人類牙髓幹細胞誘導分化成視網膜細胞之研究

Abstract

中樞神經系統受損或視網膜的退化，皆會造成視覺功能的障礙，甚至是眼盲。除了過去傳統的支持性治療及神經營養因子（neurotrophins）注射療法，幹細胞的植入會是一個有潛力的治療方式。在過去的研究中證實了幹細胞可以有效促進受傷或退化的視網膜細胞、視神經進行修復與再生。相對於人類之胚胎幹細胞及骨髓幹細胞，牙髓幹細胞（dental pulp stem cells, DPSCs）的取得相對比較簡單，也比較沒有道德倫理上的爭議。牙髓幹細胞源於神經脊（neural crest），已被證實可以有效分化成脂肪細胞、軟骨細胞、成骨細胞、肌細胞、神經細胞等。另外，過去許多實驗中顯示牙髓幹細胞對於保護神經免於傷害以及促進神經修復都有著優異的表現，因此我們相信牙髓幹細胞會是一個很有潛力的幹細胞來源。 人類視網膜色素上皮細胞（retinal pigmented epithelium, RPE）位於視網膜的外圍，在胚胎發育時眼球和視網膜形成過程中扮演了重要的角色。Sonic hedgehog（Shh）調控因子參與眼球早期發育的過程，也是前腦發育中不可或缺的因子。本實驗的假說為，當人類牙髓幹細胞被誘導分化成神經前驅細胞之後，接著與視網膜色素上皮細胞一起共同培養，並加入Sonic hedgehog因子，會被誘導分化成視網膜細胞。實驗中將誘導牙髓幹細胞分化成神經前驅細胞（neural progenitor cells），接著把神經前驅細胞與視網膜色素上皮細胞進行共同培養（co-culture）或是加入Sonic hedgehog以促進其分化成視網膜細胞，此時實驗設計分成四組，分別是R+S組、R組、S組及C組，C為控制組，R代表RPE，S代表Sonic hedgehog因子。 細胞免疫螢光染色和即時聚合酵素連鎖反應的結果顯示，R+S組、R組、S組在第14天的時候都表現了S-Opsin、Pax6和PKC的蛋白標記，並且都有顯著的基因表現。特別的是，S組的Pax6於第14天的基因和蛋白表現比其他組別更顯著，在細胞型態的部分也有明顯的差異。本實驗的結論為人類牙髓幹細胞和視網膜色素上皮細胞之共同培養及加入Sonic hedgehog因子，兩者皆會促進視網膜細胞的分化。單獨加入Sonic hedgehog因子的組別，在分化成視網膜神經節細胞（retinal ganglion cells）和無長突細胞（amacrine cells）的能力更優於其他的組別。

Injury of central nervous system and neurodegenerative diseases may contribute to retina cell loss and degeneration, resulting in functional deficits and even blindness. In addition to traditional supportive treatment and neurotrophins injection, stem cell transplantation is a promising therapeutic approach to reduce damage and promote retinal regeneration. Comparing to human embryonic stem cells（ESCs）and bone marrow stem cells （BMSCs）, dental pulp stem cells（DPSCs）are easier to obtain with lesser ethical concerns, and also becoming an alternative source of stem cells. Since dental pulp stem cells（DPSCs）are originated from cranial neural crest, which showed great potential of differentiating into adipogenic, chondrogenic, osteogenic, myogenic and neurogenic lineage. Several in vitro and in vivo studies demonstrating that dental pulp stem cells（DPSCs）protect against neural damage and promote neural repairing and regeneration. Retinal pigment epithelium（RPE）plays a role in eye development and morphogenesis of neural retina. Besides, Sonic hedgehog factor is essential for embryonic forehead and eye development. We hypothesized that DPSC-derived neural progenitor cells would differentiate into retinal cells by Sonic Hedgehog factor and/or co-culture with RPE under neuronal inductive conditions in vitro. Firstly, we induced dental pulp stem cells（DPSCs）to differentiate into neural progenitor cells, following by retinal differentiation by adding Sonic Hedgehog factor and/or co-culturing with RPE under neural inductive conditions. In retinal differentiation, neural progenitor cells were divided into four groups as C，R，S and R+S groups, which C represent control group which was only DPSCs without any induction, R represent RPE co-culturing and S represent Sonic hedgehog treatment. Our real time polymerase chain reaction（RT-PCR）and immunocytochemistry staining results indicated that R, S and R+S groups showed both gene and protein expression of S-Opsin, Pax6 and PKC on day 14. Furthermore, S groups showed unique cell morphology from other groups, and S groups also showed greater gene expression and protein expression of Pax6 than other groups on day 14. Our results indicated that dental pulp stem cells（DPSCs）co-cultured with retinal pigment epithelium（RPE）and Sonic hedgehog treatment, both can induce retinal differentiation of dental pulp stem cells. S group which is individual Sonic hedgehog treatment could induce dental pulp stem cells differentiate into retinal ganglion cells and amacrine cells more significantly than other groups.