APC-Cdh1在体内外神经元缺氧损伤中的作用
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摘要
研究背景和目的
缺血缺氧性中枢神经系统损伤在临床常见,包括心搏骤停、失血性休克等引起的全脑缺血,以及栓塞、血栓形成引起的局灶性脑缺血等。神经元对缺氧缺血十分敏感,缺血即时损伤和继发的免疫炎症反应、钙超载、自由基损伤等可引起神经元轴突损伤及凋亡,影响神经功能,甚至造成死亡,但目前的各项治疗手段效果有限。
细胞周期末期促进复合物(anaphase promoting complex, APC)及其调节亚基Cdhl是细胞内主要的泛素蛋白酶小体系统。Cdhl在有丝分裂晚期及G1期激活APC,使之与底物蛋白质连接并将泛素转移给底物,可使泛素化的底物被蛋白质酶体降解,在有丝分裂期向G1期转化中发挥着关键作用。
近年研究发现APC-Cdhl在成熟神经元中有大量表达,发挥着调节轴突生长、突触传递及神经元存活的作用。我们的前期研究也表明,APC-Cdhl在大鼠海马、皮层的神经元中均有表达；APC-Cdhl在神经干细胞增殖及分化为神经元的过程中发挥着重要作用；脊髓损伤后,下调Cdhl表达能够促进轴突生长及神经功能恢复。但是,目前关于APC-Cdhl在中枢神经系统缺血性损伤及疾病中的具体作用及机制,尚不清楚。
缺血性脑损伤对神经元的影响包括神经元凋亡及轴突损伤,同时引起胶质细胞激活、增殖,释放多种炎性物质及细胞因子。Cdhl在细胞周期中主要使细胞维持于G1期,我们推测Cdh1与缺血性神经元凋亡及胶质细胞激活有关。本研究中,我们拟通过体内实验观察局灶性脑缺血损伤后,Cdh1在缺血灶的表达分布,探讨缺血性损伤前后Cdh1在神经元和胶质细胞中的变化特点；体外培养大鼠神经元,观察体外缺氧损伤对神经元中Cdh1及其下游底物表达的影响；再通过慢病毒介导的Cdh1 RNA干扰技术,探讨体外下调Cdh1对神经元存活、凋亡的影响,从而进一步认识Cdh1在中枢神经系统缺血性损伤中的作用,为通过调控Cdh1治疗中枢神经系统损伤和疾病提供实验依据。
研究方法与结果
1.APC-Cdhl在大鼠局灶性脑缺血损伤中的表达分布特点
方法：雄性成年SD大鼠36只,采用线栓法建立大鼠右侧大脑中动脉缺血(Middle cerebral artery occlusion, MCAO)模型,于MCAO 0d> 1d> 3d、7d采集标本,其中对MCAO Od动物施行假手术组,即仅分离右侧颈动脉和迷走神经,不结扎血管。采用实时荧光定量PCR检测不同时间点缺血侧(右侧)和非缺血侧(左侧)脑组织Cdh1 mRNA的表达,免疫荧光双标法检测缺血前后Cdhl在神经元和胶质细胞中的分布变化情况。采用SPSS13.0软件进行统计学分析,计量资料采用x±s表示,组间比较采用t检验,组内比较采用方差分析,P<0.05为差异具有统计学意义。
结果：术后各时点,MCAO模型组缺血损伤侧脑组织中Cdh1 mRNA表达均低于非损伤组织(P<0.05)。免疫荧光双标法检测显示,缺血损伤后损伤区大量神经元细胞结构改变,Cdh1在缺血性损伤及正常神经元中均有大量表达；正常脑组织中星形胶质细胞仅极少数表达Cdh1,缺血损伤后可见缺血灶周围星形胶质细胞明显激活,且多见Cdh1阳性的星形胶质细胞。
2.原代培养神经元缺氧性损伤对Cdh1及其下游底物表达的影响
方法：取出生24h内乳鼠,无菌条件下分离其大脑皮质,体外培养原代神经元。培养第7日采用神经元特异性标志物NeuN进行免疫荧光鉴定神经元所占百分比。使用无糖Earle's液替代细胞培养液,利用三气培养箱充以氮气,使O2浓度维持在1%,CO2浓度为5％,37℃处理1h后复氧,建立原代神经元缺氧缺糖模型。实时荧光定量PCR检测复氧后各时点Cdh1及其下游底物Skp2、CyclinB1的表达。采用SPSS13.0软件进行统计学分析,计量资料采用x±s表示,不同细胞间比较采用方差分析,P<0.05为差异有统计学意义。
结果：成功培养了大鼠原代神经元,NeuN免疫荧光鉴定神经元约占90%。建立了体外神经元缺氧缺糖模型。体外缺氧损伤后3天、7天,原代神经元Cdh1及其下游底物Cyclin B1表达上调(P<0.05),而Skp2在缺血后各时间点表达均下调(P<0.05)。
3.慢病毒介导的Cdh1 RNAi对原代神经元凋亡的影响
方法：取出生24h内SD乳鼠大脑皮质,使用24孔细胞培养板培养神经元。神经元体外培养至第7天,按病毒与细胞的比例(MOI值=10、50和100)加入含GFP的慢病毒Cdh1 RNAi载体。感染后72h采用荧光显微镜观察GFP的表达,计算感染效率,确定慢病毒感染神经细胞的合适MOI。之后进行慢病毒感染,实时定量PCR检测慢病毒介导的Cdh1 RNAi后Cdh1的表达改变。慢病毒感染神经元后第7天,TUNEL法检测慢病毒感染组及对照组神经元凋亡情况。不同细胞间比较采用方差分析,P<0.05为差异有统计学意义。
结果：采用慢病毒感染神经元,MOI为100时能够满足后续的研究。感染后各时间点LV-Cdh1 RNAi组较未感染组Cdhl表达降低(P<0.05)。TUNEL法检测显示慢病毒感染后神经元凋亡无明显影响(P>0.05)。
研究结论
1. APC-Cdhl在正常大鼠脑组织中有大量表达,且主要定位于神经元；大鼠局灶性脑缺血性损伤后,神经元内仍有Cdhl表达。正常大鼠脑组织星形胶质细胞少见Cdh1阳性,而缺血损伤后星形胶质细胞大量激活,且部分呈Cdh1阳性。
2.体外实验表明缺氧缺糖损伤后,神经元中Cdh1表达升高,其下游底物Cyclin B1上调,而Skp2表达下调。
3.本实验组构建的Cdh1 RNAi慢病毒载体可在体外下调原代培养神经元Cdh1的表达,但慢病毒介导的Cdh1 RNAi短期内对神经元凋亡无明显影响。
研究总结
本课题首先通过体内外实验揭示缺氧缺血性损伤后,Cdh1在不同神经细胞中的差异性表达。在正常脑组织中,Cdh1主要表达在神经元,在星形胶质细胞中表达较少。局灶性缺血性脑损伤后,缺血组织Cdh1 mRNA的表达下降,缺血灶内大量神经元细胞结构改变,推测其已发生凋亡或死亡,幸存神经元内仍见Cdh1表达；缺血灶周边星形胶质细胞大量激活,且部分呈Cdh1阳性,提示Cdh1可能参与缺血后神经元凋亡及胶质细胞激活的过程。
通过培养原代大鼠皮层神经元,并构建体外神经元缺氧缺糖模型,观察体外神经元缺氧损伤对Cdh1 mRNA表达的影响,发现损伤后Cdh1的表达上调,其下游底物Cyclin B1表达趋势与Cdh1相近,Skp2表达下调,推测Cyclin B1表达上调可能通过负反馈作用使得Cdh1在神元中表达升高,而Cdh1可能作用于Skp2参与缺血性轴突损伤及神经元凋亡等病理过程。通过慢病毒介导的RNAi技术体外下调原代神经元Cdh1表达,短期内对细胞凋亡未见明显影响。本研究加深了对Cdh1在中枢神经系统缺血性损伤中作用的认识,表明其在中枢神经系统缺血损伤的病理生理过程中可能发挥着重要作用,为下一步探讨Cdh1在神经损伤后再生修复及基因治疗奠定了实验基础。
Background and objective
Anaphase-promoting complex (APC) is the major intracellular ubiquitin ligase that controls the cell cycle. Cdh1 is one of its co-activators, which is required for APC activity during late mitosis and G1. APC is activated by Cdh1 at late mitosis and the G1 phase, APC-Cdh1 is connected to the substrate proteins and transfed to ubiquitin, enableing ubiquitination of the substrate degradation by a protein enzyme body.This procession plays a key role in transformation from mitosis to G1 phase. Recent studies showed that APC-Cdh1 also expressed in mature neurons, it could regulate axon growth, synaptic transmission and neuronal survival. Our primary study showed, that APC-Cdh1 played an important role in the process in which neural stem cells differentiate into neurons; Reducing Cdh1 expression after spinal cord injury can promote axonal growth and neural function recovery. However, the role of APC-Cdh1 in the central nervous system injury and disease and its specific mechanism is not clear yet.
