背景:间充质干细胞(MSCs)移植是一种新开发的治疗急性肝衰竭(ALF)的策略。尽管如此,MSCs移植后存活率低,对受损组织归巢不良,限制了MSCs的临床应用。该研究评估了缺氧预处理(HPC)是否可以提高人羊膜间充质干细胞(hA-MSCs)的生物学活性。促进它们对ALF小鼠肝脏的归巢能力,影响肝组织修复。
方法:流式细胞术,CCK8,Transwell,和蛋白质印迹试验进行,以评估低氧预处理对表型的影响,扩散,研究了hA-MSCs的迁移以及c-Met和CXCR4基因表达水平的变化。评价缺氧预处理hA-MSCs移植对D-氨基半乳糖(D-GalN)/LPS诱导的ALF归巢和修复的影响。通过添加c-Met阐明了机制,CXCR4特异性阻断剂(SU11274和AMD3100)。
结果:缺氧预处理(1%氧气体积分数)后,hA-MSCs保持粘附和涡旋集落生长的形态学特征,并且显示高CD44、CD90和CD105表达水平和低CD31、CD34和CD45表达水平。hA-MSCs的低氧预处理显著增加其增殖和迁移,并高表达c-Met和CXCR4基因。体内和体外,c-Met特异性阻断剂SU11274抑制了这种促迁移作用.在急性肝衰竭小鼠模型中,HGF在肝脏中的表达水平明显高于血清中的表达水平,肺和肾.缺氧预处理hA-MSCs的移植显著改善了ALF小鼠的肝功能和存活率,增强了肝细胞的抗凋亡能力。c-Met特异性阻断剂SU11274抑制了低氧预处理的抗凋亡增强作用。观察到低氧hA-MSC施用比施用正常氧-hA-MSC后记录的肝脏中的荧光细胞显著增加。添加c-Met抑制剂SU11274后,与低氧预处理后记录的相比,肝荧光细胞数量显着减少,而c-Met抑制剂SU11274对正常氧-hA-MSCs的影响不显著。
结论:低氧预处理对人羊膜间充质干细胞的形态和表型特征无影响,但是它可以促进它们的扩散,迁移,抗凋亡作用,和归巢率,提高急性肝衰竭的修复,这可能是由HGF/c-Met信号轴介导的。
BACKGROUND: Transplantation of mesenchymal stem cells (MSCs) is a newly developed strategy for treating acute liver failure (ALF). Nonetheless, the low survival rate of MSCs after transplantation and their poor homing to damaged tissues limit the clinical application of MSCs. The research assessed whether hypoxic preconditioning (HPC) can improve the biological activity of human amniotic mesenchymal stem cells (hA-MSCs), promote their homing ability to the liver of mice with ALF, and influence liver tissue repair.
METHODS: Flow cytometry, CCK8, Transwell, and Western blotting assays were conducted to assess the effects of hypoxic preconditioning on the phenotype, proliferation, and migration of hA-MSCs and the changes in the c-Met and CXCR4 gene expression levels were studied. To evaluate the effects of the transplantation of hypoxic preconditioning of hA-MSCs on the homing and repair of D-galactosamine (D-GalN)/LPS-induced ALF, the mechanism was elucidated by adding c-Met, CXCR4-specific blockers (SU11274 and AMD3100).
RESULTS: After hypoxia pretreatment (1% oxygen volume fraction), hA-MSCs maintained the morphological characteristics of adherence and vortex colony growth and showed high CD44, CD90, and CD105 and low CD31, CD34, and CD45 expression levels. Hypoxic preconditioning of hA-MSCs significantly increased their proliferation and migration and highly expressed the c-Met and CXCR4 genes. In vivo and in vitro, this migration-promoting effect was suppressed by the c-Met specific blocker SU11274. In the acute liver failure mouse model, the HGF expression level was considerably elevated in the liver than that in the serum, lungs and kidneys. The transplantation of hypoxic preconditioned hA-MSCs introduced a remarkable improvement in the liver function and survival rate of mice with ALF and enhanced the anti-apoptosis ability of liver cells. The anti-apoptotic enhancing effect of hypoxic preconditioning was suppressed by the c-Met specific blocker SU11274. Hypoxic hA-MSCs administration was observed to have considerably increased the fluorescent cells in the liver than that recorded after administering normal oxygen-hA-MSCs. The number of hepatic fluorescent cells decreased remarkably after adding the c-Met inhibitor SU11274, compared to that recorded after hypoxic pretreatment, whereas the effect of c-Met inhibitor SU11274 on normal oxygen-hA-MSCs was not significant.
CONCLUSIONS: Hypoxic preconditioning depicted no impact on the morphology and phenotype features of the human amniotic mesenchymal stem cells, but it can promote their proliferation, migration, anti-apoptotic effect, and homing rate and improve the repair of acute liver failure, which might be mediated by the HGF/c-Met signaling axis.