- 學位論文
S100A8/A9預處理於人類羊膜間葉幹細胞條件培養液對心肌梗塞修復潛能之探討
Repairing myocardial infarction with S100A8/A9 pretreatment in conditioned medium from human amniotic membrane mesenchymal stem cells
摘要
心血管疾病是造成全球人口死亡之主要疾病之一，其中又以心肌梗塞占多數，在冠狀動脈阻塞後，心臟組織缺氧導致心肌細胞及心臟成纖維細胞受損，進而引發炎症及心臟重塑，將使心臟增加10%至35%之纖維組織，可能導致心臟功能受損、心律不整及收縮障礙，甚至心臟衰竭，因此探討心肌梗塞（myocardial infarction, MI）後如何維持心臟功能是刻不容緩的重要議題。 近年來許多研究開始關注細胞受損後所釋放之危險相關分子模型（danger associated molecular patterns, DAMPs）之訊息傳遞與調控，DAMPs包含HMGB1、S100A1/A8/A9及IL-1α等，具有活化常駐型巨噬細胞及增加內皮細胞通透性功能，其可招募嗜中性球及單核球使發炎反應加劇，活化成纖維細胞使其增生並累積細胞外基質。前人研究發現以S100A8/A9蛋白質預處理之人類間葉幹細胞（human mesenchymal stem cell, hMSC）可加速傷口癒合、降低疤痕組織，並對心臟具有保護作用。因此，本研究旨在探討經S100A8/A9 預處理的 hAMSCs條件培養液是否於 MI 後發揮心臟保護作用並維持心臟功能。 本研究分為體內試驗及體外試驗兩部分，第一階段，體外試驗將分別分離胎鼠心肌細胞（neonatal cardiomyocytes, nCMs）及人類羊膜間葉幹細胞（human amniotic mesenchymal stem cells, hAMSCs）進行初代培養與細胞特性分析作為本試驗細胞來源，再利用nCMs特異性磁株抗體純化後誘導其缺氧/復氧損傷（hypoxia/ reoxygenation, H/R），並以RT-PCR、qPCR進行檢測，與對照組相比，誘導H/R損傷之nCM處理組之S100蛋白、IL-1、IL-6、TNF-α和IFNγ等細胞受損、促纖維化增生及促炎症相關基因表達具有顯著差異。而人類羊膜間葉幹細胞則以含有額外添加S100A8/A9之培養液與hAMSCs培養，後以RT-PCR、qPCR確認細胞是否產生相應之基因表現，結果顯示以S100A8/A9預處理之hAMSC則於免疫調節和組織修復相關基因，包含TLR、CCR7、IL-10、GDF9、SPARCKL、MMP、TIMP等與對照組相比亦有顯著變化。此外，此研究通過在缺氧條件下更換受損 nCM 之培養液檢測與控制組相比經預處理之hAMSC條件培養液是否具有保護作用，結果顯示hAMSC來源之條件培養液組之細胞存活率顯著高於基礎培養基組，然而，未預處理之hAMSC組與S100A8/A9預處理之hAMSC組之間沒有顯著差異，除此之外，各組在某些基因表達水平之上調和下調結果則顯示hAMSC於預處理後會產生適應性反應，並調控相關之分子機制。 第二階段體內試驗，使用8週齡C57BL/6小鼠心肌梗塞/再灌流模型，以靜脈注射經S100A8/A9預處理後之hAMSC條件培養液進行治療。試驗將分為五組，分別為控制組、假手術組、伪治療組（基礎培養液）、一般治療組（hMSC條件培養液）及預處理治療組（經S100A8/A9預處理後之hMSC條件培養液），於治療後3天、1週、2週及4週以血清學檢查、心臟超音波、組織切片染色檢測心臟結構、功能與血管密度之變化，於心臟超音波結果顯示，預處理之hAMSC組之左心室射血分數和收縮分率顯著高於未預處理hAMSCs組和I/R組（P<0.05），纖維化區域之組織切片染色定量分析，亦顯示各組間纖維化具有顯著差異，並且預處理之hAMSC組雖與一般治療組並無統計上之顯著差異，然其纖維組織之定量結果仍為各處理中最少之組別。 總結，我們證實 S100A8/A9 預處理的 hAMSCs 條件培養基在體外研究和小鼠心肌 I/R 模型的治療中具有增強之修復潛力，期望此研究結果有益於受損後心臟功能之維持，為未來的心肌梗塞相關研究提供良好的治療方向與展望並貢獻於臨床治療。
並列摘要
Myocardial infarction results in tissue necrosis caused by ischemia and reoxygenation injury, which in turn to the inflammation and cardiac remodeling. Hearts’ fibrous tissue might increase and lead to irreversible cardiac functional loss, rhythm irregularities, systolic disorders, and heart failure. The transmission and regulation message of S100A8/A9 is released from injured heart tissue, which play an essential role on the intensify of inflammation, permeability and recruitment of neutrophils and monocytes, fibroblast proliferation, and accumulation of extracellular matrix through interacting with cognate pattern recognition receptors. It's urgent to explore the therapeutic options to maintain heart function and the resolution of scar formation post-MI. Based on recent study, human mesenchymal stem cells pretreated with S100A8/A9 protein can accelerate wound healing and reduce scar tissue in murine skin wound model, which represents that hMSCs can sense S100A8/A9 and perform adaptive responses such as proteolysis, macrophage phagocytosis and regulation of inflammation-related genes. However, the comprehensive hMSCs paracrine effcet of this signaling pathway hasn’t been clarified. Accordingly, we investigate that S100A8/A9 pretreated hAMSCs conditioned medium have the ability to exert the significant cardioprotective effects and preserve cardiac function after MI, which could also be used in place the cell-based therapy in clinical applications and circumvent complications. This research is divided into in vitro study and in vivo experiment. First, the mouse neonatal cardiomyocytes and hAMSCs were isolated as the experimental cell sources after purification and identification. The comparison of gene expressions related to immune regulation have significant difference between control group and hypoxia/reoxygenation treated nCMs group, including S100, IL-1, IL-6, TNF-α, and IFNγ. The immunomodulation and tissue restoration relevant genes of S100A8/A9 pretreated hAMSCs also have significant change compare to the control group such as TLR, CCR7, IL-10, GDF9, SPARCKL, MMP, and TIMP. Further, the protective effect of control and pretreated hAMSCs condition medium were tested through replacing the culture medium of injured nCMs after hypoxic condition. The cell viability of hAMSCs derived condition medium groups are significant higher than basal medium group. However, there has no significant difference between non-pretreated hAMSCs group and S100A8/A9 pretreated hAMSCs group. The up-regulation and down-regulation of several gene expression levels also represent the significant change between each group. In vivo experimental part, the echocardiography results suggest that the LV ejection fraction and fraction shortening of the pretreated hAMSCs group significantly higher than the non-pretreated hAMSCs group and I/R group （P<0.05）. The histochemistry staining also shown the significant difference of fibrotic formation between each group after quantitative analysis. In conclusion, these results confirm that the enhanced repairing potential of S100A8/A9 pretreated hAMSCs conditioned medium in the in vitro study and in the treatment of murine myocardial I/R model. These investigations will contribute to the myocardial research and therapy in the future.
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