Methods and Results
1. Changes in the Expression of APC-Cdhl in Rats with focal cerebral ischemia
Methods:36 adult male Sprague-Dawley rats were randomly divided into two groups:①sham control group (n=12);②focal cerebral ischemia model group (n= 24), on which middle cerebral artery occlusion (MCAO) was produced by insertion of a nylon thread with rounded tip at bifurcation of right common carotid artery into internal carotid artery. At different times after injury, the expression of APC-Cdhl mRNA of brain tissue was detected by Real-time fluorescence quantitative PCR, double-label immunofluorescence assays was used to detecte the expression of Cdhl in different cells.
Result:The expressions of Cdhl mRNA at each time point after surgery were lower than the sham group (P<0.05). Double immunofluorescence analysis showed that, Cdhl expressed abundantly in both injured and non-injured neurons, while Cdhl positive astrocytes were maimly observede in injured brain tissue.
2. Expression of Cdhl and its downstream substrate during hypoxic injury of Primary cultured neurons
Method:Primary neurons from cortex of postnatal 24h rat pups were cultured. Neuron was identified by NeuN immunocytochemical staining. The mRNA of neuron was extracted by Trizol method. The expression of Cdhl was examined by quantitive real time PCR. Using sugar-free Earle's solution and the three gas incubator Filled with nitrogen to maintain O2 concentration 1%, CO2 concentration of 5%,37℃for 1h. After reoxygenation treatment, Real-time quantitative PCR was used to detect the mRNA expression of Cdhl and its downstream substrates Skp2, Cyclin B1.
Results:Rat primary neurons were uccessfully cultured, NeuN immunofluorescence showed about 90% of the cultured cells were neurons. OGD model of neuronal cells was established in vitro. At 3 days,7 days after the hypoxic injury in vitro, the expression of Cdhl and its downstream substrate Cyclin B1 in primary neurons were increased (P<0.05), while Skp2 expressions were lower at different time points after injury(P<0.05).
3. Effects of lentivirus-mediated Cdhl RNAi in primary neuron survival
Method:Primary neurons from cortex of postnatal 24h rat pups were cultured in the 24-hole cell culture plate.7days later, they were infected with lentivirus containing GFP in different MOI. Seventy-two hours later, the appropriate MOI for neuuons was determined by examining GFP expression with fluorescent microscope. Real-time quantitative PCR was used to detecte the expression of Cdhl. In this study, two groups were involved, uninfected group, and LV-Cdhl RNAi group. Cell apoptosis was evaluated TUNEL at 7dth after viral infection.
Results:The appropriate lentivirus MOI for Primary cultured neurons was 100. The expression of Cdhl was significant reduced in LV-Cdhl RNAi group compaired to the uninfected group (P<0.05). TUNEL assay showed there was no significant difference in the absorption between the LV-Cdhl RNAi group and uninfected group (P>0.05)
4. Statistical Analysis
All of the statistical analyses were performed by SPSS 12.0 software. Measurement data were presented as means±SD. The differences between two groups were analyzed by t-test. The differences among three groups were analyzed by one-way ANOVA. Categorical data were presented as rate. The differences between different groups were analyzed by Chi-square test. P<0.05 was considered statistically significant.
Conclusions
1. APC-Cdhl in the normal rat brain tissue expressis mainly in neurons.After cerebral ischemic injury, the expression of neuronal Cdhl remains. Normal rat brain astrocytes are rare Cdhl-positive, while after ischemic injury, a large number of astrocytes were activated and were Cdhl-positive.
2. In vitro experiments showed that OGD injury increased expression of neuronal Cdhl and its downstream substrate Cyclin B1, while Skp2 expression was reduced.
3. Cdhl expression of Cdhl RNAi neuron group were reduced in vitro, but the lentivirus-mediated Cdhl RNAi didn't induce significant neuronal apoptosis.
Research Summary
The issue first revealed the expression of Cdhl in different nerve cells by in vivo experiments with hypoxic ischemic injury, that in normal brain tissue, Cdhl expressed mainly in neurons, but rare in astrocytes. Cdhl mRNA expression in ischemic tissue was decreased. A large number of astrocytes were activated and parts of them were Cdhl-positive. Then rat cortical neurons were cultured, and neuronal hypoxia model of neuronal hypoxia in vitro was built the in vitro. The Cdhl and Cyclin B1 mRNA expressions were found increased after injury, while the expression of Skp2 was decreased, suggesting that upregulation of Cyclin B1 might have induce the expression of Cdhl increasing by negative feedback. By lentivirus-mediated RNAi technology reduced the expression of neuronal Cdhl, no significant effect on cell survival in vitro was observed in short term. This study extended the understanding of Cdhl function in ischemic neural injury. It will be very useful for exploring the additional function of APC-Cdhl in nervous system injury and regeneration and gene therapies for neurological diseases.
