【作者】 何丹；

【导师】 吕海芹；

【作者基本信息】 东南大学 ， 人体解剖学与组织胚胎学， 2015， 硕士

【摘要】 目的：1.建立大鼠脑缺血/再灌注模型,探讨碱性成纤维细胞生长因子(bFGF)预处理对脑缺血再灌注损伤(IR)的神经保护作用。2.探究bFGF预处理对脑缺血/再灌注损伤(IR)大鼠神经干细胞增殖、分化的影响,为临床早期应用bFGF预防和治疗缺血性脑血管病提供理论和实验依据。方法：90只健康雄性SD大鼠,随机分成假手术组、脑缺血/再灌注对照组、bFGF预处理组。在制备脑缺血/再灌注模型前向bFGF预处理组大鼠侧脑室注射含有50ug/ml bFGF的溶液5u1,向假手术组和缺血/再灌注对照组大鼠侧脑室注射等体积的无菌生理盐水,注射2小时后建立脑缺血/再灌注动物模型。分别于术后1天、3天、7天进行mNSS神经功能评分、TTC染色观察梗死灶面积；术后1天采用甲苯胺蓝染色和TUNEL染色技术观察缺血/再灌注区域神经细胞受损程度和凋亡情况；免疫荧光单标染色统计BrdU阳性细胞数目；免疫荧光双标染色观察BrdU/DCX、Brdu/NeuN和Brdu/GFAP阳性细胞的表达情况。结果：1.mNSS结果显示：假手术组动物未出现神经功能损伤症状。缺血组和bFGF预处理组动物均出现缺血侧Horner征及对侧神经功能缺损症状,且在术后第1天损伤最严重,术后第3天和第7天有自发恢复的趋势。与缺血组相比,bFGF预处理组大鼠在术后不同时间点的mNSS均明显减轻。2.TTC染色观察脑缺血再灌注损伤后的梗死体积,假手术组没有发现梗死灶,组织呈现鲜红色；缺血组和bFGF预处理组大鼠在所观察的3个时间点均出现明显的苍白色梗死灶,术后第1天梗死灶面积最大,随后有减小的趋势。与缺血组相比,bFGF预处理组大鼠在各个时间点的梗死灶面积均显著减小。3.甲苯胺蓝染色结果显示：假手术组未见神经元损伤；而缺血组的损伤侧大脑皮质、纹状体和海马区出现大面积的神经元坏死和凋亡,受损区神经细胞数量显著减少。与缺血组相比,bFGF预处理组也出现神经细胞核固缩和排列错乱现象,但神经细胞的损伤变性程度明显减轻,细胞丢失情况明显减少。4.TUNEL染色结果显示：术后1天,假手术组皮质和纹状体几乎无TUNEL阳性细胞；但缺血组和bFGF预处理组的缺血侧皮质和纹状体中均可见大量的TUNEL阳性细胞。与缺血组相比,bFGF预处理组TUNEL阳性细胞数量明显减少。5.BrdU免疫荧光染色统计各组大鼠缺血侧半球(左侧)SVZ区、纹状体和皮质新增殖细胞数目。结果显示假手术组仅在SVZ区有少量的BrdU阳性细胞,纹状体和皮质中未见BrdU阳性信号。缺血组在三个区域都有BrdU阳性信号,且显著高于对照组。与缺血组相比,bFGF预处理组大鼠缺血侧的SVZ区、纹状体和皮质中BrdU阳性细胞的数目均显著增加。6.免疫荧光双标染色结果：(1)在术后第7天,大部分BrdU阳性细胞同时表达DCX, BrdU/DCX阳性细胞数约占BrdU细胞总数的70%-80%。在缺血侧SVZ区,假手术的组BrdU/DCX阳性细胞数最少;缺血组BrdU/DCX阳性细胞数为假手术组1.9倍。与缺血组相比,bFGF预处理组BrdU/DCX阳性细胞数显著增多。在缺血侧纹状体和皮质中,假手术组未见BrdU/DCX阳性信号;缺血组和bFGF预处理组均发现BrdU/DCX阳性细胞,且bFGF预处理组明显多于缺血组。(2)缺血组和bFGF预处理组大鼠的缺血侧SVZ区、纹状体和皮质都可见BrdU/GFAP阳性信号表达,BrdU/GFAP阳性细胞数约占BrdU阳性细胞总数的3%-5%。而假手术组BrdU/GFAP阳性细胞只存在于SVZ区,其他区未发现。通过实验组间比较发现：bFGF预处理组的BrdU/GFAP阳性细胞数多于缺血组,但差别无统计学意义。(3)BrdU/NeuN免疫荧光染色结果显示：缺血侧BrdU/NeuN阳性细胞数约占BrdU阳性细胞总数的5%～8%。在SVZ区,各组大鼠均未发现BrdU/NeuN阳性信号；在纹状体和皮质区,缺血组和bFGF预处理组均有少量BrdU/NeuN阳性细胞,虽然bFGF组的BrdU/NeuN阳性细胞数多于缺血组,但差别无统计学意义。结论：bFGF预处理能通过抑制皮质和纹状体缺血半暗带细胞的凋亡,缩小脑梗死体积,减轻神经细胞损伤变性程度；促进内源性神经干细胞的增殖和幼稚神经元的产生,补充缺血灶中已经死亡的神经细胞,而发挥神经保护作用,对神经功能的恢复具有重要作用。更多还原

【Abstract】 Objective:l.To establish experimental cerebral ischemia/reperfusion model in rats and to explore the neuroprotective effect of basic fibroblast growth factor (bFGF) pretreatment on cerebral ischemia-reperfusion injury.2. To explore the influence of bFGF pretreatment on the proliferation and differentiation of endogenous neural stem cells in rats with cerebral ischemia/reperfusion injury, which can provide a theoretical and experimental basis for the early application of bFGF in treating ischemic cerebrovascular disease in clinical practice.Methods:90 healthy male Sprague-Dawley(SD) rats were randomly assigned into three groups:sham-operated group, ischemia group, bFGF pretreatment group. A dose of 5 ul bFGF (50 mg/ml in saline) was administered to the animals of bFGF pretreatment group by intraventricular injection and an equal volume of sterile saline was administered to the animals of sham-operated group and ischemia group by the same method.2 hours after the injection, ischemia-reperfusion injury was induced by 2 hours of unilateral middle cerebral artery occlusion (MCAO) in the