microRNA-376b-5p对脑缺血后血管新生的调控作用及机制研究

发布时间：2018-06-12 02:14

本文选题：miR-376b-5p + 血管新生　；参考：《中南大学》2014年博士论文

【摘要】：背景和目的：脑梗死是一类发病率高、致残率高以及复发率高的急性缺血性脑血管病,占全部脑卒中的60%-80%。缺血可以导致大量神经细胞出现不可逆的变性坏死,导致严重的神经功能缺损症状。促进缺血区新生血管的形成增加新的侧枝循环,可通过改善局部脑血流供应,挽救缺血半暗带内濒临死亡的神经细胞,促进神经功能的恢复,这对脑梗死有重要的治疗作用。已有研究证明,脑缺血可诱导血管新生,而HIF-1-VEGF-NOTCH通路是缺血缺氧后调节血管新生的重要的信号通路。微小RNA(microRNA,miRNA)是一类内源性单链小分子RNA,参与了生物体的生长发育、器官形成、细胞增殖、分化、凋亡等多种生物学过程,与多种疾病密切相关。研究发现,miR-376b-5p在脑缺血后缺血区脑组织内表达降低,我们通过生物学软件分析发现HIF-1a与miR-376b-5p的序列存在配对关系,故我们推测miR-376b-5p可能参与了血管新生的调控过程,并且可能通过HIF-1-VEGF-Notch信号通路来调控血管新生。本研究通过体内外实验观察脑缺血缺氧后miR-376b-5p、 HIF-1-VEGF-Notch信号分子的表达变化和血管新生情况,并通过体外实验观察过表达和抑制miR-376b-5p对内皮细胞血管新生能力及HIF-1-VEGF-Notch信号通路分子表达的影响,探讨miR-376b-5p对血管新生的调控作用及可能作用的信号途径,以期为脑梗死的治疗提供一种新策略。方法： 1.体内实验 1.1构建大鼠永久性局灶性大脑中动脉脑缺血(Permanent middle cerebral artery occlusion,pMCAO)模型,于pMCAO后1天,3天,7天收集脑组织标本。 1.2通过神经缺损评分和TTC染色方法鉴定建模是否成功。 1.3通过免疫组化方法检测缺血脑皮质区第八因子相关抗原因子(vWF)的表达情况从而判断血管新生情况。 1.4采用实时荧光定量PCR的方法检测缺血脑皮质区miR-376b-5p、 HIF-1a mRNA、 VEGFA mRNA、 Notchl mRNA的表达变化。 1.5运用Western blot的方法观察缺血脑皮质区HIF-1α、 VEGFA、 Notchl的蛋白表达变化。 2.体外实验 2.1建立了人脐静脉内皮细胞(HUVEC)缺氧模型。 2.2运用MTT,Transwell和Matrigel基质胶实验比较常氧组和低氧组细胞的细胞增殖,细胞迁移以及小管形成情况,从而可以判断缺氧对血管新生的影响。运用实时荧光定量PCR和Western blot的方法观察常氧组和低氧组中HIF-1α、 VEGFA.Notchl在mRNA和蛋白水平的表达变化；运用实时荧光定量PCR的方法检测缺氧对细胞中miR-376b-5p表达的影响。 2.3进一步通过在缺氧HUVEC细胞中转染miR-376b-5p模拟物或抑制物使miR-376b-5p过表达或者抑制表达,观察转染后内皮细胞血管新生的能力以及细胞中HIF-1α、 VEGFA、Notchl在mRNA和蛋白水平的表达变化,从而明确miR-376b-5p对血管新生的调控作用。 2.4为了进一步研究miR-376b-5p对血管新生的调控机制,我们构建了HIF-1a shRNA表达载体,将其转染入缺氧HUVEC细胞中以敲除HIF-1α的作用,通过比较HIF-1α被抑制前后,miR-376b-5p模拟物或抑制物对内皮细胞血管新生能力的影响以及细胞中HIF-1α、 VEGFA、Notchl分子在mRNA和蛋白水平的表达变化,从而探讨miR-376b-5p是否通过HIF-1-VEGF-Notch信号通路影响血管新生。结果： 1.体内实验 1.1成功构建了大鼠永久性局灶性大脑中动脉脑缺血(pMCAO)模型,TTC染色显示：右侧大脑中动脉供血区的梗死区域呈白色,正常脑组织呈红色。 