缺血缺氧性中枢神经系统损伤在临床常见,包括心搏骤停、失血性休克等引起的全脑缺血,以及栓塞、血栓形成引起的局灶性脑缺血等。神经元对缺氧缺血十分敏感,缺血即时损伤和继发的免疫炎症反应、钙超载、自由基损伤等可引起神经元轴突损伤及凋亡,影响神经功能,甚至造成死亡,但目前的各项治疗手段效果有限。
细胞周期末期促进复合物(anaphase promoting complex, APC)及其调节亚基Cdhl是细胞内主要的泛素蛋白酶小体系统。Cdhl在有丝分裂晚期及G1期激活APC,使之与底物蛋白质连接并将泛素转移给底物,可使泛素化的底物被蛋白质酶体降解,在有丝分裂期向G1期转化中发挥着关键作用。
近年研究发现APC-Cdhl在成熟神经元中有大量表达,发挥着调节轴突生长、突触传递及神经元存活的作用。我们的前期研究也表明,APC-Cdhl在大鼠海马、皮层的神经元中均有表达；APC-Cdhl在神经干细胞增殖及分化为神经元的过程中发挥着重要作用；脊髓损伤后,下调Cdhl表达能够促进轴突生长及神经功能恢复。但是,目前关于APC-Cdhl在中枢神经系统缺血性损伤及疾病中的具体作用及机制,尚不清楚。
缺血性脑损伤对神经元的影响包括神经元凋亡及轴突损伤,同时引起胶质细胞激活、增殖,释放多种炎性物质及细胞因子。Cdhl在细胞周期中主要使细胞维持于G1期,我们推测Cdh1与缺血性神经元凋亡及胶质细胞激活有关。本研究中,我们拟通过体内实验观察局灶性脑缺血损伤后,Cdh1在缺血灶的表达分布,探讨缺血性损伤前后Cdh1在神经元和胶质细胞中的变化特点；体外培养大鼠神经元,观察体外缺氧损伤对神经元中Cdh1及其下游底物表达的影响；再通过慢病毒介导的Cdh1 RNA干扰技术,探讨体外下调Cdh1对神经元存活、凋亡的影响,从而进一步认识Cdh1在中枢神经系统缺血性损伤中的作用,为通过调控Cdh1治疗中枢神经系统损伤和疾病提供实验依据。
研究方法与结果
1.APC-Cdhl在大鼠局灶性脑缺血损伤中的表达分布特点
方法：雄性成年SD大鼠36只,采用线栓法建立大鼠右侧大脑中动脉缺血(Middle cerebral artery occlusion, MCAO)模型,于MCAO 0d> 1d> 3d、7d采集标本,其中对MCAO Od动物施行假手术组,即仅分离右侧颈动脉和迷走神经,不结扎血管。采用实时荧光定量PCR检测不同时间点缺血侧(右侧)和非缺血侧(左侧)脑组织Cdh1 mRNA的表达,免疫荧光双标法检测缺血前后Cdhl在神经元和胶质细胞中的分布变化情况。采用SPSS13.0软件进行统计学分析,计量资料采用x±s表示,组间比较采用t检验,组内比较采用方差分析,P<0.05为差异具有统计学意义。
结果：术后各时点,MCAO模型组缺血损伤侧脑组织中Cdh1 mRNA表达均低于非损伤组织(P<0.05)。免疫荧光双标法检测显示,缺血损伤后损伤区大量神经元细胞结构改变,Cdh1在缺血性损伤及正常神经元中均有大量表达；正常脑组织中星形胶质细胞仅极少数表达Cdh1,缺血损伤后可见缺血灶周围星形胶质细胞明显激活,且多见Cdh1阳性的星形胶质细胞。
2.原代培养神经元缺氧性损伤对Cdh1及其下游底物表达的影响
方法：取出生24h内乳鼠,无菌条件下分离其大脑皮质,体外培养原代神经元。培养第7日采用神经元特异性标志物NeuN进行免疫荧光鉴定神经元所占百分比。使用无糖Earle's液替代细胞培养液,利用三气培养箱充以氮气,使O2浓度维持在1%,CO2浓度为5％,37℃处理1h后复氧,建立原代神经元缺氧缺糖模型。实时荧光定量PCR检测复氧后各时点Cdh1及其下游底物Skp2、CyclinB1的表达。采用SPSS13.0软件进行统计学分析,计量资料采用x±s表示,不同细胞间比较采用方差分析,P<0.05为差异有统计学意义。
结果：成功培养了大鼠原代神经元,NeuN免疫荧光鉴定神经元约占90%。建立了体外神经元缺氧缺糖模型。体外缺氧损伤后3天、7天,原代神经元Cdh1及其下游底物Cyclin B1表达上调(P<0.05),而Skp2在缺血后各时间点表达均下调(P<0.05)。
3.慢病毒介导的Cdh1 RNAi对原代神经元凋亡的影响
方法：取出生24h内SD乳鼠大脑皮质,使用24孔细胞培养板培养神经元。神经元体外培养至第7天,按病毒与细胞的比例(MOI值=10、50和100)加入含GFP的慢病毒Cdh1 RNAi载体。感染后72h采用荧光显微镜观察GFP的表达,计算感染效率,确定慢病毒感染神经细胞的合适MOI。之后进行慢病毒感染,实时定量PCR检测慢病毒介导的Cdh1 RNAi后Cdh1的表达改变。慢病毒感染神经元后第7天,TUNEL法检测慢病毒感染组及对照组神经元凋亡情况。不同细胞间比较采用方差分析,P<0.05为差异有统计学意义。
结果：采用慢病毒感染神经元,MOI为100时能够满足后续的研究。感染后各时间点LV-Cdh1 RNAi组较未感染组Cdhl表达降低(P<0.05)。TUNEL法检测显示慢病毒感染后神经元凋亡无明显影响(P>0.05)。
研究结论
1. APC-Cdhl在正常大鼠脑组织中有大量表达,且主要定位于神经元；大鼠局灶性脑缺血性损伤后,神经元内仍有Cdhl表达。正常大鼠脑组织星形胶质细胞少见Cdh1阳性,而缺血损伤后星形胶质细胞大量激活,且部分呈Cdh1阳性。
2.体外实验表明缺氧缺糖损伤后,神经元中Cdh1表达升高,其下游底物Cyclin B1上调,而Skp2表达下调。
3.本实验组构建的Cdh1 RNAi慢病毒载体可在体外下调原代培养神经元Cdh1的表达,但慢病毒介导的Cdh1 RNAi短期内对神经元凋亡无明显影响。
研究总结
本课题首先通过体内外实验揭示缺氧缺血性损伤后,Cdh1在不同神经细胞中的差异性表达。在正常脑组织中,Cdh1主要表达在神经元,在星形胶质细胞中表达较少。局灶性缺血性脑损伤后,缺血组织Cdh1 mRNA的表达下降,缺血灶内大量神经元细胞结构改变,推测其已发生凋亡或死亡,幸存神经元内仍见Cdh1表达；缺血灶周边星形胶质细胞大量激活,且部分呈Cdh1阳性,提示Cdh1可能参与缺血后神经元凋亡及胶质细胞激活的过程。
通过培养原代大鼠皮层神经元,并构建体外神经元缺氧缺糖模型,观察体外神经元缺氧损伤对Cdh1 mRNA表达的影响,发现损伤后Cdh1的表达上调,其下游底物Cyclin B1表达趋势与Cdh1相近,Skp2表达下调,推测Cyclin B1表达上调可能通过负反馈作用使得Cdh1在神元中表达升高,而Cdh1可能作用于Skp2参与缺血性轴突损伤及神经元凋亡等病理过程。通过慢病毒介导的RNAi技术体外下调原代神经元Cdh1表达,短期内对细胞凋亡未见明显影响。本研究加深了对Cdh1在中枢神经系统缺血性损伤中作用的认识,表明其在中枢神经系统缺血损伤的病理生理过程中可能发挥着重要作用,为下一步探讨Cdh1在神经损伤后再生修复及基因治疗奠定了实验基础。
Background and objective
Anaphase-promoting complex (APC) is the major intracellular ubiquitin ligase that controls the cell cycle. Cdh1 is one of its co-activators, which is required for APC activity during late mitosis and G1. APC is activated by Cdh1 at late mitosis and the G1 phase, APC-Cdh1 is connected to the substrate proteins and transfed to ubiquitin, enableing ubiquitination of the substrate degradation by a protein enzyme body.This procession plays a key role in transformation from mitosis to G1 phase. Recent studies showed that APC-Cdh1 also expressed in mature neurons, it could regulate axon growth, synaptic transmission and neuronal survival. Our primary study showed, that APC-Cdh1 played an important role in the process in which neural stem cells differentiate into neurons; Reducing Cdh1 expression after spinal cord injury can promote axonal growth and neural function recovery. However, the role of APC-Cdh1 in the central nervous system injury and disease and its specific mechanism is not clear yet.
