慢病毒介导Wnt蛋白5a基因过表达对骨髓间充质干细胞移植治疗心肌梗死大鼠的影响  

Effects of transplantion of bone marrow mesenchymal stem cells with lentiviral vector-mediated overexpressed Wnt signaling pathway proteins 5a on myocardial infarction in rats

作　　者：王秀力 姜世强 刘颖[3] 李睿[3] 吕茜 彭珍[3] 陈东 Wang Xiuli;Jiang Shiqiang;Liu Ying;Li Rui;Lyu Qian;Peng Zhen;Chen Dong(Xuzhou Institute of Cardiovascular Disease,Xuzhou 221009,China;Department of Anesthesiology,Xuzhou Mercy Hospital,Xuzhou 221004,China;Central Laboratory of Xuzhou Central Hospital,Xuzhou 221009,China;Department of Histology and Embryology,Basic Medical College,Guangdong Medical University,Dongguan 523808,China)

机构地区：[1]徐州市心血管病研究所,221009 [2]徐州仁慈医院麻醉科,221004 [3]徐州市中心医院中心实验室,221009 [4]广东医科大学基础医学院组织学与胚胎学教研室,东莞523808

出　　处：《中华实验外科杂志》2021年第9期1720-1723,共4页Chinese Journal of Experimental Surgery

基　　金：徐州市科技发展项目(KC18029)。

摘　　要：目的探讨Wnt信号通路蛋白5a(Wnt5a)基因过表达对骨髓间充质干细胞(BMSC)移植治疗心肌梗死的影响及分子机制。方法结扎雄性SD大鼠冠脉前降支制备心肌梗死动物模型,按数字表法分为实验组(40只)和假手术组(20只),4周后超声检测大鼠血流动力学指标变化;分离大鼠BMSC并在体外培养纯化,建立Wnt5a稳定沉默(sh-Wnt5a)和过表达的(oe-Wnt5a)BMSC细胞株,采用划痕实验检测Wnt5a沉默和过表达对细胞迁移能力的影响;采用酶联免疫吸附实验(ELISA)技术检测心肌梗死大鼠模型组血清中胰岛素样生长因子1(IGF-1)、血管内皮生长因子(VEGF)、干细胞因子(SCF)和肝细胞生长因子(HGF)的含量;BMSC移植治疗心肌梗死大鼠,实验共分4组:心肌梗死对照组,生理盐水组、BMSC组和oe-Wnt5a组。移植治疗4周后,超声检测血流动力学变化后取材大鼠心肌,冰冻切片、苏木精-伊红(HE)染色后检测大鼠心肌梗死的面积;采用蛋白质印迹法(Western blot)技术检测移植后大鼠心肌组织中Wnt5a、桩蛋白(Paxillin)、干细胞因子受体(C-KIT)、Ca2+依赖的蛋白激酶Ⅱ(CaMKⅡ)和钙敏感受体(CaSR)蛋白的表达,两组和多组间比较分别采用t检验和单因素方差分析。结果 (1)成功建立Wnt5a稳定沉默和过表达的BMSC细胞模型。sh-Wnt5a组细胞迁移率显著低于对照组(6.67±1.02比18.71±3.02,t=6.542,P<0.01),oe-Wnt5a组细胞迁移率显著高于对照组(43.33±7.95比18.71±3.02,t=5.014,P<0.01)。(2)ELISA结果显示,BMSC组及oe-Wnt5a组的血清中IGF-1、VEGF、SCF和HGF的浓度明显高于心肌梗死组[(5 473.20±474.55)、(1 237.85±173.90)、(548.09±77.06)、(136.14±37.41) pg/ml比(3 799.20±384.99)、(652.32±95.34)、(352.78±65.89)、(62.92±12.93) pg/ml,t=4.745、5.114、3.337、3.204,P<0.05];oe-Wnt5a组的血清中IGF-1、VEGF、SCF和HGF的浓度明显高于BMSC组[(6 264.31±498.87)、(1 445.75±179.58)、(663.22±79.71)、(238.15±62.09) pg/ml比(5 473.20±474.55)、(1 237.85�Objective To investigate the effect of lentivirus-mediated Wnt signaling pathway proteins 5a(Wnt5a)gene overexpression on bone marrow mesenchymal stem cell(BMSC)transplantation in rats with myocardial infarction and to explore its potential molecular mechanism.Methods Animal model of myocardial infarction was established by ligation of the anterior descending coronary branch in male SD rats.According to the number table method,the rats were divided into experimental group(40 rats)and sham operation group(20 rats).BMSCs were isolated and purified in vitro,BMSC lines with stably silenced Wnt5a(sh-Wnt5a)and over-expressed Wnt5a(oe-Wnt5a)were established.The migration capacity of BMSCs was dectected by cell scratch test.The serum contents of insulin-like growth factor 1(IGF-1),vascular endothelial growth factor(VEGF),stem cell factor(SCF),hepatocyte growth factor(HGF)were determined in rats with myocardial infarction.The experimental rats were randomly assigned into 4 groups(a myocardial infarction group,a normal saline group,a BMSC group,a oe-Wnt5a group).The expression of Wnt5a,Paxillin,stem cell factor receptor(C-KIT),Ca2+dependent protein kinasesⅡ(CaMKⅡ)and calcium-sensitive receptor(CaSR)protein in the myocardium was detected by Western blotting.T-test and one-way analysis of variance were used respectively for comparison between two groups and multiple groups.Results BMSC lines with stably silencing or over-expression of Wnt5a were established.The migration rate in sh-Wnt5a group was significantly lower than that in the control group(6.67±1.02 vs.18.71±3.02,t=6.542,P<0.01),and that in oe-Wnt5a group was significantly higher than that in control group(43.33±7.95 vs.18.71±3.02,t=5.014,P<0.01).Enzyme linked immunosorbent assay(ELISA)results showed that the levels of IGF-1,VEGF,SCF and HGF in BMSC group were significantly higher than those in myocardial infarction group[(5473.20±474.55),(1237.85±173.90),(548.09±77.06),(136.14±37.41)pg/ml vs.(3799.20±384.99),(652.32±95.34),(352.78±65.89),(62.92±12.93

关 键 词：WNT信号通路 骨髓 间充质干细胞移植 细胞因子 心肌梗死 

分 类 号：R542.22[医药卫生—心血管疾病]