【摘要】：背景和目的：細胞周期蛋白依賴性激酶5(Cyclin-dependent kinase,Cdk5)是Cdk家族中的一員,并被視為調節(jié)神經(jīng)細胞生存與死亡的關鍵因子。盡管許多文獻已經(jīng)證明Cdk5參與了多種神經(jīng)系統(tǒng)疾病,如阿爾茨海默病、帕金森病和缺血性腦卒中等,但Cdk5在蛛網(wǎng)膜下腔出血(SAH)后早期腦損傷(EBI)中的作用仍不明確。本實驗的目的是研究Cdk5在大鼠SAH后大腦皮質的表達及細胞分布,探討其在SAH后EBI中的作用,為SAM的治療提供新的思路。實驗方法：1.Cdk5在大鼠蛛網(wǎng)膜下腔出血后大腦皮質中的表達將雄性SD大鼠52只隨機分為假手術組(n=12)和SAH組(下設SAH后6h、12h、24h、2d、3d組,每組8只)。通過向大鼠視交叉前池注射自體血來制備大鼠SAM模型。應用western blot、免疫組化法測定大鼠腦皮質Cdk5,p25和Cdk5-pTyr15的表達變化。免疫熒光染色方法檢測Cdk5蛋白在腦組織中的細胞分布情況。2.Cdk5抑制劑roscovitine對SAH大鼠的神經(jīng)保護作用研究將雄性SD大鼠隨機分為4組：假手術+二甲基亞砜(dimethyl sulf oxide, DMSO),向視交叉前池注射0.9%生理鹽水,并接受20%DMSO作為溶劑。SAH+DMSO組,SAH后接受相同劑量20% DMSO. SAH+Roscovitine(50μg)組,SAH后側腦室注射50μg roscovitine。SAH+Roscovitine (100 μg)組SAH后側腦室注射100 μ groscovitine。測量腦組織含水量評價腦水腫程度,TUNEL染色法檢測腦組織凋亡情況。應用Garcia神經(jīng)功能評分法評價SAH大鼠神經(jīng)功能障礙。結果：1.western blot及免疫組化結果顯示：假手術組Cdk5和Cdk5-pTyr15表達量皆較低。SAH后Cdk5和Cdk5-pTyr15蛋白表達逐漸升高,Cdk5蛋白在12h明顯升高,1d達高峰。Cdk5-pTyr15蛋白在6h明顯升高,12h達高峰。p25蛋白的表達趨勢與Cdk5和Cdk5-pTyr15相似,SAH也逐漸升高,1 d達到高峰。2.免疫熒光結果顯示：Cdk5在假手術組與SAH后1d的神經(jīng)元中皆有表達,在sham組,Cdk5主要表達于神經(jīng)元細胞漿,而SAH組中Cdk5向神經(jīng)元細胞核中移位；同時,Cdk5在sham組及SAH組中與GFAP也存在共定位。3.與假手術組相比,SAH后大鼠腦組織水腫明顯加重,神經(jīng)功能障礙明顯。實驗中應用Cdk5抑制劑roscovitine可明顯減輕腦組織水腫,改善神經(jīng)功能障礙。4.尼氏染色發(fā)現(xiàn)SAH后1 d腦皮質神經(jīng)元損傷相比假手術組顯著增加,而在SAH+Roscovitine(100μg)組,受損神經(jīng)元比例減少。5.TUNEL染色結果顯示,SAH后1d大鼠腦皮質凋亡細胞較假手術組明顯增加,應用roscovitine后可顯著減少凋亡細胞比例。結論：SAH可上調Cdk5,p25和Cdk5-pTyr15的蛋白表達,提示Cdk5在SAH后的腦組織中被激活。應用Cdk5抑制劑可明顯減輕SAH大鼠腦組織水腫,減少神經(jīng)細胞凋亡比例,改善神經(jīng)功能障礙。以上結果都提示Cdk5在SAH后EBI發(fā)病機制中起到重要作用,Cdk5有可能成為EBI的新的治療靶點,為SAH后EBI的治療提供新的思路。
[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.
【學位授予單位】：南京大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R743.35

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