Methods and Results
1. Changes in the Expression of APC-Cdhl in Rats with focal cerebral ischemia
Methods:36 adult male Sprague-Dawley rats were randomly divided into two groups:①sham control group (n=12);②focal cerebral ischemia model group (n= 24), on which middle cerebral artery occlusion (MCAO) was produced by insertion of a nylon thread with rounded tip at bifurcation of right common carotid artery into internal carotid artery. At different times after injury, the expression of APC-Cdhl mRNA of brain tissue was detected by Real-time fluorescence quantitative PCR, double-label immunofluorescence assays was used to detecte the expression of Cdhl in different cells.
Result:The expressions of Cdhl mRNA at each time point after surgery were lower than the sham group (P<0.05). Double immunofluorescence analysis showed that, Cdhl expressed abundantly in both injured and non-injured neurons, while Cdhl positive astrocytes were maimly observede in injured brain tissue.
2. Expression of Cdhl and its downstream substrate during hypoxic injury of Primary cultured neurons
Method:Primary neurons from cortex of postnatal 24h rat pups were cultured. Neuron was identified by NeuN immunocytochemical staining. The mRNA of neuron was extracted by Trizol method. The expression of Cdhl was examined by quantitive real time PCR. Using sugar-free Earle's solution and the three gas incubator Filled with nitrogen to maintain O2 concentration 1%, CO2 concentration of 5%,37℃for 1h. After reoxygenation treatment, Real-time quantitative PCR was used to detect the mRNA expression of Cdhl and its downstream substrates Skp2, Cyclin B1.
Results:Rat primary neurons were uccessfully cultured, NeuN immunofluorescence showed about 90% of the cultured cells were neurons. OGD model of neuronal cells was established in vitro. At 3 days,7 days after the hypoxic injury in vitro, the expression of Cdhl and its downstream substrate Cyclin B1 in primary neurons were increased (P<0.05), while Skp2 expressions were lower at different time points after injury(P<0.05).
3. Effects of lentivirus-mediated Cdhl RNAi in primary neuron survival
Method:Primary neurons from cortex of postnatal 24h rat pups were cultured in the 24-hole cell culture plate.7days later, they were infected with lentivirus containing GFP in different MOI. Seventy-two hours later, the appropriate MOI for neuuons was determined by examining GFP expression with fluorescent microscope. Real-time quantitative PCR was used to detecte the expression of Cdhl. In this study, two groups were involved, uninfected group, and LV-Cdhl RNAi group. Cell apoptosis was evaluated TUNEL at 7dth after viral infection.
Results:The appropriate lentivirus MOI for Primary cultured neurons was 100. The expression of Cdhl was significant reduced in LV-Cdhl RNAi group compaired to the uninfected group (P<0.05). TUNEL assay showed there was no significant difference in the absorption between the LV-Cdhl RNAi group and uninfected group (P>0.05)
4. Statistical Analysis
All of the statistical analyses were performed by SPSS 12.0 software. Measurement data were presented as means±SD. The differences between two groups were analyzed by t-test. The differences among three groups were analyzed by one-way ANOVA. Categorical data were presented as rate. The differences between different groups were analyzed by Chi-square test. P<0.05 was considered statistically significant.
Conclusions
1. APC-Cdhl in the normal rat brain tissue expressis mainly in neurons.After cerebral ischemic injury, the expression of neuronal Cdhl remains. Normal rat brain astrocytes are rare Cdhl-positive, while after ischemic injury, a large number of astrocytes were activated and were Cdhl-positive.
2. In vitro experiments showed that OGD injury increased expression of neuronal Cdhl and its downstream substrate Cyclin B1, while Skp2 expression was reduced.
3. Cdhl expression of Cdhl RNAi neuron group were reduced in vitro, but the lentivirus-mediated Cdhl RNAi didn't induce significant neuronal apoptosis.
Research Summary
The issue first revealed the expression of Cdhl in different nerve cells by in vivo experiments with hypoxic ischemic injury, that in normal brain tissue, Cdhl expressed mainly in neurons, but rare in astrocytes. Cdhl mRNA expression in ischemic tissue was decreased. A large number of astrocytes were activated and parts of them were Cdhl-positive. Then rat cortical neurons were cultured, and neuronal hypoxia model of neuronal hypoxia in vitro was built the in vitro. The Cdhl and Cyclin B1 mRNA expressions were found increased after injury, while the expression of Skp2 was decreased, suggesting that upregulation of Cyclin B1 might have induce the expression of Cdhl increasing by negative feedback. By lentivirus-mediated RNAi technology reduced the expression of neuronal Cdhl, no significant effect on cell survival in vitro was observed in short term. This study extended the understanding of Cdhl function in ischemic neural injury. It will be very useful for exploring the additional function of APC-Cdhl in nervous system injury and regeneration and gene therapies for neurological diseases.
引文
1. Bus 1 KM, Greer DM. Hypoxic-ischemic brain injury:pathophysiology, neuropathology and mechanisms. NeuroRehabilitation. 2010,26:5-13.
2. Vogel G. Nobel Prizes. Gold medal from cellular trash, science,2004, 306:400-401.
3. Pfleger CM, Kirschner MW. The KEN box:an APC recognition signal distinct from the D box targeted by Cdhl. Genes Dev,2000,14: 655-665.
4. Littlepage LE, Ruderman JV. Identification of a new APC/C recognition domain, the A box, which is required for the Cdhl-dependent destruction of the kinase Aurora-A during mitotic exit. Genes Dev,2002,16:2274-2285.
5. Reis A, Levasseur M, Chang HY, et al. The CRY box:a second APCcdhl-dependent degron in mammalian cdc20. EMBO Rep,2006,7: 1040-1045.
6.姚文龙,柳璐,张传汉.Cdhl-APC在中枢神经系统中的作用.中风与神经疾病杂志.2006,23：751-752.
7. Yamashita YM, Nakaseko Y, Samejima I, et al.20S cyclosome complex formation and proteolytic activity inhibited by the Camp/PKA pathway. Nature,1996 384:276-279.
8. Kotani S, Tugendreich S, Fujii M, et al. PKA and MPF-activated polo-like kinase regulate anaphase-promoting complex activity and mitosis progression. Mol Cell,1998,1:371-380.
9. Rudner AD, Murray AW. Phosphorylation by Cdc28 activates the Cdc20-dependent activity of the anaphase-promoting complex. J Cell Biol,2000,149:1377-1390.
10. Kraft C, Herzog F, Gieffers C, et al. Mitotic regulation of the human anaphase-promoting complex by phosphorylation. EMBO J,2003, 22:6598-6609.
11. Lukas C, SΦrensen CS, Kramer E, et al. Accumulation of cyclin B1 requires E2F and cyclin-A-dependent rearrangement of the anaphase-promoting complex. Nature,1999,401:815-818.
12. Zachariae W, Schwab M, Nasmyth K, et al. Control of cyclin ubiquitination by CDK-regulated binding of Hctl to the anaphase promoting complex. Science,1998,282:1721-1724.
13. Jaspersen SL, Charles JF, Morgan DO. Inhibitory phosphorylation of the APC regulator Hctl is controlled by the kinase Cdc28 and the phosphatase Cdc14. Curr Biol,1999,9:227-36.
