【作者】 王晓楠；

【导师】 张建宁； 刘丽； 江荣才；

【作者基本信息】 天津医科大学 ， 外科学， 2010， 硕士

【摘要】 [目的]内皮祖细胞(endothelial progenitor cells, EPCs)不仅维持正常脉管系统的生理功能,而且主导病理状态下的血管再生与修复。但目前对于颅脑创伤后的血管生成研究尚不多见。重组人粒细胞集落刺激因子(granulocyte colony stimulating factor, rhG-CSF)具有动员骨髓内皮祖细胞进入外周血,增殖分化为内皮细胞的前体细胞,参与损伤组织血管的修复和再生的作用。因此rhG-CSF在神经系统病方面的研究成为热门。本实验通过观察rhG-CSF对颅脑创伤(traumatic brain injury, TBI)后对神经功能的保护作用,以期探讨颅脑创伤治疗中的可能新途径。[材料与方法]1.健康成年雄性Wistar大鼠36只(体重300-350g,鼠龄7周),随机分为假手术(Sham)组、盐水(NS)组和粒细胞集落刺激因子(rhG-CSF)组,每组12只。TBI模型建立：在颅骨前囟后4.4mm,矢状缝侧方2.4mm钻直径4mm的骨孔,应用大鼠液压打击仪(Fluid-Percussion injury, FPI)以2.0～2.5 atm的打击力度致伤。Sham组只磨骨孔,不打击。rhG-CSF组在打击后3小时内,给予皮下注射rhG-CSF (50μg/kg),每天1次,连续注射5天。NS组于打击后给予等量生理盐水。各组分别于打击后6小时、1天、3天和6天取大鼠内眦球后静脉丛血液约0.5ml,采用Ficoll密度梯度离心法分离大鼠循环血单个核细胞,以CD34、CD133双抗体阳性作为EPCs标志,应用流式细胞仪(flow cytometer, FCM)测定大鼠循环血中EPCs数量。各组分别于TBI后1天、4天、7天、14天和21天进行改良神经功能评分(modified Neurological Severity Score, mNSS)。在TBI后21-25天进行Morris水迷宫(Morris Water Maze, MWM)试验。2.健康成年雄性Wistar大鼠56只,随机分为NS组和rhG-CSF组,每组28只。TBI后4天、7天、14天和21天,NS组和rhG-CSF组分别随机取7只大鼠,取脑行冰冻切片免疫组化,计数创伤区周围(Boundary Zone, BZ)和海马CA3区的CD31微血管(CD31-MVD)数量。[结果]1.大鼠脑创伤后6小时,循环血中CD34/CD133双阳性的EPCs明显超过正常水平,1天以后逐渐回落到正常水平。伤后皮下注射rhG-CSF,可以显著提高大鼠循环血中EPCs数量,随着用药时间的延长,EPCs数量逐渐增多。2. mNss检测发现,TBI后大鼠神经功能受到严重损害。经rhG-CSF治疗的大鼠,神经功能的改善明显优于NS组(P<0.05)。3.MWM检测发现,TBI后大鼠空间学习记忆功能障碍。经rhG-CSF治疗的大鼠,空间学习记忆功能明显优于NS组。伤后24、25天rhG-CSF组逃避潜伏期明显比NS组减少(P<0.05),同时象限百分率增高(P<0.05)。4.TBI后BZ及伤侧海马CA3区,NS组CD31-MVD于伤后7天达到峰值,随后有所下降；应用rhG-CSF后,伤后14、21天BZ及伤侧海马CA3区CD31-MVD明显高于NS组(P<0.05)。[结论]1.脑创伤后EPCs从骨髓动员入循环血中,rhG-CSF可以显著增加脑创伤后大鼠外周血EPCs的动员。2.脑创伤使大鼠的神经功能和空间学习记忆能力障碍,rhG-CSF可以明显促进神经功能恢复和改善空间学习记忆能力。3. rhG-CSF可以通过动员和促进EPCs的归巢,从而增强损伤脑组织的血管再生和促进神经功能的恢复。更多还原

