Cdh1-APC在缺血缺氧性神经元凋亡中的作用及机制
发布时间：2018-11-28 07:33
【摘要】：[目的]探讨Cdhl-APC在全脑缺血大鼠海马组织中的表达及活性变化。 [方法]24只成年雄性SD大鼠被随机分为3组：对照组、脑缺血/再灌注1天组和脑缺血/再灌注3天组,每组8只大鼠。脑缺血组采用四血管结扎法建立大鼠全脑缺血再灌注损伤模型,对照组为假手术组。脑缺血组在再灌注后1、3天时将大鼠在麻醉状态下处死,并取海马组织进行后续研究。采用TUNEL法评估全脑缺血再灌注后大鼠海马区神经元的凋亡情况；采用Western blot法检测Cdhl及其下游底物SnoN、Skp2的表达变化。 [结果]与对照组相比,脑缺血/再灌注1、3天组的大鼠大脑海马区均出现大量TUNEL阳性细胞；免疫组织化学染色显示,对照组Cdhl高表达于海马神经元核内,脑缺血/再灌注1、3天组海马神经元内的Cdhl出现核外转移现象,并且其蛋白表达显著减少(P0.05)；与此同时,与对照组相比,脑缺血组再灌注后1、3天Cdhl-APC的下游底物SnoN、Skp2的表达升高,差异有统计学意义(P0.05)。 [结论]大鼠全脑缺血再灌注后Cdhl-APC的表达及活性下降,此变化可能与缺血性神经元凋亡的过程相关,此发现为进一步探讨Cdhl-APC在中枢神经系统损伤中的作用提供了新的线索。 [目的]探讨Cdhl-APC在维持正常神经元糖代谢特征中的作用；探讨神经元OGD损伤后糖代谢的变化情况,并进一步通过慢病毒上调OGD神经元内Cdhl水平,探讨其对恢复神经元糖代谢特性,抗神经元凋亡的作用。 [方法]体外培养大鼠原代皮层神经元,并随机分为4组：对照组、OGD/R组、OGD/R+Cdhl'慢病毒干预组、OGD/R+空病毒干预组。采用OGD法建立离体神经元OGD/R模型；采用重组克隆技术,构建特异性上调Cdh1基因的野生型慢病毒和对照空病毒。采用Western-blot法检测OGD后神经元Cdhl及其下游底物SnoN、Skp2的表达变化情况及各组糖酵解、磷酸戊糖途径关键酶的变化；免疫组化与TUNEL双标法检测各组神经元凋亡情况。 [结果]Western-blot结果显示,与对照组相比,OGD/R神经元Cdhl表达明显下降,与此同时其下游底物SnoN、Skp2表达明显增高(P0.05)；正常神经元感染野生型Cdhl慢病毒后,出现Cdhl-GFP融合蛋白的表达,而被对照空病毒干预的神经元无此现象；对照组神经元糖酵解关键酶Pfkfb3无表达,与此相比,OGD/R组、OGD/R+Cdh1空病毒干预组糖酵解关键酶Pflcfb3表达明显升高(P0.05),这一现象在OGD/R+Cdhl慢病毒干预组被显著抑制(O0.05)；与对照组相比,OGD/R组、OGD/R+Cdhl空病毒干预组的PPP关键酶G6PD表达明显降低(P0.05),而这一现象在OGD+Cdh1慢病毒干预组被显著抑制(P0.05)；此外,与OGD/R组、OGD/R+Cdhl空病毒干预组相比,OGD+Cdhl'慢病毒干预组神经元凋亡率显著降低。 [结论] OGD损伤后神经元糖代谢平衡被打破,糖酵解被激活,PPP被抑制,此机制参与神经元的凋亡；通过慢病毒上调Cdhl活性,使糖酵解关键酶Pfkfb3泛素化降解,维持神经元正常糖代谢状态(低糖酵解率,高PPP率),抑制神经元凋亡。
[Abstract]:[objective] to investigate the expression and activity of Cdhl-APC in hippocampus of rats with global cerebral ischemia. [methods] Twenty-four adult male SD rats were randomly divided into three groups: control group, 1 day cerebral ischemia / reperfusion group and 3 day cerebral ischemia / reperfusion group with 8 rats in each group. The rat model of global cerebral ischemia-reperfusion injury was established by four-vessel ligation in the cerebral ischemia group and the sham operation group was used as the control group. Rats in the cerebral ischemia group were killed under anaesthesia for 3 days after reperfusion, and hippocampal tissues were taken for follow-up study. The apoptosis of hippocampal neurons in rats after global cerebral ischemia-reperfusion was evaluated by TUNEL method, and the expression of Cdhl and SnoN,Skp2 was detected by Western blot method. [results] compared with the control group, a large number of TUNEL positive cells were found in the hippocampal area of the rats in the cerebral ischemia / reperfusion group for 1 to 3 days. Immunohistochemical staining showed that Cdhl in the control group was highly expressed in the hippocampal neuron nucleus, and the Cdhl in the hippocampal neurons of the cerebral ischemia / reperfusion group showed extranuclear metastasis, and its protein expression was significantly decreased (P0.05). At the same time, compared with the control group, the expression of SnoN,Skp2 in the downstream substrates of Cdhl-APC in the cerebral ischemia group was significantly higher than that in the control group on the 3rd day after reperfusion (P0.05). [conclusion] the expression and activity of Cdhl-APC decreased after global cerebral ischemia-reperfusion in rats, which may be related to the process of apoptosis of ischemic neurons. The findings provide a new clue to further explore the role of Cdhl-APC in central nervous system injury. [objective] to investigate the role of Cdhl-APC in maintaining the characteristics of glucose metabolism in normal neurons. To investigate the changes of glucose metabolism in neurons after OGD injury, and to explore the role of lentivirus in restoring the characteristics of glucose