肝孤核受体激动剂对间充质干细胞的抗缺氧复氧损伤作用及机制

目的探讨肝孤核受体(LXR)激动剂对脂肪间充质干细胞(AD-MSCs)缺氧损伤的保护作用及可能机制。方法酶消化法分离稳定表达萤火虫荧光素酶(Fluc)报告基因的小鼠AD-MSCs,流式细胞术检测CD90、CD44、CD34、CD45细胞表面标记物。3代AD-MSCs分为7组:对照组;缺氧6 h/复氧2 h组(缺氧复氧组);缺氧/复氧+DMSO组(DMSO组);缺氧复氧+不同浓度LXR激动剂T0901317干预组(1μmol/L组、5μmol/L组、10μmol/L组、15μmol/L组)。运用Fluc报告基因生物发光成像技术对AD-MSCs细胞增殖进行定量评价,免疫印迹法检测细胞NF-κB表达水平,ELISA法检测AD-MSCs的白细胞介素6(IL-6)、TNF-α分泌水平。结果流式细胞术结果显示.AD-MSCs呈CD44(+)、CD90(+)、CD34(-)、CD45(-)。光学成像结果显示,AD-MSCs细胞数与Fluc平均生物发光信号强度呈正相关(r~2=0.97)。与对照组比较,缺氧复氧组AD-MSCs分泌TNF-α、IL-6、NF-κB明显升高;与缺氧复氧组比较,10μmol/L组AD-MSCs分泌TNF-α、IL-6、NF-κB明显减少(P<0.05,P<0.01)。结论 LXR激动剂可以促进缺氧复氧损伤后AD-MSCs的存活,并能通过NF-κB信号途径抑制炎性因子IL-6、TNF-α的释放,可为干细胞移植治疗心肌缺血再灌注损伤提供新的细胞保护方法。

Liver X receptor agonist prevents adipose-derived mesenchymal stem cells from hypoxia reoxygenation injury

Objective To investigate the effects and possible mechanisms of liver X receptor(LXR) agonist on adipose-derived mesenchymal stem cells(AD-MSCs) after hypoxia reoxygenation (H/R) injury.Methods AD-MSCs stably expressing firefly luciferase(Flue) were isolated fromβ-actin-Fluc transgenic mice and characterized by flow cytometry.Third passage AD-MSCs were divided into seven groups:control;hypoxia 6 hrs/reoxygen 2 hrs;H/R + DMSO;H/R+ LXR agonist T0901317(1μmol/L,5μmol/L,10μmol/L,15μmol/L,respectively).Cell survival conditions were assessed by bioluminesence imaging(BLI) via IVIS system.The protein expression of NF-κB was analyzed by Western blot.The secretion of TNF-αand IL-6 was tested by ELISA in each group.Results The AD-MSCs were both CD44 and CD90 assayed by flow cytometry.BLI proved that the firefly luciferase expression of AD-MSCs positively correlated with cell number (r~2=0.97),and the pretreatment with LXR agonist could improve the survival of AD-MSCs from H/R injury(P<0.05).The levels of NF-κB.TNF-αand IL-6 in H/R group were much higher than those in control group(P<0.01).The levels of NF-κB,TNF-αand IL-6 in 10μmol/L LXR agonist group were lower than those in H/R group(P<0.05,P<0.01).Conclusion LXR agonist has protective effects on the survival of AD-MSCs under H/R environment by suppressing NF-κB signaling.