【作者】 王娟；

【导师】 刘亢丁；

【作者基本信息】 吉林大学 ， 神经病学， 2016， 博士

【摘要】 背景：在中国,每年有约700万脑血病患者,近70%为脑梗死。EPCs既具有干细胞特性,也具有ECs特性,在缺血性疾病中,参与血管生成。EPCs通过参与血管网络结构的重建和生长因子或细胞因子的旁分泌,刺激局部ECs迁移、生长和功能实现,增强脑缺血后神经修复。VEGF在缺血组织中,不仅促进血管新生血管生成,还趋化循环内皮前体细胞归巢到缺血性病灶,并且原位分化血管发生。在MCAO大鼠模型中,EPCs通过趋化因子基质细胞衍生因子1(SDF-1)以及其受体C-X-C趋化因子受体4型(CXCR4)组成的生物轴,参与神经血管新生。组织氧含量降低,HIF-1α上调表达,提供细胞保护和生血管作用。目的：VEGF基因修饰的EPCs治疗脑缺血模型大鼠,探索EPCs和VEGF参与脑缺血的血管新生的效果和机制。方法：通过RT-PCR方法从雄性Wistar大鼠血液的单个核细胞中获取VEGF164,构建pLVX-VEGF164-IRES-ZsGreenl,进行酶切、测序鉴定,及mRNA和蛋白水平上的功能鉴定。采用梯度分离法从雄性Wistar大鼠的骨髓中分离并体外培养EPCs；观察EPCs细胞形态学和免疫荧光染色标记CD133、CD34和PKDR。将携带VEGF的慢病毒载体感染EPCs,获得VEGF基因修饰的EPCs (EPCs-VEGF)。立体定位移植PBS、EPCs和EPCs-VEGF到MCAO模型大鼠的侧脑室。通过mNSS、体重指数等神经功能评估,免疫组织化学染,免疫荧光双染,Tunel染色和Vestern blotting方法分析,比较第1,3,7,14天PBS治疗组和EPCs治疗组的病理变化和治疗效果,并探讨和分析MCAO模型建立第7天时,PBS、EPCs和EPCs-VEGF治疗组中HIF-1α, VEGF, MMP-9, CD34, SDF-1和CXCR4的蛋白表达情况结果：(1)构建了pLVX-VEGF164-IRES-ZsGreenl,在293T细胞中成功包装了携带VEGF164的慢病毒载体,并在mRNA和蛋白水平稳定表达。(2)采用梯度离心,体外培养EPCs,免疫荧光标记CD133, CD34和KDR阳性表达鉴定EPCs。(3)携带VEGF164的慢病毒载体感染EPCs,感染后的EPCs (EPCs-VEGF)绿色荧光表达量70%以上,Western blotting结果EPCs-VEGF的VEGF蛋白表达上调,与EPCs具有统计学差异(P<0.05)。(4)PBS和EPCs立体定位注射到MCAO大鼠的侧脑室,两组大鼠在第7天mNSS、体重比有显著差异(P<0.01)。免疫组织化学和Western blotting结果显示VEGF在第7天表达量达到高峰,CD34逐渐升高,半暗带区的细胞凋亡随时间延长而改善,而HIF-la在缺血早期高表达,两组间在第3,7,14天存在统计学差异(P<0.05)。(5)PBS、EPCs和EPCs-VEGF立体定位注射到MCAO大鼠的侧脑室,第7天的mNSS,体重比,各组间有显著差异(P<0.05)。免疫组织化学和Western blotting结果显示VEGF, HIF-1α, CD34, SDF-1和CXCR-4在EPCs-VEGF治疗组表达量最最高,MMP-9在PBS组表达量最大,存在统计学差异(P<0.05)。结论：(1)构建了pLVX-VEGF164-IRES-ZsGreen1质粒,并成功包装成携带VEGF的慢病毒载体,慢病毒在RNA和蛋白水平有效表达VEGF。(2)获得骨髓来源的EPCs,并对EPCs进行了鉴定,获得了符合实验要求细胞。(3)成功构建了过表达VEGF164基因的EPCs,通过MCAO模型鼠的治疗,验证了EPCs和VEGF通过HIF-VEGF和SDF-1/CXCR4信号途径,参与大鼠脑梗死的血管新生过程,改善了神经功能,缩小梗死灶体积,促进功能恢复。更多还原

