【作者】 陈柏龄；

【导师】 张志坚；

【作者基本信息】 福建医科大学 ， 神经病学， 2007， 博士

【摘要】 脑梗死是人类致死的三大疾病之一,其高致残性给患者、家庭和社会带来沉重的负担。长期以来,人们认为中枢神经损伤后引起神经细胞不可逆的死亡,损伤的神经元只能被胶质细胞所填充,形成胶质疤痕,从而丧失了神经功能。近年来,骨髓间充质干细胞(mesenchymal stem cells,MSCs)移植治疗脑梗死已成为研究热点。MSCs作为种子细胞,在体内可向多种急性损伤后的组织迁移并定位,在局部微环境的调控下分化为不同的组织细胞。MSCs为组织结构和功能修复提供了细胞来源,且易通过血-脑屏障,少有免疫排斥反应,已成为脑缺血性疾病治疗的理想靶细胞。血管生成素-1 ( Angiopoietin-1,Ang-1)是一类重要的血管生长因子,特异性地作用于内皮细胞,参与血管生成的调控过程,Ang-1通过干预内皮细胞、平滑肌细胞和周细胞之间的相互作用促进血管生成和重塑,在缺血等病理状态下可作为一种关键的血管生成调节剂。它可通过促进缺血组织周围侧支循环形成、稳定血管、延缓血管退化;加强内皮细胞间的紧密连结,维持血脑屏障的完整性;保护神经元、抑制凋亡等多重作用机制,从而明显增加缺血脑组织血流灌注,挽救半暗带神经元。Ang-1已成为缺血性脑血管病基因治疗新的靶点。慢病毒载体具有可感染分裂细胞及非分裂细胞、转移基因片段容量较大、目的基因表达时间长、不易诱发宿主免疫反应等优点,已成为当前基因治疗中载体研究的热点。因此,我们设想通过慢病毒载体的介导,将Ang-1基因转移至MSCs中,并将基因修饰的MSCs通过静脉移植至大脑中动脉栓塞模型大鼠中,利用MSCs可向急性损伤病灶趋化聚集的特点,期待MSCs能够携带Ang-1基因迁移至损伤部位,发挥Ang-1和MSCs的协同治疗作用。本课题分为三部分。第一部分:血管生成素-1基因重组慢病毒载体质粒的构建及鉴定从Genbank获得Ang-1基因序列,结合载体上的酶切位点需要,设计上下游引物,通过RT-PCR方法扩增目的基因片段,通过TA克隆筛选正确的重组体,再通过双酶切和基因重组构建Ang-1重组慢病毒载体质粒pNL-Ang-IRES2-EGFP,经抗生素筛选后,进行酶切鉴定和测序。结果显示我们获得长度为1512bp的Ang-1目的基因片段,质粒pNL-Ang-IRES2-EGFP经双酶切后凝胶电泳鉴定正确,测序结果与Genbank报道序列一致。第二部分:构建血管生成素-1基因修饰的骨髓间充质干细胞首先通过密度梯度离心和贴壁培养相结合的方法在体外提取、扩增培养大鼠骨髓间充质干细胞(rMSCs),并经流式细胞学检测各代CD29、CD34、CD45的表达情况。通过脂质体介导慢病毒三质粒pNL-Ang-IRES2-EGFP(或pNL-IRES2-EGFP,做为空载体对照)、pVSVG、pHELPER共转染293T细胞,收集病毒上清进行超速离心浓缩。PCR检测病毒基因组中目的基因Ang-1的插入。将病毒上清感染rMSCs,通过荧光显微镜观察目的基因的表达,细胞RT-PCR检测Ang-1的mRNA表达,细胞免疫化学和Western blot检测Ang-1蛋白的表达情况。然后通过鸡胚绒毛尿囊膜(chorioallantoic membrane,CAM)血管形成实验和内皮细胞条件培养检测所表达Ang-1蛋白的生物学活性。结果显示我们通过密度梯度离心和贴壁培养相结合的方法获得了比较纯的rMSCs,慢病毒感染后Ang-1可整合至rMSCs基因组中,并可正确转录和表达,所表达的Ang-1蛋白具有促进血管生长的生物学活性。第三部分:血管生成素-1基因修饰的骨髓间充质干细胞静脉移植对脑梗死的疗效及其迁移、分化的观察线栓法建立大鼠大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)模型,通过体征的观察和TTC染色验证模型的成功与否。然后通过股静脉移植基因修饰的rMSCs,通过modified Neurological Severity Scores(mNSS)评分法进行大鼠行为学观察,脑片HE染色观察组织学变化。利用载体自带的EGFP报告基因,在荧光显微镜下观察移植细胞的迁移和分布,并通过NF200、GFAP、NSE抗体的免疫荧光染色观察移植细胞在脑中的分化情况。结果显示,线栓法建立的大鼠MCAO模型稳定可靠,可很好地模拟人类急性脑缺血的病理生理变化。经rMSCs移植治疗的MCAO大鼠神经功能和组织学的恢复都好于对照组,而Ang-1和rMSCs联合治疗的MCAO大鼠神经功能和组织学观察则显示优于单纯rMSCs治疗组,证实了Ang-1和rMSCs的协同治疗作用。另外我们观察到移植的rMSCs可迁移定居至脑损伤部位,并且免疫荧光染色发现部分的rMSCs表达神经元的特异标志物NSE、NF、以及星形胶质细胞标志物GFAP,在移植3个月后仍可观察到此现象。以上结果说明了rMSCs可携带治疗基因至脑损伤部位,并长期存活,分化表达各种神经细胞标志物,很可能在脑缺血损伤后的恢复中发挥促进作用。更多还原

