Cdk5在大鼠蛛网膜下腔出血后早期脑损伤中的表达及作用研究
发布时间：2018-09-17 17:11
【摘要】：背景和目的：细胞周期蛋白依赖性激酶5(Cyclin-dependent kinase,Cdk5)是Cdk家族中的一员,并被视为调节神经细胞生存与死亡的关键因子。尽管许多文献已经证明Cdk5参与了多种神经系统疾病,如阿尔茨海默病、帕金森病和缺血性脑卒中等,但Cdk5在蛛网膜下腔出血(SAH)后早期脑损伤(EBI)中的作用仍不明确。本实验的目的是研究Cdk5在大鼠SAH后大脑皮质的表达及细胞分布,探讨其在SAH后EBI中的作用,为SAM的治疗提供新的思路。实验方法：1.Cdk5在大鼠蛛网膜下腔出血后大脑皮质中的表达将雄性SD大鼠52只随机分为假手术组(n=12)和SAH组(下设SAH后6h、12h、24h、2d、3d组,每组8只)。通过向大鼠视交叉前池注射自体血来制备大鼠SAM模型。应用western blot、免疫组化法测定大鼠脑皮质Cdk5,p25和Cdk5-pTyr15的表达变化。免疫荧光染色方法检测Cdk5蛋白在脑组织中的细胞分布情况。2.Cdk5抑制剂roscovitine对SAH大鼠的神经保护作用研究将雄性SD大鼠随机分为4组：假手术+二甲基亚砜(dimethyl sulf oxide, DMSO),向视交叉前池注射0.9%生理盐水,并接受20%DMSO作为溶剂。SAH+DMSO组,SAH后接受相同剂量20% DMSO. SAH+Roscovitine(50μg)组,SAH后侧脑室注射50μg roscovitine。SAH+Roscovitine (100 μg)组SAH后侧脑室注射100 μ groscovitine。测量脑组织含水量评价脑水肿程度,TUNEL染色法检测脑组织凋亡情况。应用Garcia神经功能评分法评价SAH大鼠神经功能障碍。结果：1.western blot及免疫组化结果显示：假手术组Cdk5和Cdk5-pTyr15表达量皆较低。SAH后Cdk5和Cdk5-pTyr15蛋白表达逐渐升高,Cdk5蛋白在12h明显升高,1d达高峰。Cdk5-pTyr15蛋白在6h明显升高,12h达高峰。p25蛋白的表达趋势与Cdk5和Cdk5-pTyr15相似,SAH也逐渐升高,1 d达到高峰。2.免疫荧光结果显示：Cdk5在假手术组与SAH后1d的神经元中皆有表达,在sham组,Cdk5主要表达于神经元细胞浆,而SAH组中Cdk5向神经元细胞核中移位；同时,Cdk5在sham组及SAH组中与GFAP也存在共定位。3.与假手术组相比,SAH后大鼠脑组织水肿明显加重,神经功能障碍明显。实验中应用Cdk5抑制剂roscovitine可明显减轻脑组织水肿,改善神经功能障碍。4.尼氏染色发现SAH后1 d脑皮质神经元损伤相比假手术组显著增加,而在SAH+Roscovitine(100μg)组,受损神经元比例减少。5.TUNEL染色结果显示,SAH后1d大鼠脑皮质凋亡细胞较假手术组明显增加,应用roscovitine后可显著减少凋亡细胞比例。结论：SAH可上调Cdk5,p25和Cdk5-pTyr15的蛋白表达,提示Cdk5在SAH后的脑组织中被激活。应用Cdk5抑制剂可明显减轻SAH大鼠脑组织水肿,减少神经细胞凋亡比例,改善神经功能障碍。以上结果都提示Cdk5在SAH后EBI发病机制中起到重要作用,Cdk5有可能成为EBI的新的治疗靶点,为SAH后EBI的治疗提供新的思路。
[Abstract]:Background and objective: cyclin dependent kinase 5 (Cyclin-dependent kinase,Cdk5) is a member of the Cdk family and is considered to be a key factor in regulating the survival and death of nerve cells. Although many literatures have shown that Cdk5 is involved in many neurological diseases, such as Alzheimer's disease, Parkinson's disease and ischemic stroke, the role of Cdk5 in early brain injury (EBI) after (SAH) is still unclear. The purpose of this study was to study the expression and cell distribution of Cdk5 in rat cerebral cortex after SAH, to explore the role of Cdk5 in EBI after SAH, and to provide a new idea for the treatment of SAM. Methods: 1. Expression of Cdk5 in cerebral cortex of rats after subarachnoid hemorrhage 52 male SD rats were randomly divided into two groups: sham operation group (n = 12) and SAH group (n = 8 in each group). The rat SAM model was established by injecting autologous blood into the anterior cistern of optic chiasma. The expression of Cdk5,p25 and Cdk5-pTyr15 in rat brain cortex was determined by western blot, immunohistochemical method. Distribution of Cdk5 protein in brain tissue by immunofluorescence staining. 2.The neuroprotective effect of CDK5 inhibitor roscovitine on SAH rats; male SD rats were randomly divided into 4 groups: sham operation dimethyl sulfoxide (dimethyl sulf oxide, DMSO), direction 0.9% saline was injected into the anterior cistern of optic chiasma. 