【Abstract】 Background:In China, there are about 7 million patients with cerebrovascular disease each year, nearly 70% for cerebral ischemia. EPCs have characteristics of stem cells and endothelial cells, which are involved in angiogenesis of ischemic diseases. EPCs participate in the reconstruction of vascular network and secrete growth factors or cytokines, stimulate endothelial cells migration, growth and function to enhance nerve regeneration after cerebral ischemia.In ischemic tissue, VEGF promote angiogenesis, to stimulate endothelial progenitor cells homing the ischemic injure in circulating blood.In MCAO rat models, EPCs take part in angiogenesis by the biological axis of chemokine stromal cell-derived factor-1 (SDF-1) and C-X-C and its receptor Chemokine receptor type 4 (CXCR4). With the hypoxia, HIF-la increases to provide the protection of cells and angiogenesis.Objectives:To explore the effects and mechanisms of EPCs and VEGF to promot angiogenesis in cerebral ischemia via EPCs modified by lentiviral vector containing VEGF.Methods:VEGF 164 was obtained by RT-PCR in blood mononuclear cells from male Wistar rats. pLVX-VEGF164-IRES-ZsGreenl was constructed and identified by restriction enzyme digestion analysis, RT-PCR, the sequencing and western blotting, which could achieve the functionality at mRNA and protein levels. EPCs were separated from the bone marrow in male Wistar rats by gradient separation and cultured to determine EPCs by surface markers CD133, CD34, and KDR in immunofluorescence staining. EPCs modified by Lentiviral vectors containing VEGF (EPCs-VEGF) were obtained. PBS, EPCs and EPCs-VEGF were transplantated into lateral ventricle of MCAO rats model. Through neurological assessment of the mNSS, body mass index, immunohistochemical staining, immunofluorescence double staining, Tunel staining and Western blotting analysis, the indexes of EPCs treatment group were different from which of PBS treatment group on the 3rd,7th,14th days in the pathology and therapeutic effect of MCAO rats model, and the protein expression of HIF-1α, VEGF, MMP-9, CD34, SDF-1 and CXCR4 were analysed and explored in the 7th day of MCAO models among the PBS, and EPCs and EPCs-VEGF treatment groups for cerebral infarction.Results:(1) pLVX-VEGF164-IRES-ZsGreenl was built and packaged successfully in the 293T cells. Lentiviral vector with VEGF 164 could express VEGF stablely at mRNA and protein levels. (2) Using density gradient centrifugation and EGM-2, EPCs were cultured and marked by immunofluorescence identification of CD133,CD34 and KDR. (3) Lentiviral vectors with VEGF 164 infected EPCs. Then,70% of EPCs modified by Lentiviral vectors containing VEGF (EPCs-VEGF) expressed green fluorescent protein, and EPCs-VEGF made VEGF expressed upregulatedly in western blotting (P<0.05). (4) PBS and EPCs were injected into the lateral ventricle of MCAO rats, and mNSS and the weight ratio were different significantly between the two groups in the 7th day (P<0.01). There were results of immunohistochemistry and western blotting showed that VEGF protein expressed in the 7 day at peaked, CD34 gradually increased, apoptosis of penumbra improved, and HIF-la in early ischemic expressed highly, (P<0.05). (5) PBS, EPCs and EPCs-VEGF were injected into lateral ventricles in MCAO rats, and the 7th day of mNSS and body weight ratio among the groups had significant difference (P< 0.05). In immunohistochemistry and western blotting, VEGF, HIF-1, CD34, SDF-1 and CXCR-4 in the EPCs-VEGF treatment group had the highest expression. MMP-9 in the PBS group was the highest (P<0.05).Conclusions:(1) The pLVX-VEGF164-IRES-ZsGreenl plasmid was constructed and packaged successfully. The lentiviral vector carrying VEGF infected cells to express VEGF at RNA and protein level. (2) Bone marrow derived EPCs were obtained and identified, satisfying with the requirements of the experimental cells. (3) EPCs modified by Lentiviral vectors containing VEGF improved the ischemia of MCAO rat models to by HIF-VEGF and SDF-1/CXCR4 signaling pathways to involve in angiogenesis, reduce cerebral infarction volume, and promote nerve functional recovery.更多还原