metabolism and preventing neuronal apoptosis by upregulating the level of Cdhl in OGD neurons. [methods] Primary cortical neurons of rats were cultured in vitro and randomly divided into four groups: control group, OGD/R Cdhl' lentivirus intervention group and OGD/R empty virus intervention group. OGD method was used to establish OGD/R model of isolated neurons, and recombinant clone technique was used to construct wild type lentivirus and control virus which specifically up-regulated Cdh1 gene. Western-blot method was used to detect the expression of Cdhl and its substrate SnoN,Skp2 in neurons after OGD and the changes of glycolysis and the key enzymes of pentose phosphate pathway in each group, and the apoptosis of neurons in each group was detected by immunohistochemistry and TUNEL double labeling method. [results] Western-blot results showed that the expression of Cdhl in OGD/R neurons was significantly lower than that in the control group, while the expression of SnoN,Skp2 in the downstream substrates was significantly increased (P0.05). The expression of Cdhl-GFP fusion protein was found in normal neurons infected with wild type Cdhl lentivirus, but not in neurons treated with control empty virus. Compared with the control group, the Pflcfb3 expression of glycolytic key enzyme Pflcfb3 in OGD/R group and OGD/R Cdh1 empty virus intervention group was significantly higher than that in control group (P0.05). This phenomenon was significantly inhibited in OGD/R Cdhl lentivirus intervention group (O0. 05). Compared with control group, the expression of PPP key enzyme G6PD in OGD/R Cdhl empty virus intervention group was significantly lower than that in control group (P0.05), but this phenomenon was significantly inhibited in OGD Cdh1 lentivirus intervention group (P0.05). In addition, the apoptosis rate of neurons in OGD Cdhl' lentivirus group was significantly lower than that in OGD/R group and OGD/R Cdhl empty virus intervention group. [conclusion] after OGD injury, the balance of glucose metabolism was disturbed, glycolysis was activated and PPP was inhibited. This mechanism was involved in neuronal apoptosis. By upregulating the activity of Cdhl, lentivirus can degrade the key enzyme of glycolysis, Pfkfb3 ubiquification, maintain the normal glucose metabolism of neurons (low glycolysis rate, high PPP rate) and inhibit neuronal apoptosis.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R614
本文编号：2362231
[Abstract]:[objective] to investigate the expression and activity of Cdhl-APC in hippocampus of rats with global cerebral ischemia. [methods] Twenty-four adult male SD rats were randomly divided into three groups: control group, 1 day cerebral ischemia / reperfusion group and 3 day cerebral ischemia / reperfusion group with 8 rats in each group. The rat model of global cerebral ischemia-reperfusion injury was established by four-vessel ligation in the cerebral ischemia group and the sham operation group was used as the control group. Rats in the cerebral ischemia group were killed under anaesthesia for 3 days after reperfusion, and hippocampal tissues were taken for follow-up study. The apoptosis of hippocampal neurons in rats after global cerebral ischemia-reperfusion was evaluated by TUNEL method, and the expression of Cdhl and SnoN,Skp2 was detected by Western blot method. [results] compared with the control group, a large number of TUNEL positive cells were found in the hippocampal area of the rats in the cerebral ischemia / reperfusion group for 1 to 3 days. Immunohistochemical staining showed that Cdhl in the control group was highly expressed in the