血漿外質體在遠端缺血前制約訓練下能減緩肝臟的缺血再灌流傷害

Abstract

前言：缺血再灌流傷害(ischemia-reperfusion injury, IR injury)指的是缺血的器官或組織在恢復血液供給後加劇傷害的現象。近年越來越多數據指出遠端缺血前制約訓練(Remote ischemia precondition, RIPC)能緩解再灌流傷害，但其保護效果的機制仍不明確。近來外質體(Exosome)作為細胞間分子運送的角色與其後續影響細胞的生理功能等研究越趨多樣，因此本研究首先探討經過遠端缺血前制約訓練後血漿中的外質體是否對大鼠肝臟上皮細胞株(Clone 9)產生保護效果，再進一步分析外質體中是否含有某些特殊小分子核醣核酸(microRNA)，進而產生保護細胞免於缺血再灌流傷害的效果。 材料與方法：外質體的收集部分是以七週大的Wistar大鼠做為實驗動物，隨機分為兩組，分別是進行右股動脈的遠端缺血前制約訓練(RIPC-exosome)及僅暴露股動脈而不做任何處理(Sham-exosome)。從大鼠血漿中分離出以上兩組外質體後，以奈米粒徑分析技術(Nanoparticle analysis, NTA)與西方墨點法(Western Blot)確保外質體的純度與濃度，再以攝取實驗(Uptake assay)證實外質體能成功被大鼠肝上皮細胞(Clone 9)攝取至細胞質內，且設計不同長度的培養時間，篩選出最佳條件做為後續預先處理(Pretreatment)參考。細胞的缺血再灌流傷害模式是以剝奪氧氣與葡萄糖的方式(Oxygen-glucose deprivation, OGD)進行。隨後測量結晶紫(Crystal violet)與細胞培養液中乳酸脫氫酶(Lactate Dehydrogenase, LDH)的吸光值來評估加入RIPC-exosome前後細胞的存活與受傷害程度，並以螢光染色、流式細胞儀(Flow cytometry)及西方墨點法檢測細胞凋亡(Apoptosis)相關蛋白。最後利用次世代定序(Next-Generation Sequencing, NGS)與資料庫分析外質體中差異表現的小分子核醣核酸，作為未來實驗的參考方向。 結果：根據外質體樣本的粒徑分析與西方墨點法結果可確保外質體的品質與濃度，加上胞吞實驗的螢光圖可證實Clone 9能成功攝入外質體，並且在共同培養三小時後到達攝入比例的高點。以結晶紫與LDH評估細胞存活率與傷害程度的實驗結果中得知：在O/R傷害後加入RIPC-exosome能提升細胞存活且降低缺血再灌流傷害，並達統計學上的顯著差異。相同的結果也顯示於螢光染色、流式細胞儀檢測及西方墨點法中：與細胞凋亡相關數值如：Annexin V、PI、cytochrome C、cleaved caspase 3、cleaved caspase 9及Apaf-1在加入RIPC-exosome後皆有顯著下降，另外在流式細胞儀四象限圖的細部細胞分群中還可觀察到，RIPC-exosome相較O/R傷害組或O/R+Sham-exosome組更能有效減少Clone 9進入晚期凋亡的比例。最後透過NGS結合線上資料庫分析RIPC-exosome與Sham-exosome中差異表現的miRNA，篩選出miR-3586-3p作為後續研究對象，且經過細胞存活率測試後發現加入miR-3586-3p之抑制劑後會降低RIPC-exosome的保護效果。 結論與未來方向：遠端缺血前制約訓練下血漿中的外質體能減緩大鼠肝臟上皮細胞株的缺血再灌流傷害，而其中外質體攜帶的miR-3586-3p可能在其中扮演重要角色。未來將會利用qPCR證實miR-3586-3p在RIPC-exosome或細胞中的表現高低情形，反向驗證RIPC-exosome中miR-3586-3p是否為對抗缺血再灌流傷害的重要因子。

Background: Ischemia-reperfusion injury(IR injury) is a phenomenon in which the injury is exacerbated after the restoration of blood supply to a temporarily ischemic organ or tissue. It has been shown that remote ischemic preconditioning(RIPC) can mitigate reperfusion injury, but the protective mechanism of RIPC remains unclear. Recently, increasing studies indicated the roles of exosomes as intercellular molecular transport and their subsequent effects on physiological functions. In this study, we first explore the protective effects of plasma exosomes induced by remote ischemic preconditioning on liver cells experiencing IR injury, and then we utilize next generation sequencing(NGS) and online databases to find specific microRNAs in the RIPC-exosomes that are associated with the protective effect on liver cells against ischemia-reperfusion injury. Materials and methods: The plasma exosomes following either remote ischemic preconditioning on right femoral artery(RIPC-exosome) and exposure of right femoral artery alone(Sham-exosome) were collected from seven-week-old Wistar rats. The nanoparticle analysis(NTA) and western blot analysis were used to ensure the purity and concentration of the isolated exosomes. The uptake efficiency of exosomes by Clone 9 cells(a rat liver epithelial cell line) assay was confirmed by the uptake assay. The oxygen-glucose deprivation(OGD) with reoxygenation (O/R) was used to mimic the damage of ischemia-reperfusion injury in vitro. The viability and the degree of cell injury of Clone 9 cells undergoing O/R with or without the addition of RIPC-exosomes were examined by Crystal violet assay and the level of Lactate Dehydrogenase(LDH) in the medium. The fluorescent staining, flow cytometry and western blot were used to detect the apoptosis of the cells. NGS and online databases were used to analyze the microRNAs that were differentially expressed in RIPC-exosome and sham-exosome. Results: The quality and concentration of exosomes were confirmed by NTA and western blot. Then uptake assay confirmed that the hepatocytes could successfully uptake exosomes after three hours of co-culture of them. The crystal violet assay and LDH measurement showed that the addition of RIPC-exosome before O/R significantly increased cell survival and reduced the cell injury. The expression of released cytochrome C, cleaved caspase 3, cleaved caspase 9 and Apaf-1 were significantly mitigated in the cells with the RIPC-exosome added. The fluorescence staining and flow cytometry also showed a significant decrease of apoptotic cells in the RIPC-exosome group compared to the Sham-exosome group following O/R. Finally, among the differentially-expressed miRNAs in RIPC-exosome and Sham-exosome, miR-3586-3p was selected as a target to explore by combining NGS with online databases. Conclusion and Future works: Our study showed that plasma exosomes induced by remote ischemic preconditioning can attenuate ischemia-reperfusion injury in liver cells, and exosome-derived miR-3586-3p may play an important role in this protective effect. In the future, qPCR will be used to confirm the expression of miR-3586-3p in RIPC-exosomes or Clone 9 to investigate the protective mechanisms of RIPC-exosome on ischemia-reperfusion injury.