遠端肌肉注射羊水幹細胞之心肌梗塞小鼠治療模式

Abstract

心臟疾病為國人的主要死亡原因，心肌梗塞為其中一項最知名的致命原因。心肌梗塞會導致梗塞區域之心肌細胞因死亡，並使心室結構擴張、心臟功能逐漸下降，最終可能導致心臟衰竭。幹細胞療法已證實可對受損的心臟發揮修復作用，其治療機制來自於其分化能力或來自分泌的細胞激素。羊水幹細胞為近年來新發現之幹細胞種類，可通過低侵入性之操作方式取得、且具優秀之分化潛能與免疫調節功能，並具有於體內或體外分化成為心肌細胞之潛力。目前的研究結果顯示羊水幹細胞對心肌梗塞模式動物具治療效果，但針對其分泌能力於心肌梗塞之治療效果的研究報告仍相較缺乏。因此本試驗中將利用肌肉注射之方式，測試羊水幹細胞是否能藉其分泌之因子產生治療效果。 為測試羊水幹細胞移植是否可對心肌梗塞小鼠產生治療效果，首先將由本研究室建立之Ds-red 紅色螢光轉基因豬之羊水中分離羊水幹細胞供後續試驗使用。本試驗中取得的紅色螢光豬羊水幹細胞，經表面抗原鑑定發現所取得的紅色螢光豬羊水幹細胞不會表現CD31及CD4a二項標誌，而會表現CD44與CD90，且經繼代後仍可穩定表現紅螢光蛋白質。 為確保後續之羊水幹細胞移植實驗可順利進行，需先建立穩定的心肌梗塞小鼠模型。本試驗將以8週齡之雄性C57/B6小鼠做為模式動物，將其分為心肌梗塞組與偽手術組，心肌梗塞組之小鼠採永久性結紮之方式紮起冠狀動脈左前降枝。之後利用心臟超音波及組織切片分析檢測心臟結構及功能之變化。手術後4週，心肌梗塞組小鼠之左心室收縮末期內徑與左心室舒張末期內徑均顯著增加(p < 0.05)。手術後2週心肌梗塞小鼠之左心室射血分率降至47.82 ± 2.21%，且於手術後4週更進一步降為35.51 ± 4.13%。此外，心肌梗塞組小鼠梗塞區域之心室壁厚度顯著變薄(p < 0.05)，且於梗塞區域形成了由纖維組織構成的疤痕。以上結果證實我們成功的建立了合適的心肌梗塞小鼠模式。 為謀測試肌肉注射羊水幹細胞是否可對心肌梗塞小鼠產生治療效果，本試驗中將B6小鼠分做3組：中細胞劑量組(1 x 106 cells per leg)，高細胞劑量組(4 x 106 cells per leg) 與PBS對照組。細胞或PBS將於心肌梗塞手術完成後20分鐘，直接以肌肉注射之方式移植至左右腿的大腿肌肉中。手術後4週，中細胞劑量組(43.16 ± 2.81%)及高細胞劑量組(50.04 ± 5.37%)與PBS對照組(37.54 ± 3.98%)相較，均保持較佳的左心室射血分率 (p < 0.05)。且中細胞劑量組與高細胞劑量組其手術後4週之左心室射血分率與手術後2週之左心室射血分率保持在相同水平。移植中細胞劑量組與高細胞劑量組於梗塞區域之心室壁厚度於心肌梗塞後仍保持較厚(p < 0.05)，在高細胞劑量組中疤痕的尺寸亦較PBS對照組小(p < 0.05)。且中細胞劑量組與高細胞劑量組之左心室擴張指數均較PBS對照組為小(p < 0.05)，顯示其左心室之擴張程度較低。此外利用非侵入式活體影像系統分析由各組小鼠取得之肌肉、心、肺、腎等組織樣本後，發現只有於中細胞劑量組與高細胞劑量組小鼠之腿部肌肉中可偵測到紅色螢光蛋白質之訊號，顯示該處仍留有所植入的紅色螢光豬羊水細胞，於其它組織內並無偵測到紅色螢光蛋白質之訊號。 綜上所述，肌肉注射豬羊水幹細胞可降低小鼠心肌梗塞後左心室之疤痕尺寸、維持其心室壁厚度、減低其左心室擴張程度，且可幫助保持較佳的心臟功能。由於所移植之羊水幹細胞會留存在腿部肌肉中，可推測其對於心臟結構與功能之治療功效應來自於其分泌之因子。

Heart diseases have been one of the major causes of death in Taiwan for years. Myocardial infarction (MI) is the best-known case among heart diseases, and often causes cell death in ischemic area, results in pathological remodeling and decreasing cardiac function, even may leads to heart failure eventually. Stem cell therapy had been demonstrated that could repair damaged heart through differentiation potential and factors secreting of stem cells. Amniotic fluid stem cells (AFSCs) among stem cells can be obtained with low-invasively procedure, and possess immunosuppressive properties, and shows cardiomyoplasticity in vivo and in vitro. Previous studies showed transplantation of AFSCs has beneficial effects for MI animals, but studies focused on its secretion abilities still lacks. Hence, the aims of this study are to investigate their therapeutic potential on MI mice by using the manner of AFSCs intramuscular injection. In order to test the therapeutic potential of factors released by AFSCs, porcine AFSCs were isolated from amniotic fluid of E70 porcine fetus of Ds-red-transgenic pig. These cells express surface antigen CD44 and slight express CD90, do not express CD4a, CD31, and can stably express red fluorescence protein (RFP) after serial passage. Before starting to test the therapeutic potential of AFSCs, appropriate MI model needs to be established. For this purpose, 8-week-old male C57/B6 mice were divided into MI group and Sham group. MI was induced by ligation of the left descending coronary. Four weeks after surgery, both of left ventricular (LV) end-systolic dimension and LV end-diastolic dimension increased significantly in MI group (p < 0.05). Two weeks after surgery, ejection fraction in MI group decreased to 47.82 ± 2.21%, and then further decreased to 35.51 ± 4.13% two weeks later. In addition, wall thickness decreased in infracted region, and fibrosis scar has formed within injury sites. Above results indicated suitable MI model has been established. In order to test the effects of AFSCs intramuscular injection on MI mice, MI model mice were divided into 3 groups: medium cell dose (1 x 106 cells per leg) group Cell-M, high cell dose (4 x 106 cells per leg) group Cell-H and PBS group. Cells or PBS were directly injected into hamstring muscle 20 minutes after MI surgery. Four weeks after MI surgery, Cell-M and Cell-H groups (43.16 ± 2.81%, 50.04 ± 5.37%) when compared with PBS group (37.54 ± 3.98%) preserved significantly better LV ejection fraction (p < 0.05). And the ejection fraction in Cell-M and Cell-H groups four weeks after MI persevered in same level when compared to two weeks after MI. Infarct scar sizes in both Cell-M and Cell-H groups were smaller than in PBS group. Wall thickness in scar region preserved thicker in Cell-M and Cell-H groups when compared with PBS group. Cell-M and Cell-H groups also showed smaller LV expansion index, which indicated lower degree of LV dilation. The specimens of hamstring muscle, heart, lung, liver and kidney from mice in all groups were collected and detected by IVIS (in vivo imaging system) for RFP signals from injected or migrated cells. The result showed that only hamstring muscles from Cell-M and Cell-H groups exhibit RFP positive signals. In summary, intramuscular injection of porcine AFSCs can reduce scar size, reduce pathological remodeling, and preserve better heart function after MI bearing. Putatively, intramuscularly injected AFSCs resided in hamstring muscle, and possibly assist recovery of heart morphology and function through their secreting factors.