【作者】 陈丽；

【导师】 魏全；

【作者基本信息】 四川大学 ， 康复医学与理疗学（专业学位）， 2021， 博士

【摘要】 目的:课题组前期研究发现治疗参数为30Hz,3m T的脉冲电磁场(Pulsed Electromagnetic Fields,PEMFs)对心肌梗死小鼠的心脏保护作用与PEMFs促进缺血心肌血管生成有关,但其作用机制尚未完全阐明;此外,既往研究发现,CD44在小鼠正常心肌微血管内皮细胞中低表达或不表达,但在急性心肌梗死后1～2周,CD44表达明显增加,且梗死边缘区域形成大量新血管。因此,本研究的目的是:1)明确CD44在PEMFs(30Hz,3m T)干预后的心梗小鼠缺血心肌中的表达情况;2)如果CD44在PEMFs干预后的缺血心肌中表达有变化,则进一步明确CD44在PEMFs促缺血心肌血管生成中的作用,并探究PEMFs与CD44相关的作用机制。材料和方法:结扎小鼠冠状动脉左前降支制备心肌梗死模型。将制备成功的模型随机分为对照组和PEMFs组。PEMFs组接受PEMFs干预(30Hz,3m T,45min/天×7/14天)。小鼠超声心动图检测心脏功能;RT-q PCR检测缺血心肌中CD44的表达(实验1)。如果CD44的表达在PEMFs干预后的缺血心肌有变化,则将CD44基因敲除纯合子小鼠(KO)与野生型小鼠(WT)配对繁育出遗传背景相近的CD44WT和CD44KO小鼠用于后续实验(实验2)。将制备成功的心肌梗死模型按基因型随机分为四组:即WT+Control组、WT+PEMFs组、KO+Control组、KO+PEMFs组。WT+PEMFs组和KO+PEMFs组接受PEMFs干预,干预参数及时间同实验1。评价小鼠心脏功能、心肌梗死面积及心肌梗死边缘区血管生成数量,检测CD44及相关基因的蛋白或m RNA表达水平(实验3)。同时结合小鼠体内基质胶塞血管生成实验(实验4)及体外小鼠主动脉环出芽实验(实验5)进一步研究PEMFs干预下CD44在血管生成中的作用。结果:在实验1中,超声心动图检测结果显示PEMFs分别干预1周或2周可明显改善小鼠心肌梗死后心脏功能;心肌梗死边缘区CD44 m RNA在PEMFs干预2周时表达增加。在实验2中,成功繁育、基因鉴定出遗传背景相近的可以用于后续实验的CD44野生型(CD44WT)及基因敲除型纯合子小鼠(CD44KO)。在实验3中,我们发现PEMFs干预1周对WT+PEMFs组小鼠和KO+PEMFs组小鼠心肌梗死后心脏功能均有改善作用,但对WT+PEMFs组小鼠心脏功能的改善作用优于KO+PEMFs组小鼠。PEMFs干预2周仅对WT+PEMFs组小鼠心肌梗死后心脏功能有持续的改善作用,对KO+PEMFs组小鼠心肌梗死后心脏功能的改善作用消失。在干预1周时,四个组心肌梗死面积无显著性差异;在干预2周时,与其他三个组比较,WT+PEMFs组的左室梗死面积明显降低。干预1周时,与WT+Control组比较,KO+Control组小鼠心肌梗死边缘区血管生成数量明显下降;与WT+PEMFs组比较,KO+PEMFs组小鼠心肌梗死边缘区血管生成数量明显下降。干预2周时,与其他三个组比较,WT+PEMFs组小鼠缺血心肌边缘区毛细血管密度显著升高。进一步研究发现:1)在PEMFs干预1周时,心肌梗死边缘区组织内的CD44/AKT/VEGF信号通路被激活;2)PEMFs干预2周时,心肌梗死边缘区CD44/PKC-α/VEGF/TGF-β1/HIF-1α信号通路被激活;3)在PEMFs干预1和2周时CD44基因缺失上调心肌梗死边缘区MMP-2表达水平。在实验4和实验5中,我们发现,CD44基因敲除体内基质胶塞血管生成和体外小鼠主动脉环出芽。结论:CD44在PEMFs促进小鼠心肌梗死后缺血心肌血管生成、减小心肌梗死面积及改善心脏功能的作用中扮演了重要角色。这些作用可能与PEMFs激活CD44/AKT/VEGF,CD44/PKC-α/VEGF/TGF-β1/HIF-1α和CD44/MMP-2等有细胞保护作用和促血管生成作用的信号通路有关。更多还原

