心臟前驅細胞分泌之胞吐小體潛在的心臟保護效果

Abstract

簡介：由先前的研究，我們發現A83-01(一種抑制乙型轉化生長因子第一型受體抑制劑)在心肌梗塞受損後的小鼠心臟中，能一方面透過增加心臟前驅細胞來代償死亡的心肌細胞，另一方面也經由細胞的旁泌作用，來促進心臟的自我修復並改善心臟功能。然而受A83-01刺激之心臟前驅細胞如何藉由旁泌細胞外囊泡（特別是胞吐小體）來產生心臟保護機制仍不清楚。 目的：本研究主要目的為 (1) 評估心臟前驅細胞受 A83-01刺激與否，其所分泌之胞吐小體在體外支持心肌細胞存活的效果差異，(2)找出受A83-01刺激之心臟前驅細胞所分泌之胞吐小體中之心臟保護作用分子（包含微型核醣核酸及蛋白質），(3)釐清存在於心臟前驅細胞分泌之胞吐小體內能在活體保護受損後心臟之功能分子。 材料與方法：由Nkx2.5 enhanced-GFP 報告老鼠心臟以酵素分離心臟前驅細胞，並利用螢光激活細胞分選器純化。由心臟前驅細胞的培養液中，利用ExoQuick-TC分離出胞吐小體，利用LM10 Nanosight對其物理性質進行分析。利用微陣列對微型核醣核酸進行分析有否受A83-01刺激之心臟前驅細胞所分泌之胞吐小體中的微型核醣核酸差異表現。利用限時定量聚合酶連鎖反應驗證表現量差兩倍以上之微型核醣核酸。此外，利用液相層析-質譜-質譜儀來進行蛋白體學的分析。 利MEM為基底培養酵素分離出之成鼠心肌細胞，評估胞吐小體及其內含分子對離體心肌細胞存活率之影響。利用八周大Myh6-cre/Esr1::mTmG報告老鼠腹腔注射tamoxifen 將成熟心肌細胞標記GFP 以進行細胞命運追蹤，藉此評估心臟前驅細胞所分泌之胞吐小體及其內含分子在心肌梗塞後老鼠心臟中的心臟保護效果。 實驗結果：在體外試驗中，將心臟前驅細胞和心肌細胞共同培養，可以促進心肌細胞的存活率。心臟前驅細胞分泌之胞吐小體也可以仿效共同培養的結果，也具有促進心肌細胞存活率的效果。給予心臟前驅細胞A83-01之後，促進效果可以更進一步的提升。給予心臟前驅細胞A83-01之後，胞吐小體中的微型核醣核酸在限時定量聚合酶連鎖反應的結果顯示miR-489-3p、miR-92b-3p、miR-98-5p和mmu-let-7a-5p有顯著的增加。 結論： 心臟前驅細胞分泌之胞吐小體具有心臟保護效果，而此保護效果可以透過給予心臟前驅細胞A83-01的方式更進一步提升。心臟前驅細胞的胞吐小體中，miR-30c-5p、miR-489-3p、miR-92b-3p、miR-98-5p和mmu-let-7a-5p的表現量因為A83-01的給予而增加。我們仍須需要更進一步的研究來探討，在胞吐小體的心臟保護作用主要是透過其中的哪些分子和機轉來達成。

Background: Our previous study found that A83-01, a TGFβ receptor I (TGFβRI) inhibitor, can facilitate cardiac self-repair and improve cardiac function in post-MI mice heart through the mechanisms of expanding cardiac progenitor cells (CPC) to compensate part of the lost myocardium and CPC-mediated paracrine actions. It remains unclear in the cardioprotective mechanism mediated through the extracellular vesicles, especially exosomes, secreted from A83-01-stimulated CPC. Aim: The objectives of the present study are (1) to assess the differential benefit of the exosomes secreted from CPC treated with or without A83-01 in supporting cardiomyocyte survival in vitro, (2) to identify cardioprotective molecules, including microRNAs and proteins, in the exosomes secreted from CPC treated with A83-01, and (3) to clarify the cardioprotective benefit of the functional molecules in CPC-secreted exosomes in post-injured heart in vivo. Materials and Methods: CPCs were enzymatically isolated from transgenic Nkx2.5 enhanced-GFP reporter mice, and were purified by FASC sorter. CPC-secreted exosomes were isolated from medium by ExoQuick-TC, and were further characterized the physical properties by LM10 Nanosight. miRNAs microarray was performed to identify the candidate miRNAs with more than two-fold change in the exosome secreted from A83-01-treated CPCs. Real-time PCR was employed to validate miRNA expressions. Proteomics analysis was performed to identify the candidate proteins by LC-MS-MS. Adult mouse cardiomyocytes were enzymatically isolated and cultured in MEM-based medium for viability assay. Myh6-cre/Esr1::mTmG mice were used to do genetic fate mapping for labeling the matured cardiomyocytes with GFP via intraperitoneal injection of tamoxifen at 8-weeks-old, which could assess the cardioprotective effect of CPC-secreted exosomes and the associated molecules applied in post-MI hearts. Results: Co-culture of CPC with adult mice cardiomyocyte could significantly enhance myocyte survival in vitro. The CPC-secreted exosomes could mimic the co-culture condition in the enhancement of adult mice cardiomyocyte survival. The cardioprotective effect could be further potentiated by the exosomes secreted from A83-01-treated CPCs. miRNA microarray and qPCR validation shows the marked increase of miR-489-3p, miR-92b-3p, miR-98-5p, and mmu-let-7a-5p in the exosome secreted from A83-01-treated CPCs. Conclusion: CPC-secreted exosomes possess cardioprotective effect, and the benefit could be further potentiated by the exosomes secreted from A83-01-treated CPC. The upregulation of miR-30c-5p, miR-489-3p, miR-92b-3p, miR-98-5p, and mmu-let-7a-5p was found in the exosomes secreted from A83-01-treated CPC. It needs further investigation to clarify the function and underlying mechanism mediated by the molecules in the exosomes.