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

doi:10.3877/cma.j.issn.2095-3232.2020.04.018

中华肝脏外科手术学电子杂志 ›› 2020, Vol. 09 ›› Issue (04) : 374 -379. doi: 10.3877/cma.j.issn.2095-3232.2020.04.018

所属专题：文献；

基础研究

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

刘荣强¹, 林国桢², 代天星², 邓铭彬², 周朝荣³, 汪国营²^,^†(

)

^1. 510630　广州，中山大学附属第三医院肝脏外科肝移植中心；510030　广州医科大学附属第一医院肝胆外科
^2. 510630　广州，中山大学附属第三医院肝脏外科肝移植中心
^3. 510220　广州医科大学附属第二医院普通外科

收稿日期:2020-04-18 出版日期:2020-08-10
通信作者: 汪国营
基金资助:
国家自然科学基金(81470870，81670601，81770648); 十三五科技重大专项(2017ZX10203205-006-001); 广东省自然科学基金(2016A030313278，2015A030313038，2015A030312013); 广东省科技计划项目(2014A020211015，2017B020209004); 广州市科技计划项目(2014J4100183)

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

Rongqiang Liu¹, Guozhen Lin², Tianxing Dai², Mingbin Deng², Chaorong Zhou³, Guoying Wang²^,^†()

^1. Department of Liver Surgery, Liver Transplant Center, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China; Department of Hepatobiliary Surgery, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510030, China
^2. Department of Liver Surgery, Liver Transplant Center, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China
^3. Department of General Surgery, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510220, China

Received:2020-04-18 Published:2020-08-10
Corresponding author: Guoying Wang
About author:
Corresponding author: Wang Guoying, Email: wanggy3@126.com

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引用本文：

刘荣强, 林国桢, 代天星, 邓铭彬, 周朝荣, 汪国营. 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[J/OL]. 中华肝脏外科手术学电子杂志, 2020, 09(04): 374-379.

Rongqiang Liu, Guozhen Lin, Tianxing Dai, Mingbin Deng, Chaorong Zhou, Guoying Wang. Protection of Nrf2-overexpressing human umbilical cord mesenchymal stem cells on hepatic ischemia-reperfusion injury in mice[J/OL]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2020, 09(04): 374-379.

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1×10⁶ 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。

关键词: 间充质干细胞, 核因子E2相关因子2（Nrf2）, 缺血-再灌注损伤, 肝损伤, 细胞凋亡, 小鼠

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, 1×10⁶ GFP-HUCMSCs, and 100 μl 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 hof reperfusion, the ALT and AST levels were respectively (1 472±540), (1 592±596) U/L in the IRI+Nrf2-HUCMSCs group, which were significantly lower than (4 700±1 526), (5 464±1 210) U/L in the IRI+GFP-HUCMSCs group, and (7 452±1 637), (6 928±2 439) 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.

Key words: Mesenchymal stem cells, Nuclear factor E2-related factor 2 (Nrf2), Ischemia-reperfusion injury, Liver injury, Apotosis, Mouse

图1 HUCMSCs成脂和成骨染色

图2 Nrf2-HUCMSCs成脂和成骨染色

图3 四组小鼠ALT、AST水平

图4 四组小鼠肝组织损伤情况

图5 四组小鼠肝细胞凋亡情况

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