抗氧化物對人類間葉幹細胞抗氧化及抗衰老之影響

Abstract

間葉幹細胞(MSCs)屬於複能性幹細胞(multipotent stem cells)，具分化為多種間葉細胞的能力，如骨骼細胞、脂肪細胞、軟骨細胞及肌肉細胞等，目前已知能從多種組織內分離出MSCs，包括骨髓、脂肪組織及臍帶血等。近年來MSCs被認為能成為未來細胞治療的來源。雖然MSCs具有很強的自我再生特性，但在體外擴增培養的過程，仍會發生細胞衰老，而氧化壓力在細胞老化的過程扮演了重要角色。本論文著重在研究臍帶血間葉幹細胞(CBMSCs)及骨髓間葉幹細胞(BMMSCs)，其中添加抗氧化劑對細胞衰老與氧化狀態之關聯。本研究藉由在培養過程中加入抗氧化劑Kinetin及Eepigallocatechin-3-gallate (EGCG)，來減少細胞的氧化壓力，以了解抗氧化劑對BMMSCs 及CBMSCs生長狀態、細胞凋亡、活性氧分子(ROS)產量及脂質過氧化的影響。我們發現經過長時間Kinetin及EGCG處理的MSCs與培養在正常條件下的MSCs，其表面抗原的呈現及細胞凋亡的程度沒有顯著差異，也發現在培養過程加入Kinetin的確可以降低細胞內的氧化狀態，增加過氧化氫酶(Cat)活性，並延長細胞壽命。但是培養環境添加EGCG，則沒有顯著的影響。抗氧化劑對MSCs分化能力之影響，我們發現Kinetin能夠提升CBMSCs分化為骨骼細胞之能力，而EGCG則會降低兩種MSCs之脂肪分化能力。此外，經過長時間於培養環境添加Kinetin，能夠顯著的降低老化指標β- galactosidase的產生，添加EGCG則沒有發現相同結果。這些結果顯示，Kinetin可能做為有效減少MSCs增殖造成細胞衰老的抗氧化劑之一。

Mesenchymal stem cells (MSCs) are the multipotent stem cells that can give rise to mesenchyme-lineage cells, such as osteoblasts, adipocytes, chondrocytes and myoblasts. MSCs can be isolated from various tissues including bone marrow, adipose tissue, and umbilical cord blood etc. Recently, MSCs have been represented as a promising cell source for further cellular therapy. In spite of the strong self-renewal property, MSCs undergo cellular senescence during expanded cultivation in vitro. It is well-known that the oxidative stress plays an important role for cell aging. In this study, we focused on the relationship between cellular oxidative status and cultivated senescence in bone marrow-derived MSCs (BMMSCs) and cord blood-derived MSCs (CBMSCs). We examined the cellular oxidative stress by culturing cells with antioxidants, kinetin and epigallocatechin-3-gallate (EGCG). To understand the effects of antioxidants supplementation on MSCs, the results of growth status, reactive oxygen species (ROS) value, and lipid peroxidation were analyzed in BMMSCs and CBMSCs. We found that kinetin and EGCG treated MSCs expressed similar surface marker profiles with normal culture condition. The results also showed that the lower oxidative status, the higher catalase activity, and the longer lifespan could be found in kinetin cultivation than control condition in both types of MSCs. However, there were no obvious differences between MSCs cultures with or without EGCG addition. In differentiation test, the osteogenesis of CBMSCs could be enhanced by kinetin and the adipogenesis could be inhibited by EGCG in both types of MSCs. Moreover, kinetin but not EGCG decreased the anti-aging marker β- galactosidase in late passage of BMMSCs and CBMSCs. These results indicated that kinetin could be a useful antioxidant to reduce cell senescence during MSCs expansion.