探討脂肪幹細胞的條件培養液與外吐小體對於心肌缺血再灌流引發心肌損傷的影響

Abstract

心肌梗塞(myocardial infarction, MI)是現代化國家主要的疾病之一。根據世界衛生組織2011年的統計，每年因為急性心肌梗塞死亡的人數為九百萬人，而治療方式大多使血管有再灌流的現象產生。心臟的缺血再灌流會造成心肌細胞凋亡和細胞肥大的狀況。外吐小體(exosome)是一種直徑約為30-100 nm的小囊泡，多數的動物細胞都會分泌exosome。近年來有研究指出收集培養間質幹細胞(mesenchymal stem cell, MSC)的條件培養基(conditioned medium, CM)，具有減緩心肌缺血再灌流後所造成的傷害，且CM中的exosome佔有重要的角色。但詳細的機制仍尚未明瞭，因此我們在此論文研究當中探討來自於成人脂肪間質幹細胞(adipose-derived stem cell, ADSC)的條件培養基(ADSC-CM)與外吐小體(ADSC-exosome)對於心肌缺血後再灌流(ischemia/reperfusion, I/R)的保護作用與其相關的機轉。我們使用了藉由結紮心臟左前降支冠狀動脈 (left anterior descending coronary artery, LAD)後鬆綁再灌流做為動物模式(I/R組)。在術後3小時後取心臟組織進行後續觀察。結果顯示， ADSC-CM和ADSC-exosome處理組於WGA螢光染色的實驗中有減少細胞肥大(hypertrophy)的現象。另外在TUNEL assay的結果也可看到有降低細胞凋亡的效果。除此之外細胞肥大相關蛋白ETS1和ANP及細胞凋亡相關蛋白p-p53、PUMA在ADSC-CM和ADSC-exosome處理組皆有表現量下降的情形。其中PUMA和ETS1已被認為是miR221/222的目標基因，在RT-PCR的結果中可見，I/R組別中miR221/222減少而在ADSC-CM和ADSC-exosome處理組中有回升的情形。另外在miR221/222 knockout (KO) mice中，我們同樣觀察到I/R組會有細胞凋亡和細胞肥大的狀況，同樣的，在加入ADSC-CM和ADSC-exosome後有減緩的現象。在細胞模式的部份，我們利用H2O2處理H9c2心肌母細胞來模擬缺血再灌流的氧化壓力傷害。H2O2處理後H9c2心肌母細胞有產生細胞凋亡和細胞肥大的情形，而在ADSC-CM和ADSC-exosome治療組當中有減少的狀況，在RT-PCR部份miR221/222在H2O2處理後同樣有表現量變少的情形且ADSC-CM和ADSC-exosome會有促使其回升的效果，除此之外，ADSC-CM和ADSC-exosome也會降低H2O2所誘導的PUMA和ETS1表現。綜合上述實驗結果。我們認為ADSC-CM和ADSC-exosome能有效減緩心肌缺血後再灌流所造成的傷害且miR221/222參與在其中。

Cardiovascular disorders are the major health problem and the leading cause of death and disability in both industrialized and developing nations. Myocardial ischemia/reperfusion (I/R) leads to cardiomyocyte injury, including apoptosis and hypertrophy. Here we investigated the effects of the derivatives, conditioned media (ADSC-CM) and exosomes (ADSC-exosome), from adipose-derived stem cells on cardiac I/R mice and the underlying mechanisms. ADSC-CM and ADSC-exosome significantly attenuated I/R-induced cardiomyocyte apoptosis and hypertrophy by TUNEL and WGA staining, respectively. Moreover, the expression of the apoptosis related proteins, p-p53 and PUMA, and the hypertrophy related proteins, Ets-1 and ANP, was significantly reduced in cadiomyocytes of I/R mice with ADSC-CM or with ADSC-exosome treatment. Both PUMA and Ets-1 have been reported to be the target genes of miR221/222. I/R operation dramatically decreased miR221/222 expression, which was increased with ADSC-CM or with ADSC-exosome treatment by RT-PCR. We also observed that cardiac I/R operation remarkably increased cell apoptosis and cell hypertrophy in miR221/222 knockout (KO) mice, which was decreased with ADSC-CM or with ADSC-exosome. Furthermore, either ADSC-CM or ADSC-exosome protected H9c2 cardiomyocytes from H2O2-induced damages by decreasing the number of apoptosis and hypertrophy in vitro. H2O2-treated H9c2 cells significantly reduced miR221/222 expression whereas ADSC-CM or ADSC-Exo treatment reversed it. In addition, ADSC-CM or ADSC-exosome treatment decreased H2O2-induced PUMA and Ets-1 expression. Based on these findings, ADSC-CM and ADSC-exosome could serve as an effective therapy for the prevention of I/R-induced cardiac injury and the miR-221/222 are involved in this protection.