rats. The loss of neurological function was assessed by the modified Neurological Severity Score (mNSS) and the infarct volume was determined by TTC staining at 1,3 and 7 days after MCAO.1 day after operation, Toluidine blue staining (TBS) was performed to study the gross histological changes and TUNEL staining was performed for the detection of cell apotosis. The number of BrdU positive cells was determined by immunofluorescent staining. And imrnunofluorescence double-labelled staining was performed for detection of the BrdU positive cells expressing DCX, NeuN and GFAP.Results:1. mNSS results indicate that the sham-operated group has no neurological damage. Horner syndrome on the ipsilateral side of injury and neurological deficit on the contralateral side of injury have been found in the animals of the ischemia group and the bFGF pretreatment group.The neurological function defects are the most serious at 1 day after MCAO, and the mNSS has a tendency to improve on days of 3 and 7 after operation. Compared with the ischemia group, the mNSS of the rats of the bFGF pretreatment group significantly decreases at different observation time points after operation.2. The results of TTC staining indicate that there was no infarction in the brain of the sham-operated animals and infarcts which appear pale in TTC staining were found in animals of both the ischemia group and the bFGF pretreatment group at all of the 3 observation time points. The infarct volume is the largest at 1 day after operation and it has a tendency to decrease with time going on. Compared with the ischemia group, the cerebral infarction volumes in rats of the bFGF pretreatment group significantly reduced at each observation time point.3. No neuron damage was found by Toluidine blue staining in the rats of the sham-operated group. In the rats of the ischemia group, extensive necrosis and apoptosis of the neurons within the cerebral cortex, striatum and hippocampus on the ipsilateral side of injuery were observed, and the number of neural cells significantly reduced in the ischemic area. Although karyopyknosis and disordered arrangement of nerve cells were found in the ischemic area, the extent of the neuronal damage and the loss of neurons significantly decreased in the brains of rats of the bFGF pretreatment group compared with that of the ischemia group.4. Few of TUNEL positive cells were seen in the brain of rats of the sham-operated group at 1 day after operation. However, a large number of TUNEL positive cells were seen in the cerebral cortex and striatum of the ipsilateral side of the injury in animals of the ischemia group and bFGF pretreatment group. The numbers of TUNEL positive cells in the animals of the bFGF pretreatment group were significantly higher than those in the animals of the ischemia group.5. Cell proliferation within the subventricular zone (SVZ) region, striatum and cortex of the hemisphere with ischemic/reperfusion injury (left) was determined by BrdU labelled