14. Hsu JY, Reimann JD, SΦrensen CS, et al. E2F-dependent accumulation of hEmil regulates S phase entry by inhibiting APC(Cdh1). Nat Cell Biol,2002,4:358-366.
15. Turnell AS, Stewart GS, Grand RJ, et al. The APC/C and CBP/p300 cooperate to regulate transcription and cell-cycle progression. Nature,2005,438:690-695.
16. Listovsky T, Oren YS, Yudkovsky Y, et al. Mammalian Cdh1/Fzr mediates its own degradation. EMBO J,2004,23:1619-1626.
17. Rape M, Kirschner MW. Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry. Nature,2004,432:588-595.
18. Starborg M, Brundell E, Gell K, et al. A novel murine gene encoding a 216-kDa protein is related to a mitotic checkpoint regulator previously identified in Aspergillus nidulans. J Biol Chem,1994, 269:24133-7.
19. Giefers C, Peters BH, Kramer ER, et al. Expression of the CDH1-associated form of the anaphase-promoting complex in postmitotic neurons. Proc Natl Acad Sci USA,1999,96:11317-11322.
20.姚文龙,张传汉,钱巍,邱瑾,柳璐,祝畅,桂伶俐.APC-Cdhl在中枢神经系统中的表达分布特点.第四军医大学学报.2009,30：1161-1165.
21. Konishi Y, Stegmuller J, Matsuda T, et al. Cdhl-APC controls axonal growth and patterning in the mammalian brain. Science,2004,303: 1026-1030.
22. Zhao N, Lai F, Fernald AA, et al. Human CDC23:Cdna cloning, mapping to 5q31, genomic structure, and evaluation as a candidate tumor suppressor gene in myeloid leukemias. Genomics,1998,53:184-90.
23.姚文龙,张传汉,柳璐,祝畅,邱瑾,桂伶俐,田玉科.大鼠永生化神经前体细胞CDH1小干扰RNA真核载体的构建.中华麻醉学杂志.2008,28：919-921.
24. Yao W, Qian W, Zhu C, Gui L, Qiu J, Zhang C. Cdhl-APC is involved in the differentiation of neural stem cells into neurons. Neuroreport,2010,21:39-44.
25. Cuende J, Moreno S, Bolanos JP, et al. Retinoic acid downregulates Rael leading to APC (Cdh1) activation and neuroblastoma SH-SY5Y differentiation. Oncogene,2008,27:3339-3344.
26. Harmey D, Smith A, Simanski S, et al. The Anaphase Promoting Complex Induces Substrate Degradation during Neuronal Differentiation. J Biol Chem,2009,284:4317-4323.
27. Stegmuller J, Konishi Y, Huynh MA, et al. Cell-intrinsic regulation of axonal morphogenesis by the Cdhl-APC target SnoN. Neuron,2006,50:389-400.
28.祁月红,钱巍,李平,姚文龙,万里,张传汉.慢病毒介导RNA干扰Cdh1的表达对大鼠脊髓损伤的修复作用.第四军医大学学报,2009,30：1353-1356.
29. Becker EB, Bonni A. Cell cycle regulation of neuronal apoptosis in development and disease. Prog Neurobiol,2004,72:1-25.
30. Greene LA, Biswas SC, Liu DX. Cell cycle molecules and vertebrate neuron death:E2F at the hub. Cell Death Differ,2004,11:49-60.
31. Peters JM. The anaphase promoting complex/cyclosome:a machine designed to destroy. Nat Rev Mol Cell Biol,2006,7:644-656.
32. Almeida A, Bolanos JP, Moreno S. Cdhl/Hctl-APC is essential for the survival of postmitotic neurons. J Neurosci,2005,25: 8115-8121.
33. Konishi Y, Bonni A. The E2F-Cdc2 cell-cycle pathway specifically mediates activity deprivation-induced apoptosis of postmitotic neurons. J Neurosci,2003,23:1649-1658.
34. Kaplow ME, Korayem AH, Venkatesh TR. Regulation of glia number in Drosophila by Rap/Fzr, an activator of the anaphase-promoting complex, and Loco, an RGS protein. Genetics.2008,178:2003-16.
1. Busl KM, Greer DM. Hypoxic-ischemic brain injury:pathophysiology, neuropathology and mechanisms. NeuroRehabilitation. 2010,26:5-13.
2. Vogel G. Nobel Prizes. Gold medal from cellular trash, science,2004, 306:400-401.
3. Konishi Y, Stegmuller J, Matsuda T, et al. Cdhl-APC controls axonal growth and patterning in the mammalian brain. Science,2004,303: 1026-1030.
4. Pfleger CM, Kirschner MW. The KEN box:an APC recognition signal distinct from the D box targeted by Cdhl. Genes Dev,2000,14: 655-665.
5.姚文龙,张传汉,钱巍,邱瑾,柳璐,祝畅,桂伶俐.APC-Cdhl在中枢神经系统中的表达分布特点.第四军医大学学报.2009,30：1161-1165.
6. Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989,20:84-91.
7. Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke,2001,32:100521011.
8.钱巍,姚文龙,祝畅,柳璐,桂伶俐,刘志恒,张传汉.永生化神经前体细胞治疗大鼠局灶性脑缺血的实验研究.中华医学杂志,2008,88：867-870.
9. Harper JW, Burton JL, Solomon MJ. The anaphase-promoting complex: it's not just for mitosis any more. Genes Dev,2002,16:2179-2206.
10. Peters JM. The anaphase promoting complex/cyclosome:a machine designed to destroy. Nat Rev Mol Cell Biol,2006,7:644-656.
11. Kim AH and Bonni A. Thinking within the D box:initial identification of Cdh1-APC substrates in the nervous system. Mol Cell Neurosci,2007,34:281-287.
12. Almeida A, Bolanos JP, Moreno S. Cdhl/Hctl-APC is essential for the survival of postmitotic neurons. J Neurosci,2005,25: 8115-8121.
13. Konishi Y, Bonni A. The E2F-Cdc2 cell-cycle pathway specifically mediates activity deprivation-induced apoptosis of postmitotic neurons. J Neurosci,2003,23:1649-1658.
14. Kaplow ME, Korayem AH, Venkatesh TR. Regulation of glia number in Drosophila by Rap/Fzr, an activator of the anaphase-promoting complex, and Loco, an RGS protein. Genetics,2008,178:2003-16.
15. Nakayama KI, Hatakeyama S, Nakayama K. Regulation of the cell cycle at the G1-S transition by proteolysis of cyclin E and p27Kipl. Biochem Biophys Res Commun,2001,282:853-860.
16.姚文龙,柳璐,张传汉.Cdhl-APC在中枢神经系统中的作用.中风与神经疾病杂志.2006,23：751-752.
17. Bashir T, Dorrello NV, Amador V, et al. Control of the SCF(Skp2-Cksl) ubiquitin ligase by the APC/C(Cdh1) ubiquitin ligase. Nature,2004,428:190-193.
18. Liu W, Wu G, Li W, et al. Cdhl-anaphase-promoting complex targets Skp2 for destruction in transforming growth factor beta-induced growth inhibition. Mol Cell Biol,2007,27:2967-2979.
1 Peters JM. The anaphase-promoting complex:Proteolysis in mitosis and beyond. Mol Cell,2002,9:931-943.
2. 姚文龙,柳璐,张传汉.Cdhl-APC在中枢神经系统中的作用.中风与神经疾病杂志.2006,23：751-752.
