大鼠低氧诱导因子-1α基因载体的构建及对骨髓基质细胞培养的实验研究

发布时间：2018-01-12 01:19

本文关键词：大鼠低氧诱导因子-1α基因载体的构建及对骨髓基质细胞培养的实验研究　出处：《第四军医大学》2010年硕士论文　论文类型：学位论文

【摘要】： 脊髓缺血损伤(Spinal Cord Ischemia Injury,SCII)是由各种原因引起的脊髓外伤、脊髓压迫、胸腹主动脉外科手术中动脉的阻断、损伤或低灌注压造成的原发性损害。再灌注过程中可进一步加重神经元损害,引起再灌注损伤。再灌注损伤是在缺血性损伤的基础上发展而来的,涉及多种发病机制并影响缺血性疾病的预后,再灌注可以使可逆性缺血损伤加重,亦可能促进可逆性缺血损伤转化为不可逆性损伤。探索脊髓缺血再灌注损伤的机制,并结合其发生机制制定切实有效的治疗方案,做到既能保证尽早恢复缺血组织的血流,又减轻或防止再灌注损伤的发生,是脊髓缺血性损伤防治中亟待解决的重要课题。很多研究证实有多种因素参与和调节SCII,但它们的具体作用以及作用途径目前尚未明了。良好的血液循环是组织细胞获得充足的氧和营养物质供应并排除代谢产物的基本保证,各种原因造成的组织血液灌注减少可使细胞发生缺血性损伤,尽早恢复组织的血液灌注是减轻缺血性损伤的根本措施。转基因技术和细胞移植是近年来兴起的治疗技术,因此我们选择转基因技术与细胞移植技术结合的方法对脊髓缺血损伤进行探索性的治疗。进行转基因治疗合适目的基因和载体的选择非常重要。缺氧诱导因子-1(hypoxia inducible factor 1,HIF-1)是在1992年Semenza等[1]研究缺氧诱导的促红细胞生成素基因表达时发现的一种核转录因子,在缺氧条件下广泛存在于人类和哺乳动物细胞内,它与增强脊髓组织细胞的耐受低氧能力,促进脊髓新生血管的形成[2,3]密切相关。骨髓基质细胞(BMSC)在一定的条件下可诱导分化为神经细胞、心肌细胞、成骨细胞、脂肪细胞和肌母细胞[4]等,成为目前医学及生物学领域最有潜力的研究工具。在体移植和离体实验均证实,MSC可横向分化为神经细胞或神经胶质细胞,对治疗中枢神经系统疾病潜在的应用前景[5]。因此我们利用缺氧诱导因子-1作为治疗的目的基因并为其构建合适的表达载体,选择骨髓基质细胞作为靶细胞,并且在进一步的研究中将构建的HIF-1载体转染到BMSC中。实验一大鼠缺氧诱导因子-1α基因表达载体的构建目的:构建大鼠低氧诱导因子-1α的基因表达载体方法:首先制备大鼠脊髓缺血再灌注损伤模型,分离脊髓组织并提取HIF-1α总mRNA,利用逆转录聚合酶链式反应合成大鼠HIF-1α的cDNA;根据大鼠HIF-1α基因序列设计引物和酶切位点,并进行PCR扩增,将扩增产物纯化后连接T载体,转化感受态进行基因克隆,双酶切验证后克隆入pcDNA3.1,双酶切及测序验证结果。结果:提取的mRNA完整,纯度较高,OD=1.92;通过PCR扩增出2472bp的基因片段,与大鼠HIF-1α基因长度相符;双酶切克隆载体可以得到2472bp目的片段,测序验证无碱基的改变。结论:大鼠HIF-1α与pcDNA3.1连接正确;成功构建了大鼠HIF-1α基因的表达载体。实验二大鼠骨髓基质细胞的培养目的:培养并获得高纯度的骨髓基质细胞。方法:用淋巴细胞液分离法获取大鼠骨髓基质细胞,于培养瓶中进行原代培养,融合达90%时进行传代培养,并传至第三代;培养期间利用倒置显微镜观察骨髓基质细胞的生长特性;利用免疫细胞化学法检测细胞表面抗原CD45和CD90的表达。结果:倒置显微镜观察到骨髓基质细胞呈贴壁生长,为类成纤维细胞样,大而扁平,原代细胞形状差别较大,第三代趋于稳定,以梭形为主;免疫细胞化学法鉴定CD45呈阴性表达为3.63%,CD90表达较高为96.2%。结论:大鼠骨髓基质细胞体外培养生长良好;获得的第三代细胞纯度较高。
[Abstract]:Spinal cord ischemic injury (Spinal Cord Ischemia Injury, SCII) is caused by a variety of reasons of spinal cord injury, spinal cord compression, artery occlusion surgery of thoracic and abdominal aortic surgery, injury or low perfusion pressure caused by the primary injury. Reperfusion may further aggravate neuronal damage caused by reperfusion injury. Reperfusion injury is and on the basis of ischemia injury, and the prognosis of many involved in the pathogenesis of ischemic disease, reperfusion can cause reversible ischemic injury, may promotereversible ischemic injury into irreversible injury. To explore the mechanism of spinal cord ischemia reperfusion injury, and the injury mechanism and treatment plan effectively, so as to ensure recovery of ischemic tissue blood flow as soon as possible, and reduce or prevent the occurrence of reperfusion injury, is to be solved in the prevention of ischemic spinal cord injury An important topic. Many studies have confirmed that there are many factors involved in the regulation and SCII, but their specific role and the pathway is not clear. Good blood circulation is the basic guarantee of tissue cells to obtain sufficient supply of oxygen and nutrients and remove metabolic products, all kinds of causes of perfusion reduction can make the cell ischemic injury. Early restoration of blood perfusion of the ischemic injury is to reduce the fundamental measures.
