【作者】 袁霞；

【导师】 张世明；

【作者基本信息】 苏州大学 ， 神经外科， 2010， 博士

【摘要】 脑血管病是严重危害人类健康的一组疾病,致死率和致残率高,给社会造成巨大的负担。在世界范围内,脑血管病是引起死亡的第二位原因,在我国各城市卒中死亡人数已跃居各种死因的首位。因此,脑血管病的基础和临床研究始终是医学研究的主要课题。以往认为成熟神经系统的神经细胞是终极细胞,受损后不能再生,而目前越来越多的证据表明脑有自身修复功能,成年脑室管膜下区(subventricular zone ,SVZ)、海马齿状回颗粒下区(subgranular zone,SGZ)等区域存在具有自我更新和多种分化潜能的神经干细胞。脑缺血可诱导内源性干细胞的增殖、分化,但因数量少,由此而诱导的自身修复能力有限。神经干细胞移植虽然为缺血性损伤的治疗带来生机,但伦理、来源、移植损伤和排斥反应等因素限制了其临床应用。骨髓干细胞动员是新兴的细胞移植方法,利用细胞因子使主要位于骨髓中的干细胞进入外周血,通过血液循环到达损伤组织以达到细胞移植的目的。粒细胞集落刺激因子(granulocyte colony-stimulating factor,G-CSF)是骨髓干细胞强有力的动员剂,可以大大提高外周血干细胞数量,且可向脑缺血部位迁移,在脑缺血的特定病理环境中向神经细胞分化,以修复受损脑组织,并且还有抗炎症和抗凋亡,从而起到神经保护作用。第一部分粒细胞集落刺激因子在大鼠局灶性脑缺血中的抗凋亡作用目的探讨重组人粒细胞集落刺激因子(rhG-CSF)在大鼠局灶性脑缺血中的抗凋亡作用。方法用线栓法建立大鼠大脑中动脉缺血/再灌注(MCAO/R)模型,观察rhG-CSF对神经功能缺损评分及梗死体积的影响,用半定量逆转录-聚合酶链式反应(RT-PCR)方法测定脑缺血区半胱天冬酶-3(Caspase-3)mRNA的表达,并用流式细胞仪检测缺血区神经细胞凋亡率。结果与对照组相比,rhG-CSF治疗组大鼠的神经功能缺损评分明显降低,梗死体积缩小,治疗组脑缺血区Caspase-3 mRNA表达降低,凋亡率下降。结论rhG-CSF可缩小梗死体积,降低Caspase-3 mRNA表达,并降低神经细胞凋亡率,rhG-CSF在缺血/再灌注损伤中有抗凋亡作用。第二部分粒细胞集落刺激因子在大鼠局灶性脑缺血中的抗炎症作用目的探讨重组人粒细胞集落刺激因子(rhG-CSF)在大鼠局灶性脑缺血中的抗炎症作用。方法用线栓法建立大鼠大脑中动脉缺血/再灌注(MCAO/R)模型,用免疫组织化学方法测定脑缺血区及周边髓过氧化物酶(MPO)的表达,并用半定量逆转录-聚合酶链式反应(RT-PCR)方法测定脑缺血区白介素-1β(IL-1β)mRNA和肿瘤坏死因子-α(TNF-α)mRNA的表达。结果与对照组相比,rhG-CSF治疗组缺血区MPO的表达与对照组没有区别,而治疗组脑缺血区IL-1βmRNA和TNF-αmRNA的表达明显降低。结论rhG-CSF可降低IL-1βmRNA和TNF-αmRNA表达,在缺血/再灌注损伤中有抗炎症作用。第三部分粒细胞集落刺激因子在大鼠局灶性脑缺血中促进神经细胞分化和血管生成作用目的探讨重组人粒细胞集落刺激因子(rhG-CSF)在大鼠局灶性脑缺血中促进神经细胞分化和血管生成作用。方法用线栓法建立大鼠大脑中动脉缺血/再灌注(MCAO/R)模型,于再灌注不同时间点对治疗组和对照组大鼠进行神经功能缺损评分,并用免疫组织化学方法测定脑缺血区及周边巢蛋白(Nestin)、5-溴脱氧尿核苷(Brdu)、胶质纤维酸性蛋白(GFAP)、神经元特异性核蛋白(NeuN)和血管性血友病因子(vWF)的表达。结果与对照组相比,rhG-CSF治疗组大鼠的神经功能缺损评分明显降低,治疗组缺血区及周边的Nestin、Brdu、GFAP、NeuN、vWF表达较对照组增多。结论脑缺血/再灌注后,rhG-CSF可促进神经细胞分化和血管生成,增加神经细胞修复,具有神经保护作用。更多还原

