脂肪间充质干细胞外泌体对大鼠缺血/再灌注损伤心肌的作用及Wnt3a/β-catenin信号通路的影响

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摘要目的研究脂肪源性间充质干细胞外泌体（ADMSC-exs）对大鼠缺血/再灌注（I/R）损伤心肌的保护作用及其对Wnt3a/β-catenin信号通路的影响。方法 40只成年大鼠随机分为四组：S组（假手术组），只开胸不结扎冠状动脉左前降支（LAD）；I/R组，结扎LAD 30 min后再灌注；Ex组，结扎LAD 30 min后再灌注，再灌注5 min内尾静脉注射ADMSC-exs；Ex+X组，结扎LAD 30 min后再灌注，再灌注5 min内从尾静脉注射ADMSC-exs和XAV939。每组10只。检测肌酸激酶同工酶（CK-MB）、乳酸脱氢酶（LDH）和心肌肌钙蛋白I（cTnI）的水平，TUNEL法检测各组心肌缺血区心肌细胞凋亡指数（AI），蛋白免疫印迹法检测B淋巴细胞瘤-2基因（Bcl-2）、Bcl-2相关X蛋白（Bax）、caspase-3、Wnt3a和β-catenin。结果 I/R组LDH、CK-MB、cTnI水平高于S组，差异有统计学意义（P ＜ 0.05）。Ex组心肌酶水平低于I/R组，差异有统计学意义（P ＜ 0.05）。当尾静脉注射Wnt3a/β-catenin通路抑制剂XAV939后，Ex+X组心肌酶水平高于Ex组，差异有统计学意义（P ＜ 0.05）；与I/R组比较，差异无统计学意义（P ＞ 0.05）。S组AI低于I/R组，差异有统计学意义（P ＜ 0.05）。给予ADMSC-exs治疗后，Ex组AI低于I/R组，差异有统计学意义（P ＜ 0.05）。I/R组Bcl2/Bax低于S组，差异有统计学意义（P ＜ 0.05）。Ex组Bcl2/Bax高于I/R组，差异有统计学意义（P ＜ 0.05）。Ex组caspase-3水平低于I/R组，差异有统计学意义（P ＜ 0.05）。Ex+X组caspase-3水平明显高于Ex组，差异有统计学意义（P ＜ 0.05）。I/R组Bcl2/Bax、Wnt3a、β-catenin水平低于S组，caspase-3水平高于S组，差异有统计学意义（P ＜ 0.05）。Ex组Bcl2/Bax、Wnt3a、β-catenin水平高于I/R组，caspase-3水平低于S组，差异有统计学意义（P ＜ 0.05）。Ex+X组Bcl2/Bax、Wnt3a、β-catenin水平低于S组，caspase-3水平高于Ex组，差异有统计学意义（P ＜ 0.05）。结论 ADMSC-exs可通过上调Bcl-2和下调Bax、caspase-3的表达保护I/R后的心肌细胞，其作用机制可能与Wnt3a/β-catenin信号通路有关。

