探討脂肪幹細胞的條件培養液對心肌缺血再灌流 所引發心肌凋亡及纖維化之影響

Abstract

心血管疾病是已開發國家和發展中國家造成死亡的主要原因。心肌缺血/再灌流（ischemia/reperfusion, I / R）會導致心肌細胞損傷，包括細胞凋亡和纖維化。研究指出收集間質幹細胞(mesenchymal stem cell, MSC)的條件培養液(conditioned medium, CM)，可有效減緩I/R後所造成的傷害，脂肪間質幹細胞(adipose-derived stem cell, ADSC)是間質幹細胞其中一種。本研究主旨為探討脂肪間質幹細胞的條件培養液(ADSC-CM)對於心肌缺血再灌流(I/R)的心肌凋亡和纖維化的調節機制。藉由結紮小鼠的心臟冠狀動脈左前降支(left anterior descending coronary artery, LAD)造成心肌缺血30分鐘後再灌流，作為心肌受損的(I/R組)動物模式，將ADSC-CM注射到缺血後的瘢痕和邊界中(I/R+CM組)，並在術後3小時及3天取其心臟組織進行觀察。使用TUNEL染色計數凋亡細胞及運用免疫組織化學染色評估心肌凋亡及心臟纖維化情形。其結果顯示I/R會誘導心肌細胞凋亡及纖維化；而在ADSC-CM處理組顯著改善心肌凋亡和減少心臟纖維化情形。除此之外細胞凋亡相關蛋白PUMA在ADSC-CM處理組皆有表現量下降的情形，其中PUMA已被認為是miR221/222的目標基因，在RT-PCR的結果中可以發現，I/R組別中miR221/222表現量減少而在ADSC-CM處理組中有回升的情形。另外在miR221/222 knockout (KO) mice中，同樣觀察到ADSC-CM處理後會減緩細胞凋亡和細胞纖維化的現象。 在細胞模式方面，我們利用缺氧再灌氧環境誘導H9c2心肌細胞模擬動物缺血再灌流的傷害。缺氧再灌氧處理後H9c2心肌母細胞的確產生細胞凋亡和細胞纖維化的情形，而在ADSC-CM治療組當中與細胞凋亡及纖維化相關的蛋白表現有減少的狀況，我們認為ADSC-CM可減緩心肌細胞凋亡及纖維化的情形並保護心肌缺血後再灌流所造成的傷害且miR221/222參與在其中。

Cardiovascular disorders is the leading cause of death in both developed and developing countries. Myocardial ischemia/reperfusion (I/R) leads cardiomyocyte injury, including apoptosis and fibrosis. The present study was aimed at determining the effect and regulatory mechanism of conditioned media from adipose-derived stem cells (ADSC-CM) on cardiac apoptosis and fibrosis. The mouse myocardial I/R model was established to induce by ligating the left anterior descending coronary artery for 30 min and then reperfusion for 3 h or for 3 days (I/R group). ADSC-CM treatment significantly reduced I/R-induced cardiomyocyte apoptosis by TUNEL staining. Moreover, the expression of the apoptosis related proteins, p53 upregulated modulator of apoptosis (PUMA), and the fibrosis-related proteins, fibronectin and collagen III, was significantly reduced in cardiomyocytes of I/R mice with ADSC-CM treatment. 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 treatment by RT-PCR. We also observed that cardiac I/R operation remarkably increased cell apoptosis and cell fibrosis in miR221/222 knockout (KO) mice, which was decreased with ADSC-CM. We next established the in vitro cell model with H9c2 cells under hypoxia /reoxygenation (H/R) treatment. ADSC-CM increased cell viability of H/R-treated H9c2 cells by MTT assay. Furthermore, ADSC-CM decreased cell apoptosis by TUNEL assay. In addition, ADSC-CM treatment decreased H/R-induced PUMA and Ets-1 expression by Western blot assay. ADSC-CM decreased fibrosis-related proteins, collagen III and fibronectin expression by Western blot assay. Based on these above findings, ADSC-CM could protect myocardial I/R-induced injury against apoptosis and fibrosis.