摘要
目的研究miRNA-26a在大鼠脑梗死中的作用以及miRNA-26a对脑梗死大鼠血管生成的影响。方法将16只SPF级雄性SD大鼠随机分为假手术组和脑梗死模型组,采用MCAO法建立大鼠脑梗死模型,TTC法检测大鼠脑梗死体积,Garcia法评估大鼠神经功能。对体外培养和转染的大鼠脑微血管内皮细胞(BMEC)分为:正常组、miR-26a mimic组、miR-26a inhibitor组、mimic+VEGF inhibitor组和体外氧葡萄糖剥夺(OGD)组。正常组为正常大鼠脑微血管内皮细胞;miR-26a mimic组BMEC细胞转染miRNA-26a模拟物;miR-26a inhibitor组BMEC细胞转染miRNA-26a抑制剂;mimic+VEGF inhibitor组BMEC细胞转染miRNA-26a模拟物并添加VEGF抑制剂Sorafenib Tosylate;OGD组BMEC细胞建立体外氧葡萄糖剥夺模型。Trizol法提取细胞中的总RNA,RT-PCR法检测miRNA-26a的表达,MTT法检测细胞增殖和管壁形成情况,蛋白质免疫印迹分析检测VEGF、FGF和Ang蛋白的表达水平。结果与假手术组相比,模型组大鼠脑组织中miR-26a的表达显著上调(P<0.01)。体外的细胞培养中,与正常组相比,OGD组miR-26a的表达显著上调(P<0.01)。与正常组比较,miR-26a mimic组BMEC细胞凋亡率显著降低(P<0.01),小管的形成和长度以及细胞增殖率均显著增加(均P<0.01);与miR-26a mimic组相比,miR-26a inhibitor组中细胞凋亡率显著升高(P<0.01),小管的形成和长度以及细胞增殖率均显著降低(均P<0.01)。与正常组相比,miR-26a mimic组VEGF、FGF和Ang蛋白的表达水平均显著上调(P<0.01);与miR-26a mimic组相比,miR-26a inhibitor组VEGF、FGF和Ang蛋白的表达水平显著下调(P<0.01)。与miR-26a mimic组相比,mimic+VEGF inhibitor组小管的形成受到显著抑制(P<0.01),细胞的相对数目显著减少(P<0.01)。结论miRNA-26a通过调节VEGF的表达影响脑梗死大鼠血管内皮功能及血管的重建和细胞的增殖,其有望成为脑梗死治疗的生物靶标之一。
Objective To study the role of miRNA-26a in cerebral infarction in rats and the effect of miRNA-26a on angiogenesis in rats with cerebral infarction.Methods Sixteen SPF male SD rats were randomly divided into sham group and cerebral infarction(CI)model group.MCAO method was used to establish the rat cerebral infarction model.TTC method was used to detect rat cerebral infarction volume.Garcia method was used to evaluate rat nerve function.The cultured brain microvascular endothelial cells(BMEC)were divided into control group,miR-26a mimic group,miR-26a inhibitor group,mimic+VEGF inhibitor group,and OGD group.The normal rat brain microvascular endothelial cells were chosen as control group.BMEC cells were transfected with miRNA-26a mimics in miR-26a mimic group.BMEC cells were transfected with miRNA-26a inhibitor in miR-26a inhibitor group.BMEC cells were transfected with miRNA-26a mimic and Sorafenib Tosylate in mimic+VEGF inhibitor group.BMEC cells were induced to establish an in vitro oxygen glucose deprivation model(OGD)in OGD group.Trizol method was used to extract the total RNA from cells.The expression of miRNA-26a was detected by RT-PCR method.Cell proliferation and tube wall formation were detected by MTT method,and the expression levels of VEGF,FGF and Ang proteins were detected by Western blot analysis.Results Compared with sham group,the expression level of miR-26a was significantly up-regulated in the brain tissue in model group(P<0.01).In cell culture in vitro,compared with control group,the expression level of miR-26a was also significantly increased in OGD group(P<0.01).Compared with control group,the apoptosis rate of BMEC cells in the miR-26a mimic group was significantly reduced(P<0.01),while the formation and the length of tubules and the cell proliferation rate of BMECs were significantly increased(P<0.01).Compared with miR-26a mimic group,the apoptosis rate of BMEC cells was significantly increased in miR-26a inhibitor group(P<0.01),and the formation and the length of tubules,and the cell proliferation rate of BMECs were significantly reduced(P<0.01).Compared with control group,the expression levels of VEGF,FGF and Ang proteins in miR-26a mimic group were significantly up-regulated(P<0.01).Compared with miR-26a mimic group,the expression level of VEGF,FGF and Ang proteins were significantly down-regulated in miR-26a inhibitor group(P<0.01).Compared with miR-26a mimic group,the formation of tubules in mimic+VEGF inhibitor group was significantly inhibited(P<0.01),and the relative number of BMEC cells was also significantly reduced(P<0.01).Conclusion The miRNA-26a can affect the vascular endothelial function,the vascular remodeling and the cell proliferation by regulating the expression of VEGF.Therefore,it is expected to become one of biological targets for the treatment of cerebral infarction.
作者
职瑾
段斌
王静
王清
王虎清
ZHI Jin;DUAN Bin;WANG Jing;WANG Qing;WANG Huqing(Department of Neurology,Xi’an No.1 Hospital,Xi’an 710002,China;Nephrotic Hemodialysis Center,Shanxi Provincial People’s Hospital;Department of Neurology,Second Affiliated Hospital of Xi’an Jiaotong University)
出处
《山西医科大学学报》
CAS
2021年第5期625-631,共7页
Journal of Shanxi Medical University
基金
陕西省自然科学基础研究计划项目(2017JM8142)
西安市科技计划项目(2019114613YX001SF039(6))。