过表达Nrf2人脐带间充质干细胞对小鼠肝脏缺血-再灌注损伤的保护作用

Protection of Nrf2-overexpressing human umbilical cord mesenchymal stem cells on hepatic ischemiareperfusion injury in mice

目的探讨过表达Nrf2的人脐带间充质干细胞(HUCMSCs)对小鼠缺血-再灌注损伤(IRI)的保护作用。方法制作70%肝脏IRI模型,HUCMSCs细胞分离培养、鉴定,构建过表达Nrf2慢病毒。正常SPF级健康雄性C57小鼠40只,按随机数字表法随机分为4组,分别为Sham组、IRI组、IRI+GFP-HUCMSCs、IRI+Nrf2-HUCMSCs组,缺血90 min后IRI+GFP-HUCMSCs、IRI+Nrf2-HUCMSCs组分别应用1×10^6 GFP-HUCMSCs、Nrf2-HUCMSCs 100 μl细胞悬液从小鼠下腔静脉注射,再灌注12、24 h后处死小鼠,下腔静脉取血检测ALT、AST;取肝脏组织行HE和TUNEL染色观察肝损伤和细胞凋亡情况。各组小鼠转氨酶比较采用单因素分析和LSD-t检验。结果再灌注12 h,IRI+Nrf2-HUCMSCs组肝细胞损伤较IRI、GFP-HUCMSCs组明显减轻;再灌注24 h,IRI+Nrf2-HUCMSCs组炎症细胞浸润退去,恢复也较快。再灌注12 h,IRI+Nrf2-HUCMSCs组肝功能ALT、AST分别为(1 472±540)、(1 592±596)U/L,明显低于IRI+GFP-HUCMSCs组的(4 700±1 526)、(5 464±1 210)U/L和IRI组的(7 452±1 637)、(6 928±2 439)U/L(LSD-t=-4.460,-7.757和-6.420,-4.753;P<0.05)。再灌注12 h,IRI+Nrf2-HUCMSCs组肝细胞凋亡率为(40±6)%,明显低于IRI+GFP-HUCMSCs组的(79±6)%和IRI组的(103±6)%(LSD-t=-8.162,-13.040;P<0.05)。结论过表达Nrf2的HUCMSCs对正常小鼠肝脏IRI可能是通过减少肝细胞凋亡发挥一定的保护作用。

Objective To explore the protection of Nrf2-overexpressing human umbilical cord mesenchymal stem cells(HUCMSCs)on hepatic ischemia-reperfusion injury(IRI)in mice.Methods A model of partial(70%)hepatic IRI was constructed.HUCMSCs were isolated,cultured and identified,and Nrf2-overexpressing lentiviral was constructed.40 healthy male C57 mice of SPF grade were randomly divided into 4 groups according to the random number table:Sham group,IRI group,IRI+GFP-HUCMSCs group,and IRI+Nrf2-HUCMSCs group.For IRI+GFP-HUCMSCs and IRI+Nrf2-HUCMSCs group,1×10^6 GFP-HUCMSCs,and 100μl of Nrf2-HUCMSCs cell suspension were injected in the inferior vena cava of mice respectively after 90 min of ischemia.The mice were executed after 12 or 24 h of reperfusion,and then blood was taken from the inferior vena cava for ALT and AST test.Liver injury and cell apoptosis were observed through HE and TUNEL staining of liver tissue.Transaminases levels of each group were compared using univariate analysis and LSD-t test.Results After 12 h of reperfusion,hepatocyte damage was significantly milder in the IRI+Nrf2-HUCMSCs group than that in the IRI and GFP-HUCMSCs groups.After 24 h of reperfusion,inflammatory cell infiltration receded and recovered more quickly in the IRI+Nrf2-HUCMSCs group.After 12 h of reperfusion,the ALT and AST levels were respectively(1472±540),(1592±596)U/L in the IRI+Nrf2-HUCMSCs group,which were significantly lower than(4700±1526),(5464±1210)U/L in the IRI+GFPHUCMSCs group,and(7452±1637),(6928±2439)U/L in the IRI group(LSD-t=-4.460,-7.757 and-6.420,-4.753;P<0.05).After 12 h of reperfusion,the rate of hepatocyte apoptosis was(40±6)%in the IRI+Nrf2-HUCMSCs group,significantly lower than(79±6)%in the IRI+GFP-HUCMSCs group and(103±6)%in the IRI group(LSD-t=-8.162,-13.040;P<0.05).Conclusions HUCMSCs overexpressing Nrf2 may exert certain protective effects on hepatic IRI by reducing the hepatocyte apoptosis in healthy mice.