人類牙髓幹細胞藉由與視網膜色素上皮細胞共同培養分化為Pax6表現細胞

Abstract

視網膜對於視覺而言是關鍵構造，視網膜的受損、疾病、退化皆有可能造成視覺方面的障礙。幹細胞療法相較於過去的治療方式，例如支持性療法及神經營養因子（neurotrophins）輔助療法，也許會是更有潛力的治療方式。過去的研究中曾經提及，幹細胞對於視網膜細胞以及視神經有修復與再生的能力，對於保護神經免於傷害以及促進神經修復方面亦有功效，並且相較於人類胚胎幹細胞或是骨髓幹細胞，人類牙髓幹細胞（dental pulp stem cells, DPSCs）取得容易、在倫理上的爭議較小，並且具有分化為脂肪細胞、軟骨細胞、肌細胞等多種細胞的能力，說明了牙髓幹細胞是作為幹細胞治療的合適來源。 人類視網膜色素上皮細胞（retinal pigmented epithelium, RPE）位於視網膜外皮，在胚胎發育時參與了眼球形成的過程。神經生長因子（NGF, nerve growth factor）是一種同二聚體肽類，能夠調節細胞的生長以及促進神經分化。本研究之假說為，將人類牙髓幹細胞以神經生長因子誘導處理後，初步分化為神經前驅細胞，再與人類視網膜色素上皮細胞共同培養後，能夠表現出視網膜細胞標記，以螢光染色以及收集蛋白質測試Akt及其磷酸化型態的存在，並且加入Akt-PI3K 之抑制劑LY294002對共同培養之細胞進行前處理，來探討牙髓幹細胞的階段性分化是否與Akt生化路徑相關。 細胞免疫螢光染色的結果顯示，加入神經生長因子進行初步誘導分化的組別表現Pax6較未誘導的組別早，細胞形態方面亦有明顯差異；而西方墨點法的結果顯示，共同培養後所收集的牙髓幹細胞之蛋白質表現，在加入抑制劑LY294002的組別中，磷酸化Akt顯著地減少。本實驗的結論為人類牙髓幹細胞經過神經生長因子處理後，與視網膜色素上皮細胞共同培養，可以促進細胞分化而表現出Pax6視網膜標記，而磷酸化akt的表現在細胞間接接觸之後增加，加入抑制劑則使磷酸化akt顯著減少。

One of the key structures of human vision forming is retina. Visual impairment could be caused by damage, regression and disease of retina. Compared with previous therapies such as traditional supportive treatment or neurotrophins injection, stem cell therapy is a more promising approach. Stem cells are referred to be able to repair and regenerate the optic nerves and retinal cells and they are also effective in protecting nerves from damage in the past researches. Human dental pulp stem cells are more accessible stem cell source comparing to human embryo stem cells or bone marrow stem cells with less ethical concerns, presenting differential ability to multiple cells including adipose cells, chondrocytes and muscle cells. Retinal pigmented epithelium cells (RPE) which located in retinal epithelium is highly involved in eye development. NGF is able to regulate cell growth and facilitate neural differentiation. We hypothesized that DPSC-derived neural progenitors would differentiate to express retinal marker Pax6 after coculturing with RPE in vitro and is related to akt pathway, which were evaluated by immunocytochemistry and protein collection. Our immunocytochemistry and Western blotting results indicated that NGF-primed group required less time to express Pax6, and pakt level is reduced significantly in inhibitor LY294002 group.