【摘要】：[目的]探討Cdhl-APC在全腦缺血大鼠海馬組織中的表達及活性變化。 [方法]24只成年雄性SD大鼠被隨機分為3組：對照組、腦缺血/再灌注1天組和腦缺血/再灌注3天組,每組8只大鼠。腦缺血組采用四血管結(jié)扎法建立大鼠全腦缺血再灌注損傷模型,對照組為假手術(shù)組。腦缺血組在再灌注后1、3天時將大鼠在麻醉狀態(tài)下處死,并取海馬組織進行后續(xù)研究。采用TUNEL法評估全腦缺血再灌注后大鼠海馬區(qū)神經(jīng)元的凋亡情況；采用Western blot法檢測Cdhl及其下游底物SnoN、Skp2的表達變化。 [結(jié)果]與對照組相比,腦缺血/再灌注1、3天組的大鼠大腦海馬區(qū)均出現(xiàn)大量TUNEL陽性細胞；免疫組織化學(xué)染色顯示,對照組Cdhl高表達于海馬神經(jīng)元核內(nèi),腦缺血/再灌注1、3天組海馬神經(jīng)元內(nèi)的Cdhl出現(xiàn)核外轉(zhuǎn)移現(xiàn)象,并且其蛋白表達顯著減少(P0.05)；與此同時,與對照組相比,腦缺血組再灌注后1、3天Cdhl-APC的下游底物SnoN、Skp2的表達升高,差異有統(tǒng)計學(xué)意義(P0.05)。 [結(jié)論]大鼠全腦缺血再灌注后Cdhl-APC的表達及活性下降,此變化可能與缺血性神經(jīng)元凋亡的過程相關(guān),此發(fā)現(xiàn)為進一步探討Cdhl-APC在中樞神經(jīng)系統(tǒng)損傷中的作用提供了新的線索。 [目的]探討Cdhl-APC在維持正常神經(jīng)元糖代謝特征中的作用；探討神經(jīng)元OGD損傷后糖代謝的變化情況,并進一步通過慢病毒上調(diào)OGD神經(jīng)元內(nèi)Cdhl水平,探討其對恢復(fù)神經(jīng)元糖代謝特性,抗神經(jīng)元凋亡的作用。 [方法]體外培養(yǎng)大鼠原代皮層神經(jīng)元,并隨機分為4組：對照組、OGD/R組、OGD/R+Cdhl'慢病毒干預(yù)組、OGD/R+空病毒干預(yù)組。采用OGD法建立離體神經(jīng)元OGD/R模型；采用重組克隆技術(shù),構(gòu)建特異性上調(diào)Cdh1基因的野生型慢病毒和對照空病毒。采用Western-blot法檢測OGD后神經(jīng)元Cdhl及其下游底物SnoN、Skp2的表達變化情況及各組糖酵解、磷酸戊糖途徑關(guān)鍵酶的變化；免疫組化與TUNEL雙標法檢測各組神經(jīng)元凋亡情況。 [結(jié)果]Western-blot結(jié)果顯示,與對照組相比,OGD/R神經(jīng)元Cdhl表達明顯下降,與此同時其下游底物SnoN、Skp2表達明顯增高(P0.05)；正常神經(jīng)元感染野生型Cdhl慢病毒后,出現(xiàn)Cdhl-GFP融合蛋白的表達,而被對照空病毒干預(yù)的神經(jīng)元無此現(xiàn)象；對照組神經(jīng)元糖酵解關(guān)鍵酶Pfkfb3無表達,與此相比,OGD/R組、OGD/R+Cdh1空病毒干預(yù)組糖酵解關(guān)鍵酶Pflcfb3表達明顯升高(P0.05),這一現(xiàn)象在OGD/R+Cdhl慢病毒干預(yù)組被顯著抑制(O0.05)；與對照組相比,OGD/R組、OGD/R+Cdhl空病毒干預(yù)組的PPP關(guān)鍵酶G6PD表達明顯降低(P0.05),而這一現(xiàn)象在OGD+Cdh1慢病毒干預(yù)組被顯著抑制(P0.05)；此外,與OGD/R組、OGD/R+Cdhl空病毒干預(yù)組相比,OGD+Cdhl'慢病毒干預(yù)組神經(jīng)元凋亡率顯著降低。 [結(jié)論] OGD損傷后神經(jīng)元糖代謝平衡被打破,糖酵解被激活,PPP被抑制,此機制參與神經(jīng)元的凋亡；通過慢病毒上調(diào)Cdhl活性,使糖酵解關(guān)鍵酶Pfkfb3泛素化降解,維持神經(jīng)元正常糖代謝狀態(tài)(低糖酵解率,高PPP率),抑制神經(jīng)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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R614

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相關(guān)期刊論文 前1條

1 姚文龍;張傳漢;錢巍;邱瑾;柳璐;祝暢;桂伶俐;;APC-Cdh1在中樞神經(jīng)系統(tǒng)中的表達分布特點[J];第四軍醫(yī)大學(xué)學(xué)報;2009年13期

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