透明質酸（HA）為重要的細胞外基質組成分子，且在傷口癒合過程中扮演重要角色。本實驗室曾發現於透明質酸塗附介面上（CHA）所培養的幹細胞可維持相應的分化能力，細胞增生速率卻有減緩。本研究使用人類胎盤間葉幹細胞（PDMSCs），並探討透明質酸塗付介面CHA以及未添加透明質酸TCS兩種培養方式經由何種途徑調控幹細胞訊息傳遞。首先確認PDMSC表面的分化抗原標誌表現與間葉幹細胞一致，脂肪、軟骨、及硬骨分化也與已知的間葉幹細胞分化能力相符合。基於前人研究之CHA延長細胞週期G0/G1期的成果，將CHA和TCS介面培養的細胞選取細胞週期分布相似的時間點，統一做後續的蛋白質萃取以及蛋白質組學分析。以SILAC定量及液相色譜串聯質譜法測定短期以及長期培養的間葉幹細胞中的蛋白質表現變化。於TCS對照組別中發現，PDMSC經過長期培養之後，Ras蛋白及其相關訊息傳遞途徑的MAP2K3;MAP2K6，RND3，LIMA1，FHL1，NEFM，TPM1，以及FLNC等蛋白皆有表現量上升，或與細胞老化現象相關。而在CHA長期培養PDMSC的實驗組別中，前所述的Ras等蛋白皆有表現量下降，另有NAMPT表現量上升。NAMPT可調控細胞內多餘p53蛋白的降解，由此可延緩p53所調控之細胞老化。

Hyaluronan has been documented in important biological processes. In our lab’s previous studies, cells cultured on coated hyaluronan (CHA) substratum exhibited reduced proliferation rate yet still maintained the differentiation potentials specific for mesenchymal stem cells. Some researchers were conducted on the effects of high molecular weight hyaluronan on maintaining stem cell characteristics, yet no study has reported the effects of long-term hyaluronan treatment on stem cells. Our study aims at elucidating the effect of hyaluronan on placenta-derived mesenchymal stem cells (PDMSCs) cultured in CHA versus TCS conditions. First we characterized and established biomarkers expression profile of PDMSC. For comparison of hyaluronan-mediated protein differential expression in TCS versus CHA, time points were selected for synchronized cell cycle phase on TCS and CHA culture conditions. Cellular proteins were labeled using stable isotope labeling with amino acids in cell culture (SILAC), followed by protein identification, protein quantification, and ratio determination by using liquid chromatography tandem-mass spectrometry (LC-MS/MS). Finally, in silica prediction of differentially displayed protein patterns was generated. In our findings, in PDMSC long-term cultured on TCS, Ras protein and its related signaling pathway components, MAP2K3;MAP2K6 and actin-interacting proteins (RND3, LIMA1, FHL1, NEFM, TPM1 and FLNC), were up-regulated, and may be correlated to stress fiber formation and possibly promoted senescence and aging. In contrary, CHA long-term culture was correlated with down-regulation of Ras signaling and related proteins and up-regulation of NAMPT and molecules from the endoplasmic reticulum-associated degradation (ERAD) pathway. Notably, NAMPT up-regulation may be associated with degradation of excess p53, and thus prevented p53-mediated senescence and maintained normal phenotype of PDMSC.

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