immunofluorescence staining. In rats of the sham-operated group, only a small amount of BrdU positive cells were observed in the SVZ region, and no BrdU positive cells were found in the striatum and cerebral cortex. BrdU positive cells were observed in all of the three regions in the rats of the ischemia group, and the number of BrdU positive cells was significantly higher than that of the sham-operated group. In the bFGF pretreatment group, the number of BrdU positive cells significantly increased compared with the ischemia group.6. The results of immunofluorescence double-labelled staining indicate that (1) the majority of BrdU positive cells express DCX 7 days after operation, BrdU/DCX positive cells accounts for 70%-80% of the total BrdU positive cells.The number of BrdU/DCX positive cells are the least in the SVZ region of the sham-operated animals. The number of BrdU/DCX positive cells in the SVZ of rats of the ischemia group is of 1.9 times as high as that of the sham-operated group. The number of BrdU/DCX positive cells significantly increased in the SVZ regions of the bFGF pretreated rats compared with the ischemia group. No BrdU/DCX positive signals were detected in the striatum and cortex of the sham-operated animals. However, a large quantity of BrdU/DCX positive cells were seen inthe cortex and striatum of the ipsilateral side in rats of the ischemia and bFGF pretreatment groups.The numbers of BrdU/DCX positive cells in the bFGF pretreatment group were significantly higher than those in the ischemia group. (2) BrdU/GFAP positive cells account for 3%-5% of the total BrdU positive cells. BrdU/GFAP positive cells were found in the SVZ, striatum and cortex in animals of the ischemia group and bFGF pretreatment group but theywere only detected in the SVZ of the sham-operated rats. The number of the BrdU/GFAP positive cells in the bFGF pretreated rats were higher than that of the ischemia group, which is of no statiscally significant difference. (3) BrdU/NeuN positive cells account for 5%-8% of the total BrdU positive cells.No BrdU/NeuN positive signals were found in the SVZ of the rats of the three groups. A small amount of BrdU/NeuN positive cells were detected in the striatum and cortex of the rats of the ischemia and bFGF pretreatment groups. The number of BrdU/NeuN positive cells in the bFGF pretreated rats is higher than that in the rats of ischemia group but it hasno statistical significance.Conclusion:bFGF pretreatment plays an important role in neuroprotection on cerebral ischemia/reperfusion injury by reducing the cerebral infarct volume, alleviating the degeneration of nerve cells, inhibiting the apotosis of neurons within the ischemic penumbra of cerebral cortex and striatum. bFGF pretreatment also plays an important role in the recovery of neurological functions by promoting the proliferation of endogenous neural stem cells and the production of more immature neurons which can replace the dead nerve cells of ischemia region.更多还原