3. Gieffers C, Peters BH, Kramer ER, et al. Expression of the CDH1-associated form of the anaphase-promoting complex in postmitotic neurons. Proc Natl Acad Sci USA,1999,96:11317-11322.
4. Konishi Y, Stegmuller J, Matsuda T, et al. Cdhl-APC controls axonal growth and patterning in the mammalian brain. Science,2004,303:1026-1030.
5. Almeida A, Bolanos JP, Moreno S. Cdhl/Hctl-APC is essential for the survival of postmitotic neurons. J Neurosci,2005,25:8115-8121.
6. 姚文龙,张传汉,钱巍,邱瑾,柳璐,祝畅,桂伶俐.APC-Cdhl在中枢神经系统中的表达分布特点.第四军医大学学报.2009,30：1161-1165.
7. Yao W, Qian W, Zhu C, Gui L, Qiu J, Zhang C.Cdhl-APC is involved in the differentiation of neural stem cells into neurons.Neuroreport,2010,21:39-44.
8. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 —ΔΔCt method. Methods,2001, 25:402-408.
9. Busl KM, Greer DM. Hypoxic-ischemic brain injury:pathophysiology, neuropathology and mechanisms. NeuroRehabilitation.2010,26:5-13.
10.钱巍,姚文龙,祝畅,柳璐,桂伶俐,刘志恒,张传汉.永生化神经前体细胞治疗大鼠局灶性脑缺血的实验研究.中华医学杂志,2008,88：867-870.
11. Cao G, Xing J, Xiao X,et al.Critical role of calpain I in mitochondrial release of apoptosis inducing factor in ischemic neuronal injury. J Neurosci,2007,27: 9278-93.
12. Vogel GNobel Prizes. Gold medal from cellular trash. Science,2004,306: 400-401.
13. Almeida A, Bolanos JP, Moreno S. Cdhl/Hctl-APC is essential for the survival of postmitotic neurons. J Neurosci,2005,25:8115-8121.
14. Kim AH, Bonni A. Thinking within the D box:Initial identification of Cdhl-APC substrates in the nervous system. Mol Cell Neurosci,2007, 34:281-287.
15. Cuende J, Moreno S, Bolanos JP, et al. Retinoic acid downregulates Rael leading to APC (Cdhl) activation and neuroblastoma SH-SY5Y differentiation. Oncogene,2008,27:3339-44.
16. Liu W, Wu G, Li W, et al. Cdhl-anaphase-promoting complex targets Skp2 for destruction in transforming growth factor beta-induced growth inhibition. Mol Cell Biol,2007,27:2967-2979.
17. Almeida A, Bolanos JP, Moreno S. Cdhl/Hctl-APC is essential for the survival of postmitotic neurons. J Neurosci,2005,25:8115-8121.
1. Vogel G.Nobel Prizes. Gold medal from cellular trash. Science,2004, 306:400-401.
2. Konishi Y, Stegmuller J, Matsuda T, et al.Cdhl-APC controls axonal growth and patterning in the mammalian brain. Science,2004,303: 1026-1030.
3.姚文龙,张传汉,钱巍,邱瑾,柳璐,祝畅,桂伶俐.APC-Cdhl在中枢神经系统中的表达分布特点.第四军医大学学报,2009,13：1161-1165.
4. Yao W, Qian W, Zhu C, Gui L, Qiu J, Zhang C. Cdhl-APC is involved in the differentiation of neural stem cells into neurons. Neuroreport,2010,21:39-44
5. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2—ΔΔCt method. Methods, 2001,25:402-408.
6. Tuschl T and Borkhardt A. Small interfering RNAs:a revolutionary tool for the analysis of gene function and gene therapy. Mol Interv, 2002,2:158-167.
7. Kim D and Rossi J. RNAi mechanisms and applications. Biotechniques, 2008,44:613-616.
8. Kosaka Y, Kobayashi N, Fukazawa T, et al. Lentivirus-based gene delivery in mouse embryonic stem cells. Artif Organs,2004,28: 271-277.
9. Geraerts M, Eggermont K, Hernandez-Acosta P, et al. Lentiviral vectors mediate efficient and stable gene transfer in adult neural stem cells in vivo. Hum Gene Ther,2006,17:635-650.
10.姚文龙,张传汉,柳璐,祝畅,邱瑾,桂伶俐,田玉科.大鼠永生化神经前体细胞CDH1小干扰RNA真核载体的构建.中华麻醉学杂志.2008,28：919-921.
11. Nakayama KI, Hatakeyama S, Nakayama K. Regulation of the cell cycle at the G1-S transition by proteolysis of cyclin E and p27Kip1. Biochem Biophys Res Commun,2001.282:853-860.
12. Peters JM. The anaphase-promoting complex:proteolysis in mitosis and beyond. Mol Cell,2002,9:931-943.
13. Bashir T, Dorrello NV, Amador V, et al. Control of the SCF (Skp2-Cks1) ubiquitin ligase by the APC/C (Cdh1) ubiquitin ligase. Nature,2004.428:190-193.
14. Liu W, Wu G, Li W, et al. Cdh1-anaphase-promoting complex targets Skp2 for destruction in transforming growth factor beta-induced growth inhibition. Mol Cell Biol,2007,27:2967-2979.
15. Kim AH and Bonni A. Thinking within the D box:initial identification of Cdh1-APC substrates in the nervous system. Mol Cell Neurosci,2007,34:281-287.
16. Tatton WG, Chalmers-Redman R, Brown D, et al. Apoptosis in Parkinson's disease:signals for neuronal degradation. Ann Neurol. 2003,53:61-72.
1. Vogel G. Nobel Prizes. Gold medal from cellular trash. Science,2004, 306:400-401.
2.姚文龙,柳璐,张传汉.Cdhl-APC在中枢神经系统中的作用.中风与神经疾病杂志.2006,23：751-752.
3. Konishi Y, Stegmuller J, Matsuda T, et al. Cdhl-APC controls axonal growth and patterning in the mammalian brain. Science,2004,303: 1026-1030.
4.姚文龙,张传汉,钱巍,邱瑾,柳璐,祝畅,桂伶俐.APC-Cdhl在中枢神经系统中的表达分布特点.第四军医大学学报.2009,30：1161-1165.
5. Pfleger CM, Kirschner MW. The KEN box:an APC recognition signal distinct from the D box targeted by Cdhl. Genes Dev,2000,14: 655-665.
6. Littlepage LE, Ruderman JV. Identification of a new APC/C recognition domain, the A box, which is required for the Cdh1-dependent destruction of the kinase Aurora-A during mitotic exit. Genes Dev,2002,16:2274-2285.
7. Reis A, Levasseur M, Chang HY, et al. The CRY box:a second APCcdhl-dependent degron in mammalian cdc20. EMBO Rep,2006,7: 1040-1045.
8. Yamashita YM, Nakaseko Y, Samejima I, et al.20S cyclosome complex formation and proteolytic activity inhibited by the Camp/PKA pathway. Nature,1996,384:276-279.
9. Kotani S, Tugendreich S, Fujii M, et al. PKA and MPF-activated polo-like kinase regulate anaphase-promoting complex activity and mitosis progression. Mol Cell,1998,1:371-380.
10. Rudner AD, Murray AW. Phosphorylation by Cdc28 activates the Cdc20-dependent activity of the anaphase-promoting complex. J Cell Biol,2000,149:1377-1390.