20%DMSO was used as solvent. SAH DMSO group received the same dose of 20% DMSO.. SAH Roscovitine (50 渭 g group: posterior ventricular injection of 50 渭 g roscovitine.SAH Roscovitine (100 渭 g) SAH posterior ventricular injection of 100 渭 groscovitine. The degree of brain edema was evaluated by measuring the water content of brain tissue and Tunel staining was used to detect the apoptosis of brain tissue. The neurological dysfunction of SAH rats was evaluated by Garcia score. Results 1. The results of western blot and immunohistochemistry showed that the expression of Cdk5 and Cdk5-pTyr15 in sham-operated group was lower. The expression of Cdk5 and Cdk5-pTyr15 protein increased gradually after sham-operation. The expression of Cdk5 and Cdk5-pTyr15 protein increased significantly at 12 h, and reached the peak at 1 day. CDK5-pTyr15 protein increased significantly at 6 h and reached the peak at 12 h. P25 protein. Similar to Cdk5 and Cdk5-pTyr15, SAH expression increased gradually and reached its peak at 1 day. The results of immunofluorescence showed that the expression of CDK5 was found in the neurons of sham-operated group and 1 day after SAH. In sham group, the expression of CDk5 was mainly in the cytoplasm of neurons, while in SAH group, Cdk5 was translocated to the nucleus of neurons. At the same time, there was colocalization of CDK5 with GFAP in sham group and SAH group. Compared with sham operation group, brain edema and neurological dysfunction were significantly aggravated after SAH. In the experiment, the application of Cdk5 inhibitor roscovitine can significantly reduce brain edema and improve neurologic dysfunction. 4. Nissl staining showed that the number of injured neurons in the SAH group was significantly higher than that in the sham operation group, and the proportion of injured neurons in the SAH Roscovitine (100 渭 g group was decreased. The results of Tunel staining showed that the apoptotic cells in the cortex of the rats on the 1st day after SAH were significantly higher than those in the sham operation group. The proportion of apoptotic cells was significantly decreased after roscovitine application. Conclusion the protein expression of Cdk5,p25 and Cdk5-pTyr15 can be up-regulated by Cdk5, suggesting that Cdk5 is activated in the brain tissue after SAH. The application of Cdk5 inhibitor can obviously reduce the brain edema, reduce the proportion of neuronal apoptosis and improve the neurological dysfunction in SAH rats. These results suggest that Cdk5 may play an important role in the pathogenesis of EBI after SAH. CDK5 may become a new therapeutic target for EBI and provide new ideas for the treatment of EBI after SAH.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.35
[Abstract]:Background and objective: cyclin dependent kinase 5 (Cyclin-dependent kinase,Cdk5) is a member of the Cdk family and is considered to be a key factor in regulating the survival and death of nerve cells. Although many literatures have shown that Cdk5 is involved in many neurological diseases, such as Alzheimer's disease, Parkinson's disease and ischemic stroke, the role of Cdk5 in early brain injury (EBI) after (SAH) is still unclear. The purpose of this study was to study the expression and cell distribution of Cdk5 in rat cerebral cortex after SAH, to explore the role of Cdk5 in EBI after SAH, and to provide a new idea for the treatment of SAM. Methods: 1. Expression of Cdk5 in cerebral cortex of rats after subarachnoid hemorrhage 52 male SD rats were randomly divided into two groups: sham operation group (n = 12) and SAH group (n = 8 in each group). The rat SAM model was established by injecting autologous blood into the anterior cistern of optic chiasma. The expression of Cdk5,p25 and Cdk5-pTyr15 in rat brain cortex was determined by western blot, immunohistochemical method. Distribution of Cdk5 protein in brain tissue by immunofluorescence staining. 