Transgenic technology and cell transplantation is the rise in recent years, treatment technology, so we choose the method combined with transgenic technology and cell transplantation technology to explore the treatment of spinal cord ischemia injury. Gene therapy suitable target gene and vector selection is very important. Hypoxia inducible factor -1 (hypoxia inducible factor 1, HIF-1) is a kind of the nuclear transcription factor found in the expression of erythropoietin induced by [1] Semenza in 1992 of the hypoxia gene, under hypoxic conditions exists widely in human and mammalian cells, and enhance spinal cord tissue hypoxia tolerance ability, promote spinal cord angiogenesis. [2,3] is closely related to bone marrow stromal cells (BMSC) can the induction and differentiation of neural cells, under certain conditions, myocardial cells, osteoblasts, adipocytes and muscle cells such as [4], the medicine and Biology Study on tool field in the future. In vivo transplantation and in vitro experiments confirmed that MSC can differentiate into nerve cells and glial cells, in the treatment of diseases of the central nervous system, the potential application prospect of [5]. so we use the hypoxia inducible factor -1 as target for radical treatment and also to construct a suitable expression vector. Selection of bone marrow stromal cells as target cells, cells transfected with HIF-1 and constructed in the study will further to BMSC.
Construction of an hypoxia inducible factor -1 - alpha gene expression vector in rats
Objective: to construct the gene expression vector of hypoxia inducible factor -1 alpha in rats
Methods: first, the preparation of rat spinal cord ischemia reperfusion injury model of spinal cord tissue separation and extraction of HIF-1 alpha mRNA, using reverse transcriptase polymerase chain reaction HIF-1 synthesis of rat alpha cDNA alpha gene in rat; according to the HIF-1 primers and restriction sites, and were amplified by PCR, the amplified products were purified connected to T vector and transformed into competent gene cloning, double enzyme digestion verification after cloned into pcDNA3.1, double enzyme digestion and sequencing results.
Results: the extracted mRNA was intact and highly purified, OD=1.92. The 2472bp gene fragment was amplified by PCR, which was consistent with the length of rat HIF-1 alpha gene. Double enzyme digestion cloned vector could get 2472bp target fragment, and sequencing verified no base change.
Conclusion: the connection between HIF-1 alpha and pcDNA3.1 is correct, and the expression vector of HIF-1 alpha gene in rats is successfully constructed.
The culture of bone marrow stromal cells in experimental two rats
Objective: to cultivate and obtain high purity bone marrow stromal cells.
Methods: the rat bone marrow stromal cells with lymphocyte separation, in culture flasks were cultured, passaged fusion reached 90%, and spread to the third generation; growth characteristics during the observation of bone marrow stromal cells by inverted microscope; used immunocytochemistry to detect expression of cell surface antigen CD45 and CD90.
Results: the inverted microscope showed that bone marrow stromal cells were adherent, fibroblast like, large and flat, primary cell shape differences, the third generation of stable spindle; immunocytochemistry was used to identify the expression of CD45 was negative for 3.63%, higher expression of CD90 for 96.2%.
Conclusion: the growth of rat bone marrow stromal cells in vitro is good, and the purity of the third generation cells obtained is high.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

【相似文献】