1.2免疫组化结果显示：pMCAO1天,3天,7天组缺血脑组织第八因子相关抗原因子(vWF)表达水平与假手术组相比均上调,反映了pMCAO1天,3天,7天组缺血区中微血管密度比假手术组均增加,提示脑缺血损伤可导致反应性血管新生。 1.3实时荧光定量PCR结果显示：pMCAO后1天,3天,7天组缺血脑皮质区miR-376b-5p的表达水平与假手术组相比均降低,并且HIF-1a mRNA、 VEGFA mRNA、 NotchlmRNA的表达均增加。 1.4Western blot结果显示：pMCAO后1天,3天,7天组缺血脑皮质区HIF-1α、VEGFA、 Notchl的蛋白表达与假手术组相比均增加。 2.体外实验 2.1缺氧组与常氧组相比,miR-376b-5p的表达水平降低,而HUVEC的细胞增殖,细胞迁移以及小管形成能力增强,即血管新生能力增强,同时HIF-1α、 VEGFA、 Notchl在mRNA水平和蛋白水平的表达也增加,提示miR-376b-5p可能对内皮细胞的血管新生具有抑制作用。 2.2将miR-376b-5p模拟物转染缺氧细胞后,内皮细胞形成新生血管的能力减弱,同时HIF-1α、VEGFA、 Notchl在mRNA水平和蛋白水平的表达也降低；而miR-376b-5p抑制物转染缺氧细胞后,内皮细胞形成新生血管的能力增强,同时HIF-1α、VEGFA、 Notch1在mRNA水平和蛋白水平的表达也增加,初步明确了缺氧后miR-376b-5p可能通过HIF-1-VEGF-Notch信号通路调控了血管新生。 2.3将HIF-1α shRNA表达载体转染入缺氧HUVEC细胞敲除HIF-1α的作用后,miR-376b-5p模拟物(或抑制物)与HIF-1α shRNA共转染组与单纯miR-376b-5p模拟物(或抑制物)转染组相比,内皮细胞形成新生血管的能力发生改变,同时HIF-1α、 VEGFA、 Notchl在mRNA水平和蛋白水平的表达也产生变化,进一步明确了miR-376b-5p是通过调控HIF-1-VEGF-Notch信号通路来发挥对血管新生的负调控作用。结论： 1. miR-376b-5p对脑缺血缺氧后的血管新生具有抑制作用。 2. miR-376b-5p可能是通过调控HIF-1-VEGF-Notch信号通路来参与抑制血管新生的过程。 3.通过干预miR-376b-5p的表达,促进新生血管形成,为脑梗死的治疗提供了一种新思路。图32幅,表6个,参考文献101篇。
[Abstract]:BACKGROUND & OBJECTIVE : Cerebral infarction is a kind of acute ischemic cerebrovascular disease with high morbidity , high disability rate and high recurrence rate , which accounts for 60 % -80 % of all cerebral apoplexy . It has been proved that the expression of HIF - 1 - VEGF - NOTCH is an important signal pathway to regulate angiogenesis after cerebral ischemia . It has been found that miR - 376b - 5p is a kind of endogenous single - chain small molecule RNA . It is found that miR - 376b - 5p can participate in the regulation of angiogenesis , and it is possible to regulate angiogenesis through HIF - 1 - VEGF - Notch signaling pathway .

