【作者】 范顺太；

【导师】 李平； 王金兰；

【作者基本信息】 郑州大学 ， 神经病学， 2010， 硕士

【摘要】 背景和目的脑出血(ICH)是一种威胁人类健康的急性疾病。许多研究认为：细胞凋亡参与了神经元的继发性损伤,细胞凋亡是脑出血后神经元损伤的重要因素之一。与非糖尿病患者相比；患糖尿病的脑出血患者在住院期间病情以及预后的状况都相对较差。高血糖可能会促进神经元的凋亡并且已经在缺血性脑血管病中得到证实,而目前这方面在脑出血中的研究相对较少。本实验通过建立高血糖大鼠脑出血动物模型,利用免疫组织化学和TUNEL方法,观察血肿周围脑组织Bax、Bcl-2的表达和神经细胞的凋亡；探讨高血糖对血肿周围脑组织Bax、Bcl-2表达和神经细胞凋亡的影响,为脑出血的临床治疗提供理论依据。材料与方法健康成年雄性SD大鼠96只,随机分为4.组：假手术组、正常血糖组、高血糖组以及胰岛素干预组。每组设4个时间点：6h、24h、72h、7d。通过腹腔注射链脲菌素(STZ)制作高血糖动物模型,血糖值大>11.1mmol/L,认为该模型制作成功。在立体定向仪下于右侧尾状核区注入未凝自体动脉血建立脑出血动物模型。组织切片的制备：成功模型在相应时间点灌注、取材,以注射针道为中心做冠状切片。指标检测：Bax、Bcl-2的检测是采用免疫组织化学染色。应用图像分析系统测其免疫染色阳性区平均积分光密度,取其平均值；神经细胞凋亡采用TUNEL法染色。凋亡指数(AI)=凋亡神经元/总神经元×100%。统计学方法：所有数据均用均数±标准差(x±s)表示,采用SPSS13.0软件对数据进行统计分析,多组均数间的比较采用单因素方差分析,均数间的两两比较采用LSD检验,Bax与TUNEL的相关性采用直线相关分析,检验水准a=0.05。结果1 Bax和Bcl-2表达的动态变化在假手术组纹状体Bax和Bcl-2有少量表达,从6h至7d变化相对较小。在正常血糖组、高血糖组以及胰岛素干预组血肿周围脑组织不同时间点均有Bax和Bcl-2的表达,且从6h逐渐增高,24h达峰,7d仍有表达。高血糖组大鼠脑血肿周围组织Bax和Bcl-2的表达在相应时间点明显高于其它组(P<0.05),胰岛素干预组上述指标在相应时间点均低于高血糖组(P<0.05),而高于假手术组,与脑出血组无明显差别(P>0.05)。2神经细胞凋亡的动态变化在假手术组纹状体仅见少量的凋亡细胞,从6h至7d变化相对较小。在正常血糖组、高血糖组以及胰岛素干预组血肿周围脑组织不同时间点均有凋亡细胞的表达,且从6h逐渐增高,24h达峰,7d仍有持续存在。高血糖组大鼠脑血肿周围组织凋亡细胞在相应时间点明显明显高于其它组(P<0.05),胰岛素干预组上述指标在相应时间点均低于高血糖组(P<0.05),而高于假手术组,与脑出血组无明显差别(P>0.05)。3血肿周围细胞凋亡与Bax表达的相关性结果高血糖组同一时间点的凋亡指数与Bax表达呈正相关性(r=0.80,P<0.05)。结论1.高血糖能促进大鼠脑血肿周围组织Bax、Bcl-2的表达以及神经元的凋亡。2.高血糖组的细胞凋亡与Bax表达呈正相关性。3.胰岛素适当控制血糖可以减少神经元的凋亡更多还原

【Abstract】 Background and ObjectiveIntracerebral hemorrhage (ICH) is an emergent disease. At present, there are many research results reveal that cell apoptosis participates in the secondary injury of neurocytes. So cell apoptosis is an important factor of neurocytes injury of intracerebral hemorrhage. Compared with non-diabetics, diabetics have a worse hospital and long-term outcome after intracerebral hemorrhage. Hyperglycemia may promote the apoptotosis of neurons, which has been verified in the ischemic cerebrovascular disease. At present, it was researched rarely in the ICH.In the study, using immunohistochemistry and TUNEL methods, the neuron apoptosis and the expression of Bax and Bcl-2 were explored in rat ICH model and hyperglycemia model, which may provide strategies for clinical treatment on cerebral hemorrhage.Materials and methodsNinety-six healthy adult male SD rats were randomly divided into 4 groups: sham group, normal plasma glucose group, hyperglycemia group and insulin intervention group. Each group was divided into 4 various stages:6h,24h,3d,7d.Hyperglycemia model was induced by the injection of streptozotocin (STZ) into abdominal cavity by injection syringe. The model was successful until blood glucose was higher thanl1.1mmol/L.ICH model was induced by the injection of autologous unaggregated artery blood into the right basal ganglia of rats by stereotaxic apparatus.Preparation of the tissue section:The successful models were perfused and took tissue at the according time. The tissue was taken coronary sections before and behind the pin hole.Examine parameter:Bax and Bcl-2 were studied with immunohistochemistry. Positive area average integral optical density value was measured with image analysis system, its mean value was average integral photodensity. Nerve cell apoptosis was measured by terminal-deoxynucleotidyltransferase mediated nick end label (TUNEL) method. Apoptotic index (AI)=apoptotic neurons/total neurons×100 %.Statistic analysis:All data were expressed as mean±SD, and analyzed with the SPSS13.0 software, the means of the samples were compared using one-way analysis of variance followed by Least Significant Difference (LSD) for multiple comparisions. Correlation between Bax and TUNEL was judged by linear regression. The significant difference was judged by a=0.05.Results1. The dynamic changes of Bax and Bcl-2The expression of Bax and Bcl-2 in the striatum of sham group rats was low and changed a little from 6h to 7d. The expression of Bax and Bcl-2 in the perihematomal areas at different time points after ICH in ICH group, hyperglycemia group and insulin intervention group began at 6h and increased gradually, ultimate in 24h, still can be detected at 7d. The expression of Bax and Bcl-2 in the hyperglycemia group was significantly higher than that in the other groups at the same time point (P<0.05), the above-mentioned index of insulin intervention group were lower than that in hyperglycemia group and higher than that in the sham group at the same time point (P<0.05), and had no obvious difference with that in the normal glucose group at the same time point (P>0.05).2. The dynamic changes of neuron apoptosisApoptotic neurons were few in the striatum of of sham group rats, and AI had small change from 6h to 7d. AI in the perihematomal areas at different time points after ICH in ICH group, hyperglycemia group and insulin intervention group began at 6h and increased gradually, ultimate in 24h, still can be detected at 7d. AI in the hyperglycemia group was significantly higher than that in the other groups at the same time point (P<0.05), the above-mentioned index of insulin intervention group were lower than that in hyperglycemia group and higher than that in the sham group at the same time point (P<0.05), and had no obvious difference with that in the normal glucose group at the same time point (P>0.05).3. The result of the relationship analysis of apoptotic and expression of BaxAI positively correlates with the expression of Bax in hyperglycemia group at the same time point(r=0.80, P<0.05).Conclusion1. Hyperglycemia increases the apoptotic neurons and stimulates the expression of Bax and Bcl-2 in perifocal tissue of intracerebral hemorrhage in rats.2. Apoptotic index positively correlates with the expression of Bax in hyperglycemia group at the same time point.3. Control of blood glucose level with insulin can reduce neuron apoptosis.更多还原