11. Kraft C, Herzog F, Gieffers C, et al. Mitotic regulation of the human anaphase-promoting complex by phosphorylation. EMBO J,2003, 22:6598-6609.
12. Lukas C, SΦrensen CS, Kramer E, et al. Accumulation of cyclin B1 requires E2F and cyclin-A-dependent rearrangement of the anaphase-promoting complex. Nature,1999,401:815-818.
13.Zachariae W, Schwab M, Nasmyth K, et al. Control of cyclin ubiquitination by CDK-regulated binding of Hctl to the anaphase promoting complex. Science,1998,282:1721-1724.
14. Jaspersen SL, Charles JF, Morgan DO. Inhibitory phosphorylation of the APC regulator Hctl is controlled by the kinase Cdc28 and the phosphatase Cdc14. Curr Biol,1999,9:227-36.
15. Hsu JY, Reimann JD, SΦrensen CS, et al. E2F-dependent accumulation of hEmil regulates S phase entry by inhibiting APC(Cdh1). Nat Cell Biol,2002,4:358-366.
16.Turnell AS, Stewart GS, Grand RJ, et al. The APC/C and CBP/p300 cooperate to regulate transcription and cell-cycle progression. Nature,2005,438:690-695.
17. Listovsky T, Oren YS, Yudkovsky Y, et al. Mammalian Cdhl/Fzr mediates its own degradation. EMBO J,2004,23:1619-1626.
18. Rape M, Kirschner MW. Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry. Nature,2004,432:588-595.
19. Starborg M, Brundell E, Gell K, et al. A novel murine gene encoding a 216-kDa protein is related to a mitotic checkpoint regulator previously identified in Aspergillus nidulans. J Biol Chem,1994, 269:24133-7.
20. Zhao N, Lai F, Fernald AA, et al. Human CDC23:Cdna cloning, mapping to 5q31, genomic structure, and evaluation as a candidate tumor suppressor gene in myeloid leukemias. Genomics,1998,53:184-90.
21.Giefers C, Peters BH, Kramer ER, et al. Expression of the CDHl-associated form of the anaphase-promoting complex in postmitotic neurons. Proc Natl Acad Sci USA,1999,96:11317-11322.
22. Becker EB, Bonni A. Cell cycle regulation of neuronal apoptosis in development and disease. Prog Neurobiol,2004,72:1-25.
23. Greene LA, Biswas SC, Liu DX. Cell cycle molecules and vertebrate neuron death:E2F at the hub. Cell Death Differ,2004,11:49-60.
24. Peters JM. The anaphase promoting complex/cyclosome:a machine designed to destroy. Nat Rev Mol Cell Biol,2006,7:644-656.
25. Almeida A, Bolanos JP, Moreno S. Cdh1/Hctl-APC is essential for the survival of postmitotic neurons. J Neurosci,2005,25: 8115-8121.
26. Konishi Y, Bonni A. The E2F-Cdc2 cell-cycle pathway specifically mediates activity deprivation-induced apoptosis of postmitotic neurons. J Neurosci,2003,23:1649-1658.
27. DiAntonio A, Haghlghi AP, Portman SL, Et al. Ubiquitination-dependent mechanisms regulate synaptic growth and function. Nature,2001,412:449-452.
28. Zhao Y, Hegde AN, Martin KC. The ubiquitin proteasome systern functions as an inhibitory constraint on synaptic strengthening. Curr Biol,2003,13:887-898.
29. Speese SD, Trotta N。Rodeseh CK, et al. The ubiquitin proteasome system acutely regulates presynaptie protein turnover and synaptic efficacy. Curr Biol,2003,13:899-910.
30. Juo P, Kaplan JM. The anaphase-promoting complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of celegans. Curt Biol,2004,14:2057-2062.
31. van Roessel P, Elliott DA, Robinson IM, et al. Independent regulation of synaptic size and activity by the anaphase-promoting complex. Cell,2004,119:707-718.
32. Li M, Shin YH, Hou L, et al. The adaptor protein of the anaphase promoting complex Cdhl is essential in maintaining replicative lifespan and in learning and memory. Nat Cell Biol,2008,10: 1083-1089.
33. Moss A, Blackburn-Munro G, Garry EM, et al. A role of the ubiquitin-proteasome system in neuropathic pain. J Neurosci 2002, 22:1363-1372.
34. Ossipov MH, Bazov I, Gardell LR, et al. Control of chronic pain by the ubiquitin proteasome system in the spinal cord. J Neurosci 2007,27:8226-8237.
35. Stegmuller J, Konishi Y, Huynh MA, et al. Cell-intrinsic regulation of axonal morphogenesis by the Cdhl-APC target SnoN. Neuron,2006, 50:389-400.
36.祁月红,钱巍,李平,姚文龙,万里,张传汉.慢病毒介导RNA干扰Cdh1的表达对大鼠脊髓损伤的修复作用.第四军医大学学报,2009,30：1353-1356.
37. Wu J, Yoo S, Wilcock D, et al.Interaction of NG2(+) glial progenitors and microglia/macrophages from the injured spinal cord. Glia,2010,58:410-22.
38. Lasorella A, Stegmuller J, Guardavaccaro D, et al. Degradation of Id2 by the anaphase-promoting complex couples cell cycle exit and axonal growth. Nature,2006,442:471-4.
39. Kaplow ME, Korayem AH, Venkatesh TR. Regulation of glia number in Drosophila by Rap/Fzr, an activator of the anaphase-promoting complex, and Loco, an RGS protein. Genetics,2008,178:2003-16.
2. Vogel G. Nobel Prizes. Gold medal from cellular trash, science,2004, 306:400-401.
3. Pfleger CM, Kirschner MW. The KEN box:an APC recognition signal distinct from the D box targeted by Cdhl. Genes Dev,2000,14: 655-665.
4. Littlepage LE, Ruderman JV. Identification of a new APC/C recognition domain, the A box, which is required for the Cdhl-dependent destruction of the kinase Aurora-A during mitotic exit. Genes Dev,2002,16:2274-2285.
5. Reis A, Levasseur M, Chang HY, et al. The CRY box:a second APCcdhl-dependent degron in mammalian cdc20. EMBO Rep,2006,7: 1040-1045.
6.姚文龙,柳璐,张传汉.Cdhl-APC在中枢神经系统中的作用.中风与神经疾病杂志.2006,23：751-752.
7. Yamashita YM, Nakaseko Y, Samejima I, et al.20S cyclosome complex formation and proteolytic activity inhibited by the Camp/PKA pathway. Nature,1996 384:276-279.
8. Kotani S, Tugendreich S, Fujii M, et al. PKA and MPF-activated polo-like kinase regulate anaphase-promoting complex activity and mitosis progression. Mol Cell,1998,1:371-380.
9. Rudner AD, Murray AW. Phosphorylation by Cdc28 activates the Cdc20-dependent activity of the anaphase-promoting complex. J Cell Biol,2000,149:1377-1390.
10. Kraft C, Herzog F, Gieffers C, et al. Mitotic regulation of the human anaphase-promoting complex by phosphorylation. EMBO J,2003, 22:6598-6609.
11. Lukas C, SΦrensen CS, Kramer E, et al. Accumulation of cyclin B1 requires E2F and cyclin-A-dependent rearrangement of the anaphase-promoting complex. Nature,1999,401:815-818.
12. Zachariae W, Schwab M, Nasmyth K, et al. Control of cyclin ubiquitination by CDK-regulated binding of Hctl to the anaphase promoting complex. Science,1998,282:1721-1724.
