小鼠精原幹細胞於聚電解質多層膜之自我更新增殖研究

Abstract

近年來，逐層堆疊的聚電解質多層膜漸漸被用在幹細胞培養的研究上。藉由各種不同材料或生物分子的搭配，這種簡易又多方適用的多層膜堆疊法在表面改質上展現其無窮的潛力。在本研究中，我們利用數種多層膜修飾過後的表面培養具有多能幹細胞潛力的小鼠精原幹細胞，並且研究多層膜對幹細胞自我更新增生的能力的影響。首先，培養於pH 2.0或6.5環境下堆疊的poly(allylamine hydrochloride)/poly(acrylic acid) (PAH/PAA)多層膜上的精原幹細胞與以傳統模式laminin及TCPS培養者被用以相互比較。此外，堆疊層數由3至20.5個雙層的pH 6.5多層膜亦被研究由不同層數造成的效益。結果顯示，精原幹細胞在以pH 6.5的聚電解質 (PAH/PAA)溶液堆疊的多層膜上能形成貼附鬆散，為數眾多且型態呈完整圓形的細胞群聚。當堆疊層數不同，幹細胞群落的數量和大小也會隨之變化。經由鹼性磷酸酶(alkaline phosphatase，AP)及多能幹細胞的表現基因Octamer-4 (Oct4)二者檢驗，這些在多層膜上培養的細胞群聚仍保留在未分化的狀態，同時也證實具有分化為神經細胞的能力。在本研究的第二個部分，則應用精細管基質蛋白主成分laminin衍生出的胺基酸序列改質的多層膜表面，以及利用細胞外間質製備的多層膜。與PAH結合的SIKVAV、YIGSR及RGD被吸附於三個雙層的pH 6.5 PAH/PAA多層膜。當多層膜以胺基酸序列SIKVAV進行表面改質後，以之培養的精原幹細胞群落擁有較高度的未分化表現。接著由天然高分子膠原蛋白、明膠、幾丁聚醣與海藻酸鈉及玻尿酸搭配製備出三個雙層的多層膜，幹細胞群聚在大小與數量上皆表現不佳。總體而言，實驗結果指出，加以胺基酸序列SIKVAV輔助的PAH/PAA多層膜修飾過後的表面，是一個能夠有效率培養未分化精原幹細胞群的材料基質。

Polyelectrolyte multilayers (PEMs) by layer-by-layer deposition have recently been applied on approach to stem cells cultivation. This simple and versatile technique provides wide potential for variation of surface characters by applying diverse materials or insertion of biomolecules. In this study, enrichment of mouse germline stem cells (GSCs), which have potential for induction into pluripotent stem cells, achieved by variety of PEMs was investigated. First, GSCs were cultured on PEMs of polyelectrolyte poly(allylamine hydrochloride)/poly(acrylic acid) (PAH/PAA) assembled at either pH 2.0 or 6.5 in comparison with laminin and TCPS used as controls. An adjustment of deposited layer pairs from 3 to 20.5 bilayers was following proceeded on pH 6.5 films. We found that GSCs formed a greater number of loosely attached spherical colonies on pH 6.5 films than pH 2.0 or conventional culture. The number and size of colonies varied with the numbers of deposited PEM layers. Alkaline phosphatase (AP) activity and expression of Octamer-4 (Oct4) in these colonies indicated their potential as pluripotent stem cells. In the meanwhile, the ability of neuron-lineage differentiation suggested their great potential as well. In the second part of this study, the potential of immobilizing laminin-derived peptides on PEMs and altering assembled polyelectrolyte to promote the self-renewal of GSCs were assessed. SIKVAV, YIGSR and RGD were conjugated to PAH and incorporated on a 3 bilayers of PAH/PAA films assembled at pH 6.5. Enhancement of undifferentiated GSCs compared with pristine PEMs was found on the SIKVAV-conjugated films. Three bilayers of PEMs fabricated by natural polymers such as collagen (COL), gelatin (GEL), chitosan (CHI), alginate (ALG) and hyaluronic acid (HA) were utilized as substrates for GSC culture as well. However, colonies grew poorly on these films. Together, our results showed pH 6.5 PAH/PAA films incorporated with SIKVAV peptide provide a versatile substratum for self-renewal of GSC-colonies.