出生後小鼠心臟前驅細胞分泌之外吐小體的功能特性

Abstract

根據先前的研究，本實驗團隊發現注射A83-01(乙型轉化生長因子第一型受體抑制劑) 進入心臟衰竭的老鼠，可以防止其心臟功能喪失，一方面透過增加心臟前驅細胞（Nkx2.5+ CPC）數量來增加新生心肌細胞以補償心臟受傷後心肌缺損；另一方面，可增加心臟前驅細胞旁泌支持心肌細胞存活，改善心臟功能。然而其旁泌產生之心臟保護機制尚未明瞭。 本篇研究目的為比較A83-01處理過之心臟前驅細胞分泌的外吐小體和控制組的外吐小體，對於活體或離體心肌細胞存活率以及心臟前驅細胞增生的影響；並鑑別出能增進心肌細胞存活之外吐小體中的微小分子核醣核酸(microRNA)；檢驗注射外吐小體進入心臟受損後的老鼠是否能改善心臟功能。 結果顯示在體外試驗中，A83-01處理過之心臟前驅細胞所分泌的外吐小體，在進行分離後直接給予心肌細胞，能夠增加心肌細胞的存活率，並可促進心臟前驅細胞增生。接著檢驗直接給予外吐小體中的微小分子核醣核酸miR-30c-5p, miR-489-5p, miR-92b-3p, miR-98-5p, and mmu-let-7a-5p是否影響離體心肌細胞的存活，結果顯示miR-30c-5p和miR-92b-3p能在體外增加心肌細胞存活率，並具有劑量相關性。動物實驗中，透過連續七天施打異丙腎上腺素(ISO)造成動物心臟受損，並於第八天靜脈注射心臟前驅細胞分泌之外吐小體，實驗證實外吐小體能改善心臟受損後之功能恢復。 總結，本實驗發現A83-01處理過之心臟前驅細胞分泌之外吐小體能夠顯著地增加離體心肌細胞的存活率，並提升離體及活體心臟前驅細胞自我更新，能貢獻在改善受損後心臟之功能失常。

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 Nkx2.5+ cardiac progenitor cells (Nkx2.5+ CPC) to compensate part of the lost myocardium and CPC-mediated paracrine actions. It still remains unclear in the paracrine actions of A83-01-stimulated CPC on cardioprotective mechanism. In this study, the aims are (1) to compare the different effects of the exosomes secreted from Nkx2.5+ CPC treated with DMSO [Exo(Nkx+/DMSO)] or A83-01 [ Exo(Nkx+/A83) ] in the survival of adult cardiomyocyte and the proliferation of Nkx2.5+ CPC in vitro and in vivo, (2) to identify the exosomal miRNAs that could potentially increase cardiomyocyte viability, and (3) to examine whether the treatment of either Exo(Nkx+/DMSO) or Exo(Nkx+/A83) in post-injured hearts could improve cardiac function. Our results have demonstrated that Exo(Nkx+/A83) could markedly increase cell viability of adult mice cardiomyocyte cultured in vitro for 3 days, and could also increase Nkx2.5+ CPC proliferation. The direct effect of Exo(Nkx+/A83) exosomal miRNAs, including miR-30c-5p, miR-489-5p, miR-92b-3p, miR-98-5p, and mmu-let-7a-5p, in cardiomyocyte viability was further examined in vitro, and found that miR-30c and miR-92b could dose-dependently increase myocyte viability in vitro. In isoprenaline (ISO)-injection induced cardiac injury mice model, it was found that intravenous injection of Exo(Nkx+/A83) on the next day after the last dose of ISO could significantly improve cardiac function at the end point on day 7. In conclusion, the present study found that Exo(Nkx+/A83) could produce significant benefit in supporting myocyte survival and enhancing Nkx2.5+ CPC self-renewal in vitro and in vivo, which may contribute to the amelioration of cardiac dysfunction in post-injured hearts.