MicroRNA-296对脑缺血梗死后血管新生的调控机制研究

发布时间：2018-01-20 13:26

  本文关键词： miR-296 血管新生 脑缺血 VEGF Notch　出处：《中南大学》2014年博士论文　论文类型：学位论文

【摘要】：背景与目的：侧枝循环的建立是改善脑梗死后脑组织血流供应的有效途径,治疗性血管生成为缺血性脑卒中血运重建提供了一种新的治疗策略。微小RNA (microRNA, miRNA)是近年发现的一类内源性非编码小分子RNA,在转录后水平调控细胞增殖分化、凋亡、分裂及器官的发育,与人类生命活动的多种疾病密切相关,已成为医学领域的研究热点。在脑肿瘤实体及体外实验中已证实miR-296通过直接抑制其靶基因HGS表达,呈现出促进血管新生的作用。本研究体内实验拟通过构建大鼠大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)脑缺血梗死模型,检测缺血梗死后脑组织中miR-296及其靶基因HGS的表达变化,同时动态观察脑缺血梗死后血管新生的变化,初步明确miR-296是否参与脑缺血梗死后血管新生过程；进一步在体外环境下培育人脐静脉内皮细胞(human umbilical vein endothelial cell, HUVEC),利用重组腺病毒技术过表达miR-296于HUVEC内,观察血管内皮细胞中HGS及VEGF-Notch通路相关分子VEGF、VEGFR2、DLL4和Notchl的mRNA及蛋白表达水平的变化,明确miR-296对VEGF-Notch信号通路的调控作用,深入探讨阐明miR-296参与调控脑缺血梗死后血管新生的分子机制,为缺血性脑卒中治疗提供新的靶点。 研究内容与方法： 1.体内实验 1.1构建大鼠MCAO模型,行TTC染色以鉴定脑缺血梗死区域。于缺血梗死后第1天,第3天,第7天留取脑组织标本行相关检测。 1.2采用qRT-PCR方法检测脑缺血梗死后不同时间点大鼠缺血脑皮质区miR-296的表达水平,明确脑缺血梗死后miR-296动态表达变化。 1.3采用Western blot法检测脑缺血梗死后不同时间点大鼠缺血脑皮质区HGS的蛋白表达,明确脑缺血梗死后miR-296的靶基因HGS的蛋白表达变化。 1.4采用免疫组化染色法检测脑缺血梗死后不同时间点缺血脑皮质区CD105标记的新生微血管以判断血管新生的情况。 2.体外实验 2.1采用贴壁法培养人脐静脉内皮细胞HUVEC-12。构建腺病毒载体,将包装AdV-miR-296-GFP及AdV-GFP的重组腺病毒分别转染至HUVEC-12;荧光显微镜下观察GFP绿色荧光的阳性率。实验分为miR-296过表达组(AdV-miR-296-GFP)和对照组(AdV-GFP)。 2.2行内皮细胞小管形成实验,比较miR-296过表达组与对照组内皮细胞血管形成数,明确miR-296对血管内皮细胞血管形成能力的影响。 2.3采用qRT-PCR法比较miR-296过表达组与对照组中miR-296的表达水平,以明确AdV-miR-296-GFP上调miR-296表达的水平。 2.4采用RT-PCR、Western Blot法分别比较miR-296过表达组与对照组中HGS及VEGF-Notch信号通路相关分子VEGF、VEGFR2、DLL4、 Notch1的mRNA和蛋白表达水平,明确miR-296对VEGF-Notch信号通路的调控途径。 结果： 1.体内实验 1.1成功构建大鼠MCAO模型。TTC染色可见左侧大脑中动脉供血区的梗死灶呈白色,正常脑组织呈红色。 1.2qRT-PCR结果显示,假手术组脑组织中可见miR-296的表达,脑缺血梗死后各时间点实验组大鼠缺血脑皮质区miR-296表达均高于假手术组(各P0.05),miR-296在脑缺血梗死后第1天表达即开始上调(2.25±0.36),第三天表达量继续上调(5.35±0.35),7天时最明显(7.91±0.21),表明正常非缺血梗死脑组织中存在miR-296的表达,且脑缺血损伤可刺激miR-296表达水平的逐渐上调。 1.3Western blot结果显示检测脑缺血梗死后Id,3d,7d实验组大鼠缺血脑皮质区HGS的蛋白表达逐渐下降,验证了脑缺血梗死后过表达的miR-296可抑制其靶基因HGS的蛋白表达。 1.4免疫组化染色结果显示,脑缺血梗死后第一天即出现CD105阳性细胞(1.8±0.84),第3天时阳性细胞数增多(6.8±2.17),第7天可见更多的新生血管(10±2.17),与miR-296表达水平呈正相关(r=0.95,P0.05),提示miR-296促进脑缺血梗死后血管新生过程。 2.体外实验 2.1成功构建腺病毒载体,将包装AdV-miR-296-GFP及AdV-GFP的重组腺病毒转染至HUVEC-12细胞,荧光显微镜下可见绝大部分细胞均表达GFP绿色荧光,GFP阳性细胞率达90%以上。 2.2qRTHPCR结果显示,与对照组比较,miR-296过表达组miR-296上调了320±30倍。 2.3内皮细胞小管形成实验结果显示,与对照组比较,miR-296过表达组HUVEC-12细胞形成血管管腔数明显增多(28±1.5vs.8±2.5,P0.05),表明miR-296可促进血管内皮细胞形成管腔样结构。 2.4RT-PCR、western blot结果显示,与对照组比较,miR-296过表达组HUVEC-12细胞中HGS mRNA的表达水平下降,其蛋白水平则显著下降。与此同时,VEGF、VEGFR2的mRNA和蛋白表达水平均显著上调而DLL4、Notchl的mRNA和蛋白表达水平均显著下降(各P0.05)。 结论： 1.大鼠正常(非梗死)脑组织中存在miR-296的表达。 2.miR-296促进大鼠脑缺血梗死后血管新生的过程。 3.miR-296通过抑制其靶基因HGS的表达,上调VEGF-VEGFR2信号通路的活性,促进血管新生的过程。 4.促血管生成因子miR-296抑制DLL4-Notchl信号通路的活性,其机制有待进一步研究。图16幅,表2个,参考文献116篇。
[Abstract]:Background and purpose: the establishment of collateral circulation is an effective way to improve cerebral blood supply, provides a new therapeutic strategy for the treatment of angiogenesis in ischemic stroke revascularization. Micro RNA (microRNA, miRNA) is a kind of newly discovered endogenous small molecule non encoding RNA, apoptosis at the post transcriptional level the regulation of cell proliferation, differentiation, proliferation and organ development, a variety of diseases and is closely related to the activities of human life, has become a hot research topic in the field of medicine. In the brain tumor and in vitro experiments have demonstrated that miR-296 through direct inhibition of HGS gene expression, showing a role in