應用與優化脂肪幹細胞條件培養基之心肌保護效果

Abstract

研究背景與目的 冠狀動脈阻塞導致缺氧的心肌短時間內大量死亡，稱為心肌梗塞 (myocardial infarction)，當前臨床處置主要為經皮冠狀動脈介入術，然而缺血再灌注傷害，致使部分病人預後不佳，可能併發心臟衰竭。 脂肪幹細胞 (adipose-derived stem cell, ASC) 可透過旁分泌生物活性因子，調控梗塞處的心肌發炎反應並進行再生修復，具有縮減缺血再灌注氧化壓力及救援瀕死的心肌細胞之潛能。然而，心肌梗塞病人多半有代謝症候群的共病，自體ASC功能減損，因此需發展強化ASC的方法，並應用其條件培養基 (conditioned media, CM) 於心肌保護。 研究方法與結果 本研究第一部份以心肌細胞建立模擬缺血再灌注 (simulated ischemia reperfusion, SIR) 的療效測試平台，分析收集自代謝症候群疾病模式小鼠Tsumura, Suzuki, obese diabetes (TSOD) 的ASC-CM之心肌保護效果，結果指出ASCTSOD-CM有效回復HL-1心肌細胞的代謝活性 (48% vs 33%, p < 0.05)，卻會加劇心肌細胞總體凋亡 (2.53倍 vs 1.86倍, p < 0.05)。因此，第二部分研究進一步發展強化ASC心肌保護潛能的方法開發，分別以6種化合物合併雙重細胞激素預處理收集多種ASC-CM，稱為優化CM，經由定性與定量分析所含的滋養因子，篩選候選CM，未經預處理的CM則稱為基礎CM (ASCBALB/c-CTL CM)。功能性測試結果指出基礎CM及優化CM均能有效回復損傷心肌細胞之代謝活性 (CTL CM = 63%, BA 750 CM = 59%, SAHA 0.5 CM = 55%, p < 0.05)，減少再灌注時細胞初期和總體凋亡比例 (SIR = 3.2 倍, CTL CM = 1.93 倍, BA 750 CM = 1.85 倍, SAHA 0.5 CM = 2.18 倍, p < 0.05)。進一步我們利用mtSOX螢光染劑標定粒線體的超氧化物，以高通量影像系統所偵測的螢光強度定量超氧化物的生成量，結果顯示特定組別之條件培養基可降低粒線體超氧化物的生成 (SIR = 24393, CTL CM = 23438, SAHA 0.5 CM = 23463, p < 0.05)。 結論 本研究以ASCTSOD-CM療效分析結果推導出具代謝症候群共病之病人自體ASC的治療潛能不佳，因此開發ASC策略，優化CM於模擬缺血再灌注時可有效救援心肌細胞，然而，基礎CM與優化CM療效並無顯著差異。本研究可作為應用及優化ASC條件培養基於開發幹細胞治療產品之參考，而未來應將發展重心放在提高CM的濃度與療效穩定性，並可深入探討預處理策略調控ASC功能的機轉，提升促存活因子之分泌。

Background and Research Aim The blockage of the coronary arteries leads to the massive death of the ischemia myocardium in a short period called myocardial infarction (MI). The dominant treatment, percutaneous coronary intervention, can temporarily treat MI. However, ischemia and reperfusion injury results in a poor prognosis among some patients, which may contribute to complicated heart failure. Adipose-derived stem cells (ASCs) and their paracrine factors simultaneously modulate the inflammatory responses and repair the infarcted area. ASCs may exert their potential when reducing oxidative stress induced by a reperfused blood flow to salvage dying cardiomyocytes. Patients with MI often have comorbidities such as metabolic syndrome or other cardiovascular disorders, which are responsible for insufficient ASCs. Consequently, researchers must devise methods to optimize autologous ASC function and use ASC-conditioned media (CM) for cardioprotection. Methods and Results In the first part of our study, we developed a simulated ischemia-reperfusion (SIR) platform. We analyzed the cardioprotective effect of ASC-CM derived from Tsumura, Suzuki, obese diabetes (TSOD) mice in vitro. We named ASC-CM derived from TSOD mice ASCTSOD-CM. The ASCTSOD-CM could significantly restore the metabolic activity of HL-1 cardiomyocytes (48% vs 33%, p < 0.05), but exacerbated the total apoptosis (2.53 vs 1.86, p < 0.05). Therefore, in the second part of the study, we demonstrated that the ASCBALB/c could protect human umbilical vein endothelial cells from high-intensity oxidative injury. To further enhance the cardioprotective function of ASCs, we primed the ASCs with dual cytokines and six compounds through high throughput screening. The optimized CM was selected after qualitative and quantitative analyses of trophic factors. We called the ASC-CM derived from BALB/c mice without priming ASCBALB/c-CTL CM, abbreviated CTL CM. The CTL CM and priming CM could significantly restore the metabolic activity of injured cardiomyocytes (CTL CM = 63%, BA 750 CM = 59%, SAHA 0.5 CM = 55%, p < 0.05). Moreover, the application of CM attenuated the excessive apoptosis caused by ischemia and reperfusion injury (SIR = 3.2, CTL CM = 1.93, BA 750 CM = 1.85, SAHA 0.5 CM = 2.18, p < 0.05). After labeling the mitochondrial superoxide with mtSOX dye, we detected and quantified the mtSOX fluorescence using a high-content imaging system. The result indicated that the specific groups of CM significantly reduced the generation of mitochondrial superoxide (SIR = 24393, CTL CM = 23438, SAHA 0.5 CM = 23463, p < 0.05). Conclusion ASCs from MI donors complicated with metabolic syndrome have limited utility; thus, we developed an ASC priming strategy. The optimized CM efficiently rescued the damaged cardiomyocytes; however, the priming CM was not superior to the original CMs. Moreover, our findings could be applied to develop novel ASC-based products. Future research should improve the concentration and effectiveness of ASC-CM, and explore the mechanism underlying the effects of pro-survival factors.