一體外平台，用以模擬中腦多巴胺神經細胞（midbrain dopaminergic neurons, mDA neurons）的發育過程，得以藉此篩選出具有刺激神經細胞新生潛力的植化素，並擬探討此最具有刺激神經細胞新生功效之活性成分的作用機制。結果顯示，確可透過一個五階段（Stage I-V）的分化流程，成功使H9細胞株分化形成類mDA的神經細胞，其形成TH+細胞的分化率為32.8-53.6%，並進一步透過mDA相關因子如En1、Lmx1a、TH（tyrosine hydroxylase）、AADC（aromatic L-amino acid decarboxylase）與GIRK2等之mRNA表現分析，以及相關的特定蛋白如nestin、TH、DAT（dopamine transporter）與β-III tubulin之免疫螢光染色分析，證明所分化的細胞確為類似mDA神經細胞。另外，透過與維持神經電生理相關的離子通道之mRNA表現分析與多巴胺分泌量的測定，確立了分化而成的細胞具有神經功能性，並發現此流程可使細胞產生具mDA特定性的Kv10.1（Kv: voltage gated-potassium channel）表現。在確立了分化流程後，我們更進一步利用過去文獻發現具有刺激神經新生的藥物多巴胺受器促進劑bromocriptine、7-OH-DPAT與抑制劑sulpiride驗證此平台的效果，其方法為分析兩DA神經細胞之特定蛋白TH與GIRK2的qPCR表現量，以此分別確立了上述正、負控藥物的有效濃度為：20 μM bromocriptine、10 μM 7-OH-DPAT與10 μM sulpiride。最後，在植化素成分的篩選上，發現綠茶兒茶素效果與其結構上的差異相關，而EGCG（epigallocatechin gallate）為其中最具效果的食品成分，其最佳處理濃度為12.5 μM；而人參皂苷Rb1的處理結果則顯著優於綠茶兒茶素，當其處理濃度為6.25 μM時，可大幅提升TH與GIRK2的表現量分別達12與14倍，顯示其在促成mDA神經細胞分化形成上的潛力。而在相關機制探討中發現，Rb1可能是透過促使周圍環境的星狀細胞分泌相關的神經滋養因子BDNF（brain derived neurotrophic factor）與NT-3（neurotrophin-3），以及誘使Wnt-1/ frizzled-1/β-catenin傳導途徑的產生，而具有促類似mDA神經細胞分化的能力。

This study aimed to establish an in-vitro model using hESC (human embryonic stem cell) H9 cell line, which is expected to simulate the development of midbrain dopaminergic neurons (mDA neurons). Thus, through the model, we expected to screen phytochemicals with potential on stimulating mDA neurogenesis to improve Parkinson's disease (PD). As a result, we successfully established a five-stage process which could differentiate hESC H9 cell line into mDA-like neurons, and the differentiation rate of TH+ cells was 32.8-53.6%. Moreover, the final differentiated DA neurons truly expressed mature DA neuron-related factors like En1, Lmx1a, TH (tyrosine hydroxylase), AADC (aromatic L-amino acid decarboxylase), GIRK2 and other factors. Through the analysis of immunefluorescence staining, the in-vitro model also showed specific protein expression such as nestin, TH, β-III tubulin and DAT (dopamine transporter). These results demonstrated these differentiated cells were certainly mDA-like neurons. Besides, these cells showed mRNA expression of several voltage-gated ion channels, especially the mDA-specific one, Kv 10.1. In addition, the dopamine release (7.28 ± 1.30 ng/ml) by the differentiated cells was also detected. After that, according to the qPCR analysis of two marker genes, TH and GIRK2, we determined bromocriptine and 7-OH-DPAT as positive control drugs and sulpiride as negative control suitable for this model. Their effective dosages are presented as the following: 20 μM bromocriptine, 10 μM 7-OH-DPAT and 10 μM sulpiride. Finally, we tested green tea catechins (GTCs), including EGCG, ECG, EGC and EC as well as ginsenoside Rg1, Rg3 and Rb1 using the established model. We found that the abilities of GTCs might be related to their differences in structure. EGCG, which possesses both galloyl moiety and tri-hydroxyl substitutes on the B-ring, significantly increased the TH and GIRK2 expression at 12.5 μM. Moreover, better than GTCs, at 6.25 μM, ginsenoside Rb1 greatly enhanced the TH and GIRK2 expression to 12 and 14 folds, respectively. Thus, it appeared that Rb1 was the most potent phtyochemical to have potential of stimulating the differentiation of mDA-like neurons. Its possible mechanism might include stimulating the BDNF (brain-derived neurotrophic factor) and NT-3 (neurotrophin-3) secretion from surrounding astrocytes and activting the Wnt-1/ frizzled-1/β-catenin signaling pathway.