【Abstract】 ObjectiveIn our previous study,we found that pulsed electromagnetic fields(PEMFs)of 30 Hz,3m T could improve cardiac function that may be contributed by increased angiogenesis in mice with myocardial infarction.However,its mechanism needs to be elucidated.In addition,previous studies have found that CD44 was low or not expressed in microvascular endothelial cells in mouse normal myocardium,but was highly expressed and a large number of new vessels were formed in the infarct area within 1 to 2 weeks after acute myocardial infarction.Therefore,the purposes of this study were: 1)to investigate the expression pattern of CD44 in the ischemic myocardium of MI mice after PEMFs(30 Hz,3m T)intervention,2)If the expression of CD44 in ischemic myocardium changes after PEMFs intervention,the role of CD44 protein in PEMFs promoting angiogenesis in ischemic myocardium should be further clarified,and the possible mechanism should be explored.Materials and MethodsThe mouse MI model was prepared by left anterior descending coronary artery ligation.The successfully prepared model was randomly divided into the control group and the PEMFs group which received PEMFs intervention(30Hz,3m T,45min/day for 7/14 days).Cardiac function is evaluated by echocardiography.The expression of CD44 in ischemic myocardium was detected by RT-q PCR(Experiment 1).If the CD44 expression is increased in ischemic myocardium after PEMFs intervention,CD44 knockout homozygous mouse(CD44KO)will be paired with local CD44 wild-type(CD44WT)mouse to breed homozygous CD44 WT and CD44 KO mice with similar genetic background for the following experiments(Experiment 2).The successfully prepared MI models were randomly divided into four groups according to their genotypes: WT+Control,WT+PEMFs,KO+Control and KO+PEMFs group.WT+PEMFs and KO+PEMFs group received the same PEMFs intervention as mentioned in experiment 1.Cardiac function,MI size and angiogenesis were evaluated,and the protein or m RNA expression levels of CD44 and related genes were detected(Experiment 3).At the same time,the effect of CD44 on angiogenesis under PEMFs intervention will be further studied using the matrigel angiogenesis model in mice(Experiment 4)and in-vitro mouse aortic ring angiogenesis model(Experiment 5).ResultsIn Experiment 1,echocardiography showed that PEMFs intervention for 1 or2 weeks significantly improved the cardiac function of MI mice.CD44 m RNA expression was increased in the myocardial peri-infarcted area after 2 weeks of PEMFs intervention.In Experiment 2,CD44 wild-type and knockout homozygous mice were successfully bred and be genotyped for subsequent experiments.In Experiment 3,we found that PEMFs intervention for 1 week had improved cardiac function both in WT+PEMFs mice and KO+PEMFs mice,and the improvement in cardiac function in WT+PEMFs mice was more significant than that in KO+PEMFs mice.After 2 weeks of PEMFs intervention,only WT+PEMFs mice had sustained improvement in cardiac function after myocardial infarction,while KO+PEMFs mice no longer had improvement in cardiac function.There was no significant difference in the myocardial infarct size among the four groups after 1 week PEMFs intervention.Comparing to the other three groups,left ventricular infarct size was significantly reduced in the WT+PEMFs group after 2 weeks of intervention.At 1 week after intervention,comparing to WT+Control group,angiogenesis in the myocardial peri-infarcted area was significantly decreased in KO+Control group;comparing to WT+PEMFs group,peri-infarcted area angiogenesis was significantly decreased in KO+PEMFs group.After 2 weeks of PEMFs intervention,Comparing to the other three groups,the capillary density in the myocardial peri-infarcted area in WT+PEMFs group was significantly increased.With the detection of protein and m RNA levels,we found that: 1)CD44/AKT/VEGF signalings pathways were activated in the myocardial peri-infarcted area after 1 week of PEMFs intervention.2)CD44/PKC-α/VEGF/TGF-β1/HIF-1α signalings pathways were activated after 2 weeks of PEMFs intervention.3)CD44 gene deletion upregulates the expression of MMP-2 in the myocardial peri-infarcted area both after 1 and 2 weeks of PEMFs intervention.In experiments 4 and 5,we found that CD44 knockout inhibited in vivo matrix plug angiogenesis and in vitro aortic ring angiogenesis.ConclusionCD44 plays an important role in promoting ischemic myocardial angiogenesis,preventing the enlargement of myocardial infarction area and improving cardiac function in MI mice treated with PEMFs.These effects may be contributed by PEMFs-induced activation of CD44/AKT/VEGF,CD44/PKC-α/VEGF/TGF-β1/HIF-1α and CD44/ MMP-2 related signaling pathways that may have protective effects and pro-angiogenic effects on cells.更多还原