In this study , the expression of miR - 376b - 5p , HIF - 1 - VEGF - Notch signaling molecule and angiogenesis were observed in vivo and in vivo , and the effects of miR - 376b - 5p on angiogenesis in endothelial cells and the expression of HIF - 1 - VEGF - Notch signaling pathway were observed through in vitro experiments , and the effects of miR - 376b - 5p on angiogenesis and possible signaling pathways were investigated in order to provide a new strategy for the treatment of cerebral infarction .

Method :

1 . In vivo experiment

1.1 Rat permanent middle cerebral artery occlusion ( TIA ) model was constructed , and the brain tissue samples were collected on 1 day , 3 days and 7 days after the operation .

1.2 Identification of the success of the modeling by the neural defect score and TTC staining method .

1.3 The expression of eighth factor related antigen factor ( VWF ) in ischemic cerebral cortex was detected by immunohistochemistry .

1.4 The expression of miR - 376b - 5p , HIF - 1a mRNA , VEGFA mRNA and Notchl mRNA was detected by real - time fluorescence quantitative PCR .

1.5 The expression of HIF - 1伪 , VEGFA and Notchl was observed by Western blot .

2 . In vitro experiments

2.1 The hypoxia model of human umbilical vein endothelial cells ( HUVEC ) was established .

2.2 The expression of HIF - 1伪 , VEGFA and Notchl in normal oxygen and hypoxic groups was determined by real - time fluorescence quantitative PCR and Western blot .
The effect of hypoxia on the expression of miR - 376b - 5p in cells was detected by real - time fluorescence quantitative PCR .

2.3 Further , miR - 376b - 5p overexpression or inhibition of miR - 376b - 5p was observed or inhibited by transfection of miR - 376b - 5p in an anoxic HUVEC cell , and expression of HIF - 1伪 , VEGFA , Notchl in the cells was observed to change in mRNA and protein levels , thereby defining the regulation of miR - 376b - 5p on angiogenesis .

2.4 In order to further study the regulation mechanism of miR - 376b - 5p on angiogenesis , we constructed the expression vector of HIF - 1a shRNA , transfected into anoxic HUVEC cells to knock out HIF - 1伪 . After the inhibition of HIF - 1伪 , the expression of HIF - 1伪 , VEGFA and Notchl on endothelial cells and the expression of HIF - 1伪 , VEGFA and Notchl on endothelial cells were investigated .

Results :

1 . In vivo experiment

1.1 The rat permanent focal cerebral artery ischemia model was successfully constructed . TTC staining showed that the infarct area of the right middle cerebral artery supply area was white , and the normal brain tissue was red .

1.2 Immunohistochemical results showed that the expression level of eighth factor related antigen factor ( VWF ) in ischemic brain tissue was up - regulated in 3 days and 7 days after pMCAO1 , which reflected the increase of microvessel density ( MVD ) in the ischemic area of 7 days after the treatment of pMCAO1 , 3 days and 7 days , which suggested that cerebral ischemia injury could lead to reactive angiogenesis .

1.3 The expression level of miR - 376b - 5p in ischemic brain cortex was lower than that in sham operation group , and the expression of HIF - 1a mRNA , VEGFA mRNA and Notchloral mRNA increased 1 day , 3 days and 7 days after the operation .

1 . Western blot showed that the expression of HIF - 1伪 , VEGFA and Notchl in the ischemic brain cortex increased in 1 day , 3 day and 7 days after the treatment .

2 . In vitro experiments

2.1 The expression level of miR - 376b - 5p decreased compared with the normal oxygen group , while the cell proliferation , cell migration and small tube formation ability of HUVEC were enhanced , that is , the ability of angiogenesis was enhanced , while the expression of HIF - 1伪 , VEGFA and Notchl increased at mRNA level and protein level , suggesting that miR - 376b - 5p could inhibit angiogenesis in endothelial cells .

2.2 After transfection of hypoxia cells with miR - 376b - 5p , the ability of endothelial cells to form new blood vessels decreased , while HIF - 1伪 , VEGFA and Notchl decreased at mRNA level and protein level ;
At the same time , the expression of HIF - 1伪 , VEGFA , Notch1at mRNA level and protein level increased , and the expression of HIF - 1伪 , VEGFA and Notch1at mRNA level and protein level was also increased . After hypoxia , the expression of miR - 376b - 5p could regulate angiogenesis through HIF - 1 - VEGF - Notch signaling pathway .

2.3 After the HIF - 1伪 shRNA expression vector was transfected into the hypoxia HUVEC cells to knock out the HIF - 1伪 , the ability of the miR - 376b - 5p mimetic ( or inhibitor ) to co - transfected with the HIF - 1伪 shRNA was changed , and the expression of HIF - 1伪 , VEGFA and Notchl also changed at the mRNA level and protein level , and further clarified that miR - 376b - 5p plays a negative regulatory role in angiogenesis by regulating the HIF - 1 - VEGF - Notch signaling pathway .

Conclusion :

1.miR - 376b - 5p has an inhibitory effect on angiogenesis after cerebral ischemia .

2 . miR - 376b - 5p may be involved in inhibiting angiogenesis by modulating the HIF - 1 - VEGF - Notch signaling pathway .

3 . The expression of miR - 376b - 5p is intervened to promote the formation of neovascularization , which provides a new idea for the treatment of cerebral infarction .
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.33

【参考文献】