2.The neuroprotective effect of CDK5 inhibitor roscovitine on SAH rats; male SD rats were randomly divided into 4 groups: sham operation dimethyl sulfoxide (dimethyl sulf oxide, DMSO), direction 0.9% saline was injected into the anterior cistern of optic chiasma. 20%DMSO was used as solvent. SAH DMSO group received the same dose of 20% DMSO.. SAH Roscovitine (50 渭 g group: posterior ventricular injection of 50 渭 g roscovitine.SAH Roscovitine (100 渭 g) SAH posterior ventricular injection of 100 渭 groscovitine. The degree of brain edema was evaluated by measuring the water content of brain tissue and Tunel staining was used to detect the apoptosis of brain tissue. The neurological dysfunction of SAH rats was evaluated by Garcia score. Results 1. The results of western blot and immunohistochemistry showed that the expression of Cdk5 and Cdk5-pTyr15 in sham-operated group was lower. The expression of Cdk5 and Cdk5-pTyr15 protein increased gradually after sham-operation. The expression of Cdk5 and Cdk5-pTyr15 protein increased significantly at 12 h, and reached the peak at 1 day. CDK5-pTyr15 protein increased significantly at 6 h and reached the peak at 12 h. P25 protein. Similar to Cdk5 and Cdk5-pTyr15, SAH expression increased gradually and reached its peak at 1 day. The results of immunofluorescence showed that the expression of CDK5 was found in the neurons of sham-operated group and 1 day after SAH. In sham group, the expression of CDk5 was mainly in the cytoplasm of neurons, while in SAH group, Cdk5 was translocated to the nucleus of neurons. At the same time, there was colocalization of CDK5 with GFAP in sham group and SAH group. Compared with sham operation group, brain edema and neurological dysfunction were significantly aggravated after SAH. In the experiment, the application of Cdk5 inhibitor roscovitine can significantly reduce brain edema and improve neurologic dysfunction. 4. Nissl staining showed that the number of injured neurons in the SAH group was significantly higher than that in the sham operation group, and the proportion of injured neurons in the SAH Roscovitine (100 渭 g group was decreased. The results of Tunel staining showed that the apoptotic cells in the cortex of the rats on the 1st day after SAH were significantly higher than those in the sham operation group. The proportion of apoptotic cells was significantly decreased after roscovitine application. Conclusion the protein expression of Cdk5,p25 and Cdk5-pTyr15 can be up-regulated by Cdk5, suggesting that Cdk5 is activated in the brain tissue after SAH. The application of Cdk5 inhibitor can obviously reduce the brain edema, reduce the proportion of neuronal apoptosis and improve the neurological dysfunction in SAH rats. These results suggest that Cdk5 may play an important role in the pathogenesis of EBI after SAH. CDK5 may become a new therapeutic target for EBI and provide new ideas for the treatment of EBI after SAH.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.35
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