- 學位論文
應用人類羊膜間葉幹細胞治療大鼠心肌缺血再灌流損傷模型之潛力
The therapeutic potential of human amnion-derived mesenchymal stem cells on ischemia/reperfusion in rats
摘要
根據世界衛生組織於2019年統計世界十大死因排行顯示，缺血性心臟病為十大死因之首。目前最直接的治療方法就是透過再灌流將阻塞處打通。惟再灌流處理後仍然無法避免心肌損傷以及後續心室重塑產生之纖維化、心室擴張及心室壁變薄。研究指出間葉幹細胞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。其中分離自人類羊膜之間葉幹細胞對大鼠心肌再灌流損傷模型 (IR) 的療效仍尚未清楚。故本研究擬建立大鼠心肌再灌流損傷疾病模式，藉由尾靜脈注射人類羊膜之間葉幹細胞來探討其治療效果。 本研究主要分成三個試驗，試驗一為人類羊膜間葉幹細胞之分離與培養，並確保其具有人類間葉幹細胞之特性，以利後續治療使用。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, CD19, CD45以及HLA-DR。並且這些細胞也具有分化成硬骨、軟骨及脂肪的能力。前述結果顯示本試驗已從人類羊膜組織成功分離出間葉幹細胞。試驗二為大鼠心肌再灌流損傷模型之建立，並確保其在心臟功能以及結構上之變化皆符合先前大鼠心肌再灌流損傷之研究結果。試驗使用8週齡雄性SD大鼠，並分成控制組、假手術組、心肌缺血/再灌流組三組。在超音波影像偵測結果顯示，經過再灌流處理後28天相較於控制組出現左心室擴張以及心臟前壁變薄之現象；心臟功能的部分能看到在再灌流處理後第7天相較於控制組左心室射血分率以及左心室縮短分率皆顯著下降 (p<0.05)，而假手術組則與控制組沒有顯著差異 (p>0.05)。心臟組織切片染色分析中也可以看到再灌流處理後相較控制組及假手術組都有明顯之心肌細胞損傷以及纖維化之現象。故上述結果顯示已成功建立大鼠心肌再灌流損傷模型。試驗三為將人類羊膜間葉幹細胞透過尾靜脈注射治療心肌再灌流損傷大鼠，目的為測試人類羊膜間葉幹細胞是否對心肌再灌流損傷大鼠具有療效。並將試驗分成控制組、假手術組、PBS對照組及細胞組四組。超音波影像偵測結果顯示，人類羊膜間葉幹細胞治療後相較於PBS對照組在第7及第28天皆顯著地 (p<0.05) 改善再灌流損傷大鼠之左心室射血分率(day7: 68.58±3.43% ; day28: 63.92±2.94% ; 平均值±標準誤差) 以及左心室縮短分率 (day7: 39.7±2.76% ; day28: 36.2±2.31% ; 平均值±標準誤差)。在心臟切片染色的結果上，人類羊膜間葉幹細胞治療後相較於PBS對照組，能顯著增加左心室前壁厚度 (1.76±0.1mm ; 平均值±標準誤差， p<0.01) 以及顯著減緩左心室纖維化疤痕占左心室肌肉面積之比例 (12.2±2.5% ; 平均值±標準誤差， p<0.05)。上述結果證實人類羊膜間葉幹細胞在大鼠心肌再灌流損傷模型中能確實幫助改善心臟收縮功能、減緩心室重塑以及降低心肌損傷範圍。 綜觀上述，本試驗之結果有助於了解人類羊膜間葉幹細胞治療大鼠心肌再灌流損傷模型可行性，未來能再深入探討人類羊膜間葉幹細胞之治療路徑與機制，期能作為治療人類心肌損傷基礎及臨床應用研究之參考。
並列摘要
According to the World Health Organization's ranking of the top ten causes of death in 2019, ischemic heart disease (IHD) was the No. 1 cause of death in the world. At present, re-opening of the occluded coronary artery is the major therapeutic goal in IHD. However, after reperfusion, myocardial injury is remained, and subsequent ventricular remodeling may cause fibrosis, ventricular dilation, ventricular wall thinning. Studies have pointed out that mesenchymal stem cells (MSCs) have unique characteristics, such as multipotency and immune tolerance, and have been widely used in stem cell therapy research. Among them, the efficacy of MSCs therapy isolated from human amniotic membrane (human amnion derived MSCs, hAMSCs) for rat myocardial reperfusion injury (IR) models is not well‐known. Therefore, this study intends to establish a IR rat model, and explore its therapeutic effect by injecting hAMSCs into the tail vein. The research is mainly divided into three parts. Part one is the isolation and culture of hAMSCs, and to ensure that they have the characteristics of human mesenchymal stem cells for subsequent treatment. The amniotic membrane was taken from caesarean section at Far Eastern Memorial Hospital. The isolated cells showed spindle-shaped MSCs and clearly observed under microscope. The cell surface antigen analysis showed that these cells were positive for CD73, CD90, CD105, and CD44, and did not express hematopoietic lineage markers such as CD19, CD11b, CD19, CD45 and HLA-DR. And these cells also have the ability to differentiate into osteoblasts, adipocytes, chondrocytes. These data demonstrated the successful isolation of hAMSCs from amniotic membrane. Part two is the establishment of IR rat model. We aimed to establish a reliable rat model of IR. Seven-week-old, male Sprague–Dawley rats were used, and they were divided into three groups: control group (pre-op), sham group, and IR group. The results showed that the left ventricular dilatation and the thinning of the anterior wall of the heart occurred in IR group compared with control group in ultrasound image. In cardiac function, left ventricular ejection fraction (LVEF%) and left ventricular fractional shortening (LVFS%) was significantly decreased (p<0.05) in the IR group compared with pre-op group, while there was no significant difference between the sham group and the pre-op group (p>0.05). In histological analysis of heart tissue, there were obvious cardiomyocytes damage and fibrosis in IR group compared with the control group and the sham group. Therefore, the above results show that showed that the rat model of IR was successfully established. The third experiment was to inject hAMSCs through the tail vein to treat IR rats. The purpose was to test whether hAMSCs had therapeutic effect in IR rats. The experiment was divided into four groups: control group (pre-op), sham group, PBS group and hAMSC group. Results showed that hAMSCs treatment significantly (p<0.05) improved the LVEF% (day7: 68.58±3.43; day28: 63.92±2.94; mean±SE) and LVFS% (day7: 39.69±2.76; day28: 36.24±2.31; mean±SE) compared with PBS group at day7 and day28. Moreover, compared with the PBS group, hAMSCs treatment can significantly increase the thickness of the left ventricular anterior wall (1.76±0.1mm; mean±SE; p<0.01) and significantly reduce the area of left ventricular fibrosis scars (12.2±2.5%; mean±SE; p<0.05). Above all, transplantation of hAMSCs provided significant improvement in a rat model of IR. In conclusion, this study helped to understand the feasibility of hAMSCs in the treatment of IR rats. In the future, we can further explore the therapeutic pathways and mechanisms of hAMSCs, which can be used as a reference for basic and clinical application research on the treatment of human myocardial injury.
參考文獻
延伸閱讀
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