HIF-lα基因修饰骨髓间充质干细胞对脑损伤的保护作用及其机制的研究

发布时间：2018-05-14 13:24

本文选题：骨髓间充质干细胞 + 分离　；参考：《第四军医大学》2012年博士论文

【摘要】：脑卒中引起的脑损伤是神经外科常见的疾病，发病率和死亡率居脑血管病之首。根据原因可分为出血性脑卒中和缺血性脑卒中，无论是什么原因引起的脑损伤，神经功能的损伤将是不可逆转的。随着科研和临床研究的不断深化和发展，抗凝治疗、溶栓治疗和介入治疗取得了良好效果，但如何防止继发性脑损伤的也是目前临床面临的难题。继发性脑损伤脑功能的恢复有两个关键因素，即血管再生与神经细胞再生。干细胞移植和基因治疗相结合，为治疗脑损伤和神经功能的恢复带来了新的希望。骨髓间质干细胞（Mesenchymal stem cells，MSCs），成为近年来研究的热点，由于其来源丰富，采集方便同时具有较小的免疫反应性，目前已广泛应用在各个领域。低氧诱导因子1α (hypoxia-inducible factor-1α, HIF-1α）是一种重要的转录因子，广泛表达于哺乳动物体内，参与并保持在体内的氧平衡，在病理条件下对缺血缺氧反应产生应答，目前已知有70多个靶基因。在这项研究中，我们使用重组腺病毒HIF-1α基因转染骨髓间充质干细胞移植到局灶性脑缺血再灌注（MCAO）模型，进一步研究HIF-1α的脑损伤保护作用。通过研究，我们发现，HIF-1α的可促进下游靶基因上调，从而促进血管新生，改善脑组织的血液供应，减少脑组织缺血、缺氧损伤。研究同时还发现，HIF-1α可促进骨髓间充质干细胞向神经干细胞（NSCs）分化和维持神经干细胞的特点。本实验分为四个部分：第一部分：骨髓间充质干细胞的分离、培养与鉴定目的：通过分离培养骨髓间充质干细胞（Mesenchymal stem cells，MSCs），鉴定其生物学特性，为组织的基因治疗提供实验依据。方法：采用密度梯度离心法分离MSCs，，倒置相差显微镜观察MSCs的形态学变化；MTT测定细胞生长曲线；细胞表型通过流式细胞仪进行鉴定；利用光镜观察分化能力。结果：密度梯度离心法能分离出较高纯度的MSCs。流式细胞仪显示：CD29(93.2%)、CD105(92.6%)、CD45(94.68%)、CD44(94.6%)、CD73(86.4)、CD34(1.5%)、CD45(2.2%)、CD19(2.0%)、CD14(1.9%)。细胞生长曲线可见细胞增殖的最高峰出现在第6至7d，其后细胞增殖速度迅速下降。光镜下观MSCs能够在体外诱导分化成为成骨细胞与脂细胞。结论：采用密度梯度离心法成功的分离出高纯度的MSCs是有效的方法。第二部分重组腺病毒载体Ad. HIF-lα转染MSCs 目的：构建重组腺病毒Ad. HIF-lα并转染MSCs，观察转染HIF-lα外源性基因及蛋白在MSCs中的表达。方法：将在目的基因HIF-lα的上游引入BamH I酶切位点，下游引入Xba I酶切位点，对质粒pcDNA3.1/HIF-lα进行双酶切鉴定，测序。体外分离、培养MSCs，腺病毒载体与HIF-lα基因连接，转染MSCs，用Western blot检测HIF-lα蛋白表达。结果：双酶切鉴定、测序和Western blot结果显示HIF-lα基因成功转入腺病毒载体，并且能够在MSCs中表达。结论：成功构建了重组腺病毒Ad. HIF-lα，并转染到MSCs中。第三部分：腺病毒介导的HIF-1α基因转染MSCs对脑损伤后血管生成、神经干细胞增殖分化的影响目的：观察低氧诱导因子-1α（hypoxia-inducible factor-1α, HIF-1α）基因对大鼠脑缺血再灌注后血管生成的影响，探讨HIF-1对神经干细胞人（Neuralstem cells，NSCs)增殖与分化的作用机制。方法:建立大鼠大脑中动脉缺血再灌注(Ischemic reperfusion injury, I/R)模型，大鼠随机分为PBS组、MSC组和Ad-HIF-1α转染MSC (Hy-MSC组)。分别将PBS、MSCHy-MSC定位注射到I/R模型大鼠脑缺血区，于7d、14d、21d进行神经功能缺失评分(Neurological deficit score,NDS)并比较。免疫组化检测HIF-1α的表达，Westentblot检测皮层VEGF、EPO和Ang-1的表达，TUNEL检测细胞的调亡情况。结果:（1）Hy-MSC组在第7、14、21d的NDS优于MSC组PBS组（P0.05）；（2）与MSC组相比, Hy-MSC组增强了各时间点HIF-1、VEGF及EPO的表达；（3）Hy-MSC组和MSC细胞数明显，但凋亡率下降，与PBS相比细胞调亡率(P0．05)。结论: HIF-1α基因可促进大鼠局灶性脑缺血后血管生成，同时促进MSCs向神经干细胞分化，并维持NSCs的特性。第四部分HIF-1α与Wnt信号途径的关系以及对NSCs特性的影响目的：观察HIF-1α对Wnt信号途径的相互关系，以及在MSCs向NSCs分化、增殖中作用。方法:体外培养大鼠NSCs以及HIF-1α转染后的骨髓间充质干细胞（Hy-MSC）,利用免疫共沉淀观察两者之间存在联系。为进一步证实上述结果，分别加入HIF-1α与Wnt的活化剂与抑制剂，通过Westernblot观察下游基因血管内皮生长因子（Vascular endothelial growth factor，VEGF）、促进细胞生成素（Erythropoietin，EPO）、淋巴增强因子（Lymphoidenhancer factor-1，LEF-1）、β-链蛋白（β-catenin）表达的变化。结果：（1）阻断HIF-1α的表达，Wnt下游基因表达减少LEF-1（P 0.01）和β-catenin（P 0.05）。（2）阻断Wnt的表达，对HIF-1α下游基因VEGF和EPO无明显影响。（3）阻断Wnt的表达, HIF-1α的表达无影响。结论： HIF-1α调节骨髓间充质干细胞向神经干细胞分化与增殖是通过调节Wnt信号转导通路实现的。