promoting angiogenesis in vivo. This study intends to by constructing a rat model of middle cerebral artery occlusion (middle cerebral artery occlusion, MCAO) model of ischemic cerebral infarction, miR-296 and target detection based brain infarction due to the expression of HGS At the same time, changes in angiogenesis of ischemic infarction after dynamic observation of brain changes, preliminary to determine whether miR-296 is involved in ischemic brain infarction angiogenesis; further cultivating human umbilical vein endothelial cells in vitro environment (human umbilical vein endothelial cell, HUVEC), using the technology of recombinant adenovirus overexpression of miR-296 in HUVEC, the observation of vascular endothelial cells in the HGS and VEGF-Notch pathway related molecules VEGF, VEGFR2, mRNA and protein expression of DLL4 and Notchl, miR-296 on the clear regulation of the VEGF-Notch signaling pathway, explore the molecular mechanism of angiogenesis to clarify miR-296 involved in the regulation of cerebral ischemia after myocardial infarction and provide a new target for the treatment of ischemic stroke.
Research contents and methods:
1 in vivo experiment
1.1 the rat model of MCAO was constructed, and TTC staining was used to identify the cerebral ischemic infarct area. The brain tissue specimens were collected at first days, third days and seventh days after the ischemic infarction.
1.2 qRT-PCR method was used to detect the expression level of miR-296 in the ischemic cortex of rats at different time points after cerebral ischemia, and to clarify the dynamic expression of miR-296 after cerebral ischemia.
1.3 Western blot method was used to detect the protein expression of HGS in the ischemic cortex of rats at different time points after cerebral ischemia, and to clarify the protein expression of target gene HGS after cerebral ischemia and infarction in miR-296.
1.4 immuno histochemical staining was used to detect the neovascularization of CD105 markers in ischemic cerebral cortex at different time points after cerebral ischemia in order to determine angiogenesis.
2. in vitro experiment
2.1 the cultured human umbilical vein endothelial cells HUVEC-12. to construct adenovirus vector, the recombinant adenovirus packaging AdV-miR-296-GFP and AdV-GFP were transfected into HUVEC-12; observe the positive rate of GFP green fluorescence under fluorescence microscope. The experiment was divided into miR-296 overexpression group (AdV-miR-296-GFP) and control group (AdV-GFP).
2.2 endothelial cell canaliculus formation experiments were performed to compare the number of endothelial cells in the miR-296 overexpressing group and the control group, and to clarify the effect of miR-296 on the angiogenesis of vascular endothelial cells.
2.3 qRT-PCR was used to compare the expression level of miR-296 in the miR-296 overexpression group and the control group, so as to determine the level of AdV-miR-296-GFP to increase the expression of miR-296.
2.4, we used RT-PCR and Western Blot to compare HGS and VEGF-Notch signal pathway related molecules VEGF, VEGFR2, DLL4, Notch1 mRNA and protein expression levels between miR-296 overexpressing group and control group, and to clarify the regulation pathway of DLL4 on the signal pathway.
Result锛，

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