hippocampal neuron nucleus, and the Cdhl in the hippocampal neurons of the cerebral ischemia / reperfusion group showed extranuclear metastasis, and its protein expression was significantly decreased (P0.05). At the same time, compared with the control group, the expression of SnoN,Skp2 in the downstream substrates of Cdhl-APC in the cerebral ischemia group was significantly higher than that in the control group on the 3rd day after reperfusion (P0.05). [conclusion] the expression and activity of Cdhl-APC decreased after global cerebral ischemia-reperfusion in rats, which may be related to the process of apoptosis of ischemic neurons. The findings provide a new clue to further explore the role of Cdhl-APC in central nervous system injury. [objective] to investigate the role of Cdhl-APC in maintaining the characteristics of glucose metabolism in normal neurons. To investigate the changes of glucose metabolism in neurons after OGD injury, and to explore the role of lentivirus in restoring the characteristics of glucose metabolism and preventing neuronal apoptosis by upregulating the level of Cdhl in OGD neurons. [methods] Primary cortical neurons of rats were cultured in vitro and randomly divided into four groups: control group, OGD/R Cdhl' lentivirus intervention group and OGD/R empty virus intervention group. OGD method was used to establish OGD/R model of isolated neurons, and recombinant clone technique was used to construct wild type lentivirus and control virus which specifically up-regulated Cdh1 gene. Western-blot method was used to detect the expression of Cdhl and its substrate SnoN,Skp2 in neurons after OGD and the changes of glycolysis and the key enzymes of pentose phosphate pathway in each group, and the apoptosis of neurons in each group was detected by immunohistochemistry and TUNEL double labeling method. [results] Western-blot results showed that the expression of Cdhl in OGD/R neurons was significantly lower than that in the control group, while the expression of SnoN,Skp2 in the downstream substrates was significantly increased (P0.05). The expression of Cdhl-GFP fusion protein was found in normal neurons infected with wild type Cdhl lentivirus, but not in neurons treated with control empty virus. Compared with the control group, the Pflcfb3 expression of glycolytic key enzyme Pflcfb3 in OGD/R group and OGD/R Cdh1 empty virus intervention group was significantly higher than that in control group (P0.05). This phenomenon was significantly inhibited in OGD/R Cdhl lentivirus intervention group (O0. 05). Compared with control group, the expression of PPP key enzyme G6PD in OGD/R Cdhl empty virus intervention group was significantly lower than that in control group (P0.05), but this phenomenon was significantly inhibited in OGD Cdh1 lentivirus intervention group (P0.05). In addition, the apoptosis rate of neurons in OGD Cdhl' lentivirus group was significantly lower than that in OGD/R group and OGD/R Cdhl empty virus intervention group. [conclusion] after OGD injury, the balance of glucose metabolism was disturbed, glycolysis was activated and PPP was inhibited. This mechanism was involved in neuronal apoptosis. By upregulating the activity of Cdhl, lentivirus can degrade the key enzyme of glycolysis, Pfkfb3 ubiquification, maintain the normal glucose metabolism of neurons (low glycolysis rate, high PPP rate) and inhibit neuronal apoptosis.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R614
【参考文献】
相关期刊论文 前1条
1 姚文龙;张传汉;钱巍;邱瑾;柳璐;祝畅;桂伶俐;;APC-Cdh1在中枢神经系统中的表达分布特点[J];第四军医大学学报;2009年13期
，本文编号：2362231
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