[Abstract]:Brain injury caused by cerebral apoplexy is a common disease in the Department of neurosurgery. The incidence and mortality rate are the first in cerebrovascular disease. According to the causes, it can be divided into hemorrhagic stroke and ischemic stroke. No matter what cause of brain injury, the damage of nerve function will be irreversible. With the continuous deepening and development of scientific research and clinical research, Anticoagulant therapy, thrombolytic therapy and interventional therapy have achieved good results, but how to prevent secondary brain injury is also a difficult problem at present. The recovery of brain function in secondary brain injury has two key factors, that is, vascular regeneration and nerve cell regeneration. Stem cell transplantation and basic therapy are combined to treat brain injury and nerve work. The restoration of energy has brought new hope.
Mesenchymal stem cells (MSCs), which has become a hot spot in recent years, has been widely used in various fields because of its rich sources, convenient collection and small immune response. Low oxygen inducible factor 1 alpha (hypoxia-inducible factor-1 alpha (HIF-1 a)) is an important transcription factor and is widely expressed in the field. In mammals, they participate in and maintain oxygen balance in the body and respond to ischemic anoxia response under pathological conditions. There are more than 70 known target genes. In this study, we used recombinant adenovirus HIF-1 alpha gene transfection into bone marrow mesenchymal stem cells to the focal cerebral ischemia reperfusion (MCAO) model and further study HIF-1 We have found that HIF-1 alpha can promote the up regulation of the downstream target gene, thus promoting angiogenesis, improving the blood supply of brain tissue, reducing brain tissue ischemia and hypoxia injury. The study also found that HIF-1 a can promote the differentiation and maintenance of neural stem cells from bone marrow mesenchymal stem cells to neural stem cells (NSCs). The characteristics of the cell. This experiment is divided into four parts:
Part one: isolation, culture and identification of bone marrow mesenchymal stem cells
Objective: to identify the biological characteristics of bone marrow mesenchymal stem cells (Mesenchymal stem cells, MSCs), and to provide experimental basis for the gene therapy of tissue. Methods: MSCs was separated by density gradient centrifugation, and morphological changes of MSCs were observed by inverted phase contrast microscope, and cell growth curve was measured by MTT; cell phenotype passed through flow. Results: the density gradient centrifugation can separate the high purity MSCs. flow cytometer: CD29 (93.2%), CD105 (92.6%), CD45 (94.68%), CD44 (94.6%), CD73 (86.4), CD34 (1.5%), CD45 (2.2%), CD19 (2%), CD14 (1.9%). The cell growth curve can see the peak of cell proliferation peak. The cell proliferation rate decreased rapidly after sixth to 7d. MSCs could be induced to differentiate into osteoblasts and fat cells in vitro under light microscope. Conclusion: the successful separation of high purity MSCs by density gradient centrifugation is an effective method.
