- 學位論文
人類羊膜幹細胞體外分化為心肌細胞之可行性評估
Assessment of the feasibility of human amniotic membrane stem cell-derived cardiomyocytes in vitro
摘要
根據世界衛生組織 (World Health Organization) 於2019年的統計，缺血性心臟病 (ischemic heart disease) 已成為全球第一大死因。現今之治療方法仍然無法避免心肌損傷所導致之心臟功能下降，器官短缺也使得缺血性心臟病的後續治療停滯不前。研究指出間葉幹細胞 (mesenchymal stem cells, MSCs) 雖具有複分化潛力及免疫耐受性，已被廣泛應用於幹細胞治療研究，惟體內移植的修復速度較緩慢，無法應付緊急需求，體外分化為心肌細胞有成效不彰之問題，因此本研究擬利用人類羊膜之MSCs (human amnion derived MSCs, hAMSCs) 在體外分化為心肌細胞，並探討其效率及功能。 本研究主要分成二個試驗，試驗一為hAMSCs之分離與培養。羊膜取自亞東醫院剖腹產之婦女，最終分離之細胞在顯微鏡下呈現MSC特有之紡錘狀。細胞表面抗原分析結果顯示這些細胞會表現人類MSCs應表現之CD105, CD73, CD90, CD44；不表現造血細胞相關之CD19,、 CD11b,、 CD34, 、CD45以及HLA-DR，且有部分表現胚幹細胞相關marker，如:SSEA1、SSEA3及SSEA4。並且這些細胞也具有分化成硬骨、軟骨、脂肪細胞的能力，且經RT-PCR分析結果顯示這些細胞會表現多能性相關之Oct4、Nanog及Rex1。前述結果顯示本試驗已從羊膜成功分離出hAMSCs。 試驗二為分化條件測試，本試驗使用BMP4、Activin A、5-azacytidine、CHIR99021及IWP2來做為誘導分化之因子，將這些因子以特定濃度加入hAMSCs之培養液，分別以BMP4/Activin A及其他三種因子的添加與否來將試驗組分為16組，後利用qPCR檢測MLC2v、Nkx2.5及MyoD基因的表現量以探討這些化學分子對心肌細胞分化的影響，結果顯示添加5ng/ml BMP4、10ng/ml Activin A、10μM 5-azacytidine、7.5μM CHIR99021及5μM IWP 2之組別在此三種心肌相關marker中分別具有第二高及最高的表現量(MLC2v：3.82±0.12；Nkx2.5：4.53±0.21；MyoD：6.99±0.66)。此試驗組在後續的免疫染色試驗中顯示有α-actinin及Troponin T的表現。 綜觀上述，本試驗之結果有助於了解hAMSCs在體外分化成心肌細胞之可行性，未來能再深入探討此誘導分化之機制，並進一步改善試驗細節，以期能提高分化效率及功能性，作為治療人類心肌損傷基礎及體外心臟藥物測試之參考。
並列摘要
According to the statistics of World Health Organization in 2019, ischemic heart disease (IHD) was the number one cause of death in the world. The treatments nowadays can not prevent the decline in heart function. Stem cell therapy may be the potential treatments for IHD. Many researches have successfully induced stem cells to differentiate into cardiomyocytes in vitro. Among several kinds of stem cells, mesenchymal stem cells (MSCs) have enormous potential for clinical applications including the immune privilege and the multipotent differentiation efficiency. However, the repair speed of in vivo transplanting is too slow to handle the urgent needs. In the other hand, differentiation in vitro is inefficient. Therefore, this study intends to explore the feasibility and efficiency of inducing the human amniotic membrane MSCs (hAMSCs) to differentiate into cardiomyocytes in vitro. This research is mainly divided into two parts. Part one is the isolation, culture and characterization of hAMSCs. The amniotic membrane was taken from caesarean section at Far Eastern Memorial Hospital. These isolated cells showed spindle-shaped MSCs which can be clearly observed under microscope. The cell surface antigen analysis showed that these cells were positive for CD73, CD90, CD105, and CD44, negative for CD19, CD11b, CD19, CD45 and HLA-DR, and positive for some embryonic stem cell markers such as SSEA-1, SSEA-3 and SSEA-4. Also, these cells have the ability to differentiate into adipocytes, chondrocytes and osteoblasts. Besides, these cells could express some pluripotent markers such as Oct4, Nanog and Rex1 with the analysis of RT-PCR. These data demonstrated the successful isolation of hAMSCs from amniotic membrane. Part two is the inductive differentiation of hAMSCs. In this research, there were five inductive chemicals, including BMP4, ActivinA, 5-azacytidine, CHIR99021 and IWP2. After inductive differentiation, we analyzed the expression level on MLC2v, Nkx2.5 and MyoD with qPCR. The qPCR results showed that the treatment with 5ng/ml BMP4, 10ng/ml Activin A, 10μM 5-azacytidine, 7.5μM CHIR99021and 5μM IWP 2 expressed highest levels of these genes (MLC2v：3.82±0.12；Nkx2.5：4.53±0.21；MyoD：6.99±0.66). Also, this group could express α-actinin and Troponin T with immunofluorescence staining. In conclusion, this study helped to understand the feasibility of the hAMSCs differentiation into cardiomyocytes in vitro. In the future, we can further explore the differentiating pathways and mechanisms of hAMSCs, and revise this protocol to improve the differentiating efficiency and the function of differentiating cells, which can be used as a reference for basic and clinical application research on the treatment of human myocardial injury and heart drug test in vitro.
參考文獻
延伸閱讀
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