外質體在遠端缺血前制約訓練對肝臟缺血性傷害的保護效應與機制

Abstract

前言：最近發現外質體的囊泡運輸為細胞間物質與訊息運輸的重要方式。本研究為了解遠端缺血-前制約訓練(Remote ischemia precondition, RIPC)是否透過Exosome增進對於缺血再灌流傷害(ischemia-reperfusion, I/R)傷害的保護，以細胞(in vitro)與動物(in vivo)的傷害模型，探討RIPC外質體對I/R injury的保護效果與機制。 材料與方法：以雄性大鼠(Wistar rat) 經由RIPC訓練後，以梯度離心收集其血中的Exosome 。本研究使用小鼠(C57BL6 mice)的部分肝臟缺血作為I/R injury的指標缺血模型。將RIPC Exosome或磷酸鹽緩衝生理食鹽水(PBS)經由脾臟注射後流至肝臟，確認RIPC Exosome是否可以減少部分肝臟缺血再灌流傷害。細胞學實驗使用大鼠肝細胞(Clone 9 cell)，剝奪其氧及葡萄糖供應(oxygen-glucose deprivation,OGD)作為缺血再灌流傷害的模擬，並給PBS或是RIPC Exosome來確認RIPC Exosome組別是否有保護效果。 結果：注射RIPC-Exosome的肝臟缺血再灌流傷害小鼠比注射PBS的小鼠丙酮酸轉胺酶(GPT)更低，並且有顯著差異(P<0.05)，而sham組GPT則遠比這兩組都來得低，有更顯著的差異(P<0.01)。給予RIPC Exosome的OGD Clone9肝細胞株組別，比給予PBS的組別存活率(MTT assay)更高(P<0.05)，而Sham組存活率為最高，並與其他兩組有顯著差異(P<0.05)。在LDH傷害測試下，RIPC-Exosome組別比PBS組別顯示的傷害更低(P<0.05)，而Sham組顯示的LDH數值最低，且與其他兩組有顯著差異(P<0.05)。RIPC-Exosome與Sham-Exosome經過次世代定序(Next-Generation Sequencing, NGS)分析，有24組基因序列有顯著差異(P<0.05)，放入KEGG與GO基因庫搜尋預測路徑，則預測有metabolism, mTOR等路徑會與該24組基因的差異有相關性。 結論：本研究顯示了RIPC Exosome對於細胞學與動物的缺血再灌流傷害具有一定的保護效果，在肝功能指數與細胞死亡數值上與控制組有顯著差異(P<0.05)。而NGS也指出RIPC-Exosome與Sham-Exosome之間有24個基因出現顯著差異，並且預測與metabolism, mTOR等路徑有關。

Background: Exosome vesicle transport has recently been discovered as an important way of transporting materials and messages between cells. This study is to understand whether remote ischemia precondition enhances the protection against ischemia-reperfusion (I/R) injury through the exosomes. Both in vitro and in vivo injury models were used to explore the protective effect and mechanism of RIPC-induced exosomes (RIPC-exosomes) on I/R injury. Materials and methods: The exosomes from the peripheral blood of male Wistar rats following RIPC were collected by gradient centrifugation. Partial liver I/R was established on C57BL6 mice as an index injury model. RIPC-exosome or phosphate buffered saline (PBS) was injected into the liver through the spleen to examine their protective effects on the liver I/R injury. Oxygen-glucose deprivation (OGD) was conducted on the Clone 9 cells (a rat liver cell line) to mimic I/R injury. RIPC-exosome group or PBS were added explore their protective effects on the clone 9 cells after OGD. Next-generation sequencing was used to detect the differential expressions of microRNAs in the exosomes with/with RIPC. Results: The serum level of pyruvate transaminase (GPT) was significantly lower in the RIPC-exosome group than that in the PBS group (641±32.462) (P<0.05) following partial liver I/R. The survival rate of Clone 9 cells following OGD, measured by MTT assay, was significantly higher in the RIPC-Exosomes group (0.604±0.02)than in the PBS group (o.449±0.01) (P<0.05). Under the Lactate dehydrogenase(LDH) test, RIPC-exosome group showed lower damage than the PBS group(P<0.05), the Sham group showed the lowest LDH level than the other two groups (P<0.05). NGS analysis showed 24 sets of gene sequences differentially expressed in RIPC-exosomes (P<0.05). The gene banks (KEGG and GO) search indicated that these genes were associated the metabolisms or mTOR pathways. Conclusion: This study shows that RIPC-exosome could mitigate the I/R-injuries. Next-Generation Sequencing (NGS) also find out that 24 differentially-expressed genes in RIPC-exosomes were related to the metabolism and mTOR pathways.