The second part is recombinant adenovirus vector Ad. HIF-l alpha transfected into MSCs.
Objective: to construct recombinant adenovirus Ad. HIF-l alpha and transfect MSCs, and to observe the expression of HIF-l - alpha exogenous gene and protein in MSCs. Methods: the BamH I enzyme cutting site was introduced into the upstream of HIF-l alpha of the target gene, and the Xba I enzyme cutting site was introduced into the downstream, and the plasmid pcDNA3.1/HIF-l alpha was identified by double enzyme cutting, sequenced and cultured in vitro. The virus vector was connected with the HIF-l alpha gene, transfected to MSCs and detected the expression of HIF-l alpha protein with Western blot. Results: double enzyme digestion identification, sequencing and Western blot results showed that HIF-l a gene was successfully transferred to adenovirus vector and could be expressed in MSCs. Conclusion: the recombinant adenovirus Ad. HIF-l alpha was successfully constructed and transfected into MSCs.
The third part: the effect of adenovirus mediated HIF-1 MSCs gene transfection on angiogenesis and proliferation and differentiation of neural stem cells after brain injury.
Objective: To observe the effect of hypoxia inducible factor -1 alpha (hypoxia-inducible factor-1 alpha, HIF-1 a) gene on angiogenesis after cerebral ischemia-reperfusion in rats and to explore the mechanism of HIF-1 on the proliferation and differentiation of neural stem cells (Neuralstem cells, NSCs). Methods: to establish the middle cerebral artery ischemia reperfusion (Ischemic reperfusion injury) in rats. I/R model, rats were randomly divided into PBS group, MSC group and Ad-HIF-1 alpha transfected MSC (Hy-MSC group). PBS, MSCHy-MSC were injected into the ischemic area of rat model of I/R model, and 7d, 14d, and 21d were compared. The expression of -1 and TUNEL were used to detect the apoptosis of the cells.
Results: (1) NDS in group Hy-MSC was better than group MSC in group MSC (P0.05); (2) compared with group MSC, Hy-MSC enhanced the expression of HIF-1, VEGF and EPO at all time points; (3) the number of Hy-MSC groups and cells was obvious, but apoptosis rate decreased and cell apoptosis rate was compared with those of MSC. Conclusion: the gene can promote the blood vessels of focal cerebral ischemia in rats. It also promotes the differentiation of MSCs into neural stem cells and maintains the characteristics of NSCs.
The fourth part is the relationship between HIF-1 alpha and Wnt signaling pathway and its effect on NSCs characteristics.
Objective: To observe the relationship between HIF-1 alpha and Wnt signal pathway, and to differentiate and proliferate from MSCs to NSCs. Methods: bone marrow mesenchymal stem cells (Hy-MSC) after transfection of NSCs and HIF-1 a were cultured in vitro, and the relationship between them was observed by immunoprecipitation. In order to further confirm the above results, HIF-1 A and Wnt were added to the above results. Activator and inhibitor, the expression of Vascular endothelial growth factor (VEGF) was observed by Westernblot, and the expression of cytokine (Erythropoietin, EPO), lymphatic enhancement factor (Lymphoidenhancer factor-1, LEF-1), beta chain protein (beta -catenin) was changed. Gene expression reduced LEF-1 (P 0.01) and beta -catenin (P 0.05). (2) blocked the expression of Wnt and had no obvious effect on the HIF-1 alpha downstream gene VEGF and EPO. (3) blocking the expression of Wnt, the expression of HIF-1 a was not affected. Conclusion: HIF-1 alpha modulate the differentiation and proliferation of bone marrow mesenchymal stem cells to neural stem cells by regulating Wnt signal transduction pathway. Yes.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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