【Abstract】 Cerebrovascular diseases are a sort of diseases that harm humans health seriously.They have high mortality and disability and bring huge load to society. Cerebrovascular disease is the second cause of death worldwide.In our country,the number of Cerebrovascular disease is the first cause of death every city.However,no effective treatment currently exists.Thus,the basic and clinical study on Cerebrovascular disease are the main task of medical study.It was considered before that the neurous of adult was final stage and could not regenerate again when injuried.But more and more evidence indicate that the brain has the function of self-repairing.In adult brain,there have nerve stem cells(NSCs) in Subventricular zone(SVZ) and dentate gyrus hippocampus Subgranular zone(SGZ).These NSCs have the potentialty of self-renewing and multidifferentiation.Cerebral ischemia can lead endogenous neurogenesis,but the amount is few.Its capacity of self-repairing is limited.Although NSCs transplantation bring us hope to treat ischemic injury,but a lot of factor such as ethics, origin,transplantation injury,reject reaction limited its clinical apply.Mobilize bone marrow stem cells is a new method of cell transplantation,the stem cells which in bone marrow enter into peripheral blood making use of cell factor and arrive injuried organization.Granulocyte Colony-Stimulating Factor(G-CSF) is a strong mobilization reagent to bone marrow stem cells.It can increase the amount of stem cells in peripheral blood ,which move to cerebral ischemia area,differentiate to nerve cells in cerebral iechemia situation,repair injuried brain and protect nerve cells from damage. PartⅠAntiapoptosis effects of recombinant human granulocyte colony-stimulating factor in focal cerebral ischemia ratsObjective To study Recombinant Human Granulocyte Colony-Stimulating Factor(rhG-CSF) has antiapoptosis effects in focal cerebral ischemia Rats.Methods A model of focal cerebral ischemia /reperfusion in rats was performed with the intraluminal filament occlusion.To investigate the effects of rhG-CSF on neurological severity score and infarct volumes. Reverse transcriptional polymerase chain reaction(RT-PCR) was used to detect Caspase-3 mRNA expression in cerebral ischemia zone.Flow cytometry(FCM) was used to detect the rate of apoptosis in cerebral ischemia zone.Results Compare with contrast group, rhG-CSF significantly reduced the neurological severity scores and infarct volumes. rhG-CSF decreased the expression of Caspase-3 mRNA and the rate of apoptosis in cerebral ischemia zone.Conclusion rhG-CSF may reduced infarct volumes , decreased Caspase-3 mRNA expression and the rate of apoptosis in cerebral ischemia zone, rhG-CSF has antiapoptsis effects in focal cerebral ischemia/reperfusion injury.PartⅡAntiinflammatory effects of recombinant human granulocyte colony-stimulating factor in focal cerebral ischemia ratsObjective To study Recombinant Human Granulocyte Colony-Stimulating Factor(rhG-CSF) has antiinflammatory effects in focal cerebral ischemia Rats.Methods A model of focal cerebral ischemia /reperfusion in rats was performed with the intraluminal filament occlusion. Using immunohistochemistry to measure the expression of myeloperoxidase(MPO).Reverse transcriptional polymerase chain reaction(RT-PCR) was used to detect IL-1βmRNA和TNF-αmRNA expression in cerebral ischemia zone.Results Compare with contrast group, the expression of MPO was no deference between two group.But rhG-CSF decreased the expression of IL-1βmRNA和TNF-αmRNA .Conclusion rhG-CSF may decrease IL-1βmRNA和TNF-αmRNA expression and has antiinflammatory effects in focal cerebral ischemia/reperfusion injury.PartⅢInducing neuronal differentiation and angiogenesis effects of recombinant human granulocyte colony-stimulating factor in focal cerebral ischemia ratsObjective To investigate Recombinant Human Granulocyte Colony-Stimulating Factor(rhG-CSF) induce neuronal differentiation and angiogenesis in focal cerebral ischemia Rats.Methods A model of focal cerebral ischemia /reperfusion in rats was performed with the intraluminal filament occlusion.To evaluate neurological severity score of rhG-CSF treated group and contrast group in different reperfusion time,and using immunohistochemistry to measure the expression of neuroepithelial stem protein(Nestin),5-bromodeoxyuridine(Brdu),glial fibrilary acidic protein(GFAP),Neuronal Nuclei(NeuN),von Willebrand factor(vWF).Results Compare with contrast group, rhG-CSF significantly reduced the neurological severity scores and increased the expression of Nestin,Brdu, GFAP, NeuN, vWF in peri-infarcted zones. Conclusion the rhG-CSF treatment can induce neuronal differentiation and angiogenesis, can enhance the repairment of neuronal cells and have neuroprotective effects.更多还原