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	陈晓佳郭子傲郭金华李林科张剑凯杨春崔晓军

关键词 ：间充质干细胞, 外泌体, 缺血/再灌注, Wnt3a/&beta, -catenin信号通路, 凋亡

Abstract：Objective To study the protective effect of adipose-derived mesenchymal stem cell exosomes (ADMSC-exs) on myocardial ischemia / reperfusion (I/R) injury in rats and the influence on the Wnt3a/β-catenin signaling pathway. Methods A total of 40 adult rats were randomly divided into four groups: S group (sham operation group), only thoracotomy without ligation of the coronary artery left anterior descending (LAD); I/R group, reperfusion after ligation of LAD 30 min; in the Ex group, LAD was ligated for 30 minutes and then reperfused, and ADMSC-exs was injected into the tail vein within five minutes of reperfusion; in the Ex+X group, LAD was ligated for 30 minutes and then reperfused, and ADMSC-exs and XAV939 were injected from the tail vein within five minutes of reperfusion. Ten rats in each group. The levels of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) and cardiac troponin I (cTnI), and myocardial cell apoptosis index (AI) in each group of myocardial ischemic area were detected by TUNEL method; Western blot was used to detect B lymphoma-2 gene (Bcl-2), Bcl-2 related X protein (Bax), caspase-3, Wnt3a, and β-catenin. Results The levels of LDH, CK-MB and cTnI in the I/R group were higher than those in the S group, and the differences were statistically significant (P ＜ 0.05). The levels of myocardial enzymes in the Ex group was lower than those in the I/R group, and the differences were statistically significant (P ＜ 0.05). When the Wnt3a/β-catenin pathway inhibitor XAV939 was injected into the tail vein, the levels of myocardial enzymes in the Ex+X group were higher than those in the Ex group, and the differences were statistically significant (P ＜ 0.05); compared with the I/R group, there was no statistical difference between two groups (P ＞ 0.05). The AI of S group was lower than that of I/R group, and the difference was statistically significant (P ＜ 0.05). After ADMSC-exs treatment, AI in Ex group was lower than that in I/R group, and the difference was statistically significant (P ＜ 0.05). The Bcl2/Bax ratio of I/R group lower higher than that of S group, and the difference was statistically significant (P ＜ 0.05). The Bcl2/Bax ratio of Ex group was higher than that of I/R group, and the difference was statistically significant (P ＜ 0.05). The level of caspase-3 in Ex group was lower than that in I/R group, and the difference was statistically significant (P ＜ 0.05). The level of caspase-3 in Ex+X group was significantly higher than that in Ex group, and the difference was statistically significant (P ＜ 0.05). The levels of Bcl2/Bax, Wnt3a, and β-catenin in the I/R group were lower than those in the S group, while the caspase-3 level was higher than that in the S group, and the differences were statistically significant (P ＜ 0.05). The levels of Bcl2/Bax, Wnt3a and β-catenin in the Ex group were higher than those in the I/R group, while the level of caspase-3 was lower than that in the S group, and the differences were statistically significant (P ＜ 0.05). The levels of Bcl2/Bax, Wnt3a, and β-catenin in the Ex+X group were lower than those in the S group, while the caspase-3 level was higher than that in the Ex group, and the differences were statistically significant (P ＜ 0.05). Conclusion ADMSC-exs can protect cardiomyocytes after I/R by up-regulating the expression of Bcl-2 and down-regulating the expression of Bax and caspase-3. Its mechanism may be related to the Wnt3a/β-catenin signaling pathway.

Key words： Mesenchymal stem cell Exosomes Ischemia/reperfusion Wnt3a/β-catenin signaling pathway Apoptosis

基金资助:广东省自然科学基金面上项目（2019A15150116 26）；
广东医科大学自然科学类重点培育项目（GDMUZ2019007）；
广东医科大学自然科学类面上项目（M2016030）。

通讯作者: 崔晓军（1975.5-），男，博士，教授，硕士生导师；研究方向：干细胞与再生医学。

作者简介: 陈晓佳（1989.5-），男，硕士；研究方向：干细胞与再生医学、脂代谢。

引用本文:

陈晓佳郭子傲郭金华李林科张剑凯杨春崔晓军. 脂肪间充质干细胞外泌体对大鼠缺血/再灌注损伤心肌的作用及Wnt3a/β-catenin信号通路的影响[J]. 中国医药导报, 2021, 18(35): 28-31,45.
CHEN Xiaojia GUO Ziao GUO Jinhua LI Linke ZHANG Jiankai YANG Chun CUI Xiaojun. Effect of adipose-derived mesenchymal stem cell exosomes on myocardial ischemia / reperfusion injury in rats and the effect of Wnt3a/β-catenin signaling pathway#br#. 中国医药导报, 2021, 18(35): 28-31,45.

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