【Abstract】 Objective:Endothelial progenitor cells (EPCs) maintain the physiologic functions of normal vascular system; they also play a pivotal role in vasculogenesis and angiogenesis in pathologic condition. However, there is rare study of EPCs related angiogenesis in traumatic brain injury (TBI). Granulocyte colony-stimulating factor (G-CSF) is well-established to mobilize EPCs which proliferate and differentiat precursor cells of endotheliocyte from bone marrow into the peripheral blood, participates repair and regeneration of injure tissues. G-CSF becomes popular in nervous system disease. To investigate the effect of G-CSF on the neurofunctional protection, we hope to find a new way to treat craniocerebral trauma.Material and Methods:1.36 of 300-350g and 7 week age adult male Wistar rats (from Academy of Military Medical of China) were randomly divided into three groups:Sham group (12), normal saline group (NS,12) and rhG-CSF group (12). rats were anesthetized with 10% chloral hydrate (0.3ml/kg) administered intraperitoneally and then placed in a stereotaxic frame. A 4.0 mm craniotomy was performed over the right parietal skull to expose the dura (4.4 mm posterior from bregma and 2.4 mm lateral to the sagittal suture). The pressure pulse of the fluid percussion device between 2.0 to 2.5 atm. Sham group grinds the skull but not to percuss. RhG-CSF group was administered G-CSF (50μg/kg; Chugai Pharmaceutical, Co., Japan) subcutaneously for 5 consecutive days after TBI. NS group mitte tales doses normal saline. The 0.5ml blood samples was collected from the rat eyes at 6 hr, 1day,3 day and 6 day after TBI. Blood samples were first subjected to a Ficoll gradient centrifugation to isolate mononuclear cells using a commercial kit. The isolated cells suspended in BSA buffer were incubated with a both PE-conjugated CD34 antibody and FITC-conjugated CD133 antibody for 10 minutes at room temperature. Then the staining cells were analyzed in a flow cytometry. The modified neurological severity score tests were carried out on preinjury and on days 1,4,7,14, and 21 after TBI. The Morris water maze test was tested on days 21-25 after TBI.2.56 of adult male Wistar rats were randomly divided into NS group and rhG-CSF group,28 rats in each group. CD31-MVD were measured by immunohistochemistry in boundary zone of lesion and the CA3 region of the hippocampus on days 4,7,14 and 21 after TBI. Random 7 rats were divided in each time piont.Results:1. There were double CD34/CD133 positive cells, which were defined as EPCs, in the circulating blood in rats. The numbers of circulating EPCs increased significantly at 6 hr after TBI, recurred to normal level after 1 day. After G-CSF mobilization, the numbers of circulating EPCs increased significantly and gradully increased with injection frequency.2. Injury in the hemisphere cortex of rats causes neurological functional deficits as measured by mNSS. The mNSS scores for the rhG-CSF group were significantly decreased at days 14 and 21 after TBI when compared with the NS group.3. The dysfunction of spatial learning were found by the Morris water maze test. the rhG-CSF group reduced the dysfunction of spatial learning caused by the brain damage in this model. The percentage of time spent in the correct quadrant was significantly higher and escape latency was significantly lower in the rhG-CSF group than the NS group during the water maze test.4. CD31-MVD was measured in boundary zone of lesion and the CA3 region of the hippocampus. The number of CD31-MVD was highest at 7 day in the NS group, significantly higher in the rhG-CSF group than the NS group at 7, 14, and 21 days after TBI.Conclusion:1. EPCs can be mobilized to the circulating blood from bone marrow after TBI. The numbers of circulating EPCs were increased significantly by G-CSF after TBI.2. Injury in the hemisphere cortex of rats causes neurological functional deficits and spatial learning disfunction. These disfunctions were recoveried by G-CSF.3. Circulating EPCs were mobilized and homing by G-CSF. The higher EPCs level conduces to augment more angiogenesis and functional recovery in the injured brain after TBI.更多还原