【Abstract】 Cerebral infarction is one of the three deadly diseases,the mutilation of which burdens the patients,family and society heavily. For a long time,it was considered that the central neurons would die nonreversibly after ischemic injury and be replaced by gliocytes which formed the glial scar,and then lost their function. In recent years, transplantation of mesenchymal stem cells(MSCs) as the therapeutic method of cerebral infarction has become the focus of research. MSCs can be used as seed cells migrating to a variety of acute injured tissues in vivo,then differentiate into different tissue cells under the control of local microenvironment. As the source of cells repairing the structure and function of ischemic focus,MSCs can easily pass the blood-brain barrier,migrate towards the ischemic focus,and rarely suffer the immunological rejection,become an ideal target cells in the treatment of ischemic diseases. Angiopoietin-1 (Ang-1) is an important vascular endothelial growth factor, which acts on endothelial cells specifically and participates in the regulation of angiogenesis. In the pathologic status of ischemia,Ang-1 is a key regulator of angiogenesis,which can promote angiogenesis and remodel vessels by interfering with the interaction among endothelial cells,smooth muscle cells and pericytes. Ang-1 can increase the perfusion of ischemic brain tissue significantly and rescue the neurons in penumbra by multiple mechanisms , such as promoting collateral circulation around the ischemic tissue , stabilizing vessels , delaying vascular degradation,strengthening the connection of endothelial cells,maintaining the integrity of the blood-brain barrier,protecting neurons and inhibiting apoptosis,and so on. Ang-1 has become a new target of gene therapy for ischemic cerebrovascular diseases. Lentiviral(LV) vector,has become the focus of vector study in gene therapy since it can infect separated and unseparated cells,transfer comparatively large gene fragment,express objective gene longer,rarely induce the host immune response and so on. Therefore,the idea that transfering the Ang-1 gene to MSCs mediated by LV vector,then injecting the gene-modified MSCs intravenously to rat suffering from middle cerebral artery occlusion(MCAO) will be a good design for the therapeutic study of cerebral infarction. Besides the characteristic migration of MSCs,the synergistic effects of MSCs and Ang-1 gene are expected.This study is consisted of three parts.Part 1:The construction and identification of lentiviral vector plasmid recombined by Ang-1 genePrimers were designed according to the Ang-1 gene sequences reported in Genbank and the restriction sites of the vector. The Ang-1 gene was obtained with RT-PCR. Established TA cloning. The recombination plasmid pNL-Ang-IRES2-EGFP was constructed with double digestion and gene recombinant,then identified with restriction endonuclease and sequencing after screening with antibiotics. The resulting 1512bp Ang-1 gene fragment was confirmed by sequencing and comparing with the sequence of the Ang-1 gene sequences reported in GenBank. The pNL-Ang-IRES2-EGFP was also identified to be correctPart 2: Construction of Ang-1 gene-modified mesenchymal stem cellsThe rat bone marrow-derived mesenchymal stem cells (rMSCs) were separated and cultured with the combination of density gradient centrifugation and adherent culture,and the expressions of CD29、CD34、CD45 in rMSCs were detected with flow cytometry. The constructed transfer plasmid pNL-Ang-IRES2-EGFP (or pNL-IRES2-EGFP,as mock group) with the packaging plasmid pHELPER and the envelop plasmid pVSVG were co-transfected into 293T cells. The supernatant was collected after transfection and was concentrated by centrifugation. Whether the Ang-1 gene was included in the replication-defective LV vector particles was identified by PCR using the genome extracted from the resulting concentrated LV vectors preparations as template. The rMSCs were infected by the LV vectors that were produced already and were identified with fluorescent microscope,RT-PCR,immunocytochemical staining,and western blot. The biologic activities of Ang-1 protein were tested with the chick chorioallantoic membrane(CAM) vasiformation experiment and the conditional culture of vascular endothelial cell. The result showed that the pure rMSCs were obtained with the combination of density gradient centrifugation and adherent culture,the Ang-1 gene was integrated into rMSCs genome,and transcribed and expressed correctly. Ang-1 protein expressed in constructed rMSCs had the biological activity of angiogenesis.Part 3:Treatment of cerebral infarction by transplantation of Ang-1 gene-modified mesenchymal stem cells and the observation on the migration and differentiation of transplanted cellsThe rat middle cerebral artery occlusion (MCAO) model was established, and the operation was validated by signs and tetrazolium chloride(TTC) stain. The gene-modified rMSCs were injected into the femoral vein, the ethology of animal models was assessed with modified Neurological Severity Scores (mNSS),the histological changes of brain slices were observed with HE stain. The migration and distribution of grafted rMSCs were observed under fluorescence microscope .The differentiation of grafted rMSCs were observed with immunohistology using the antibodies of anti- NF,anti-GFAP and anti-NSE as first antibodies. The results showed that the rat MCAO model was reliable,which can be used to simulate human pathophysiological changes in acute cerebral ischemia. The MCAO rats treated with Ang-1 gene-modified rMSCs recovered better in both function and brain histology than the control group,and than the rats treated only with rMSCs,this was the evidence for the synergistic effects of Ang-1 and rMSCs. It was also observed that the grafted rMSCs can migrate to the area of brain injury,and express the NSE and NF markers of neuron,and the GFAP marker of astrocyte until three months. This study proved that rMSCs carrying Ang-1 gene can migrate to the ischemic focus and possiblely differentiate into various kinds of neurocytes,which may promote the recovery of cerebral infarct greatly.更多还原