13. Jaspersen SL, Charles JF, Morgan DO. Inhibitory phosphorylation of the APC regulator Hctl is controlled by the kinase Cdc28 and the phosphatase Cdc14. Curr Biol,1999,9:227-36.
14. Hsu JY, Reimann JD, SΦrensen CS, et al. E2F-dependent accumulation of hEmil regulates S phase entry by inhibiting APC(Cdh1). Nat Cell Biol,2002,4:358-366.
15. Turnell AS, Stewart GS, Grand RJ, et al. The APC/C and CBP/p300 cooperate to regulate transcription and cell-cycle progression. Nature,2005,438:690-695.
16. Listovsky T, Oren YS, Yudkovsky Y, et al. Mammalian Cdh1/Fzr mediates its own degradation. EMBO J,2004,23:1619-1626.
17. Rape M, Kirschner MW. Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry. Nature,2004,432:588-595.
18. Starborg M, Brundell E, Gell K, et al. A novel murine gene encoding a 216-kDa protein is related to a mitotic checkpoint regulator previously identified in Aspergillus nidulans. J Biol Chem,1994, 269:24133-7.
19. Giefers C, Peters BH, Kramer ER, et al. Expression of the CDH1-associated form of the anaphase-promoting complex in postmitotic neurons. Proc Natl Acad Sci USA,1999,96:11317-11322.
20.姚文龙,张传汉,钱巍,邱瑾,柳璐,祝畅,桂伶俐.APC-Cdhl在中枢神经系统中的表达分布特点.第四军医大学学报.2009,30：1161-1165.
21. Konishi Y, Stegmuller J, Matsuda T, et al. Cdhl-APC controls axonal growth and patterning in the mammalian brain. Science,2004,303: 1026-1030.
22. Zhao N, Lai F, Fernald AA, et al. Human CDC23:Cdna cloning, mapping to 5q31, genomic structure, and evaluation as a candidate tumor suppressor gene in myeloid leukemias. Genomics,1998,53:184-90.
23.姚文龙,张传汉,柳璐,祝畅,邱瑾,桂伶俐,田玉科.大鼠永生化神经前体细胞CDH1小干扰RNA真核载体的构建.中华麻醉学杂志.2008,28：919-921.
24. Yao W, Qian W, Zhu C, Gui L, Qiu J, Zhang C. Cdhl-APC is involved in the differentiation of neural stem cells into neurons. Neuroreport,2010,21:39-44.
25. Cuende J, Moreno S, Bolanos JP, et al. Retinoic acid downregulates Rael leading to APC (Cdh1) activation and neuroblastoma SH-SY5Y differentiation. Oncogene,2008,27:3339-3344.
26. Harmey D, Smith A, Simanski S, et al. The Anaphase Promoting Complex Induces Substrate Degradation during Neuronal Differentiation. J Biol Chem,2009,284:4317-4323.
27. Stegmuller J, Konishi Y, Huynh MA, et al. Cell-intrinsic regulation of axonal morphogenesis by the Cdhl-APC target SnoN. Neuron,2006,50:389-400.
28.祁月红,钱巍,李平,姚文龙,万里,张传汉.慢病毒介导RNA干扰Cdh1的表达对大鼠脊髓损伤的修复作用.第四军医大学学报,2009,30：1353-1356.
29. Becker EB, Bonni A. Cell cycle regulation of neuronal apoptosis in development and disease. Prog Neurobiol,2004,72:1-25.
30. Greene LA, Biswas SC, Liu DX. Cell cycle molecules and vertebrate neuron death:E2F at the hub. Cell Death Differ,2004,11:49-60.
31. Peters JM. The anaphase promoting complex/cyclosome:a machine designed to destroy. Nat Rev Mol Cell Biol,2006,7:644-656.
32. Almeida A, Bolanos JP, Moreno S. Cdhl/Hctl-APC is essential for the survival of postmitotic neurons. J Neurosci,2005,25: 8115-8121.
33. Konishi Y, Bonni A. The E2F-Cdc2 cell-cycle pathway specifically mediates activity deprivation-induced apoptosis of postmitotic neurons. J Neurosci,2003,23:1649-1658.
34. Kaplow ME, Korayem AH, Venkatesh TR. Regulation of glia number in Drosophila by Rap/Fzr, an activator of the anaphase-promoting complex, and Loco, an RGS protein. Genetics.2008,178:2003-16.
1. Busl KM, Greer DM. Hypoxic-ischemic brain injury:pathophysiology, neuropathology and mechanisms. NeuroRehabilitation. 2010,26:5-13.
2. Vogel G. Nobel Prizes. Gold medal from cellular trash, science,2004, 306:400-401.
3. Konishi Y, Stegmuller J, Matsuda T, et al. Cdhl-APC controls axonal growth and patterning in the mammalian brain. Science,2004,303: 1026-1030.
4. Pfleger CM, Kirschner MW. The KEN box:an APC recognition signal distinct from the D box targeted by Cdhl. Genes Dev,2000,14: 655-665.
5.姚文龙,张传汉,钱巍,邱瑾,柳璐,祝畅,桂伶俐.APC-Cdhl在中枢神经系统中的表达分布特点.第四军医大学学报.2009,30：1161-1165.
6. Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989,20:84-91.
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8.钱巍,姚文龙,祝畅,柳璐,桂伶俐,刘志恒,张传汉.永生化神经前体细胞治疗大鼠局灶性脑缺血的实验研究.中华医学杂志,2008,88：867-870.
9. Harper JW, Burton JL, Solomon MJ. The anaphase-promoting complex: it's not just for mitosis any more. Genes Dev,2002,16:2179-2206.
10. Peters JM. The anaphase promoting complex/cyclosome:a machine designed to destroy. Nat Rev Mol Cell Biol,2006,7:644-656.
11. Kim AH and Bonni A. Thinking within the D box:initial identification of Cdh1-APC substrates in the nervous system. Mol Cell Neurosci,2007,34:281-287.
12. Almeida A, Bolanos JP, Moreno S. Cdhl/Hctl-APC is essential for the survival of postmitotic neurons. J Neurosci,2005,25: 8115-8121.
13. Konishi Y, Bonni A. The E2F-Cdc2 cell-cycle pathway specifically mediates activity deprivation-induced apoptosis of postmitotic neurons. J Neurosci,2003,23:1649-1658.
14. Kaplow ME, Korayem AH, Venkatesh TR. Regulation of glia number in Drosophila by Rap/Fzr, an activator of the anaphase-promoting complex, and Loco, an RGS protein. Genetics,2008,178:2003-16.
15. Nakayama KI, Hatakeyama S, Nakayama K. Regulation of the cell cycle at the G1-S transition by proteolysis of cyclin E and p27Kipl. Biochem Biophys Res Commun,2001,282:853-860.
16.姚文龙,柳璐,张传汉.Cdhl-APC在中枢神经系统中的作用.中风与神经疾病杂志.2006,23：751-752.
17. Bashir T, Dorrello NV, Amador V, et al. Control of the SCF(Skp2-Cksl) ubiquitin ligase by the APC/C(Cdh1) ubiquitin ligase. Nature,2004,428:190-193.
18. Liu W, Wu G, Li W, et al. Cdhl-anaphase-promoting complex targets Skp2 for destruction in transforming growth factor beta-induced growth inhibition. Mol Cell Biol,2007,27:2967-2979.
1 Peters JM. The anaphase-promoting complex:Proteolysis in mitosis and beyond. Mol Cell,2002,9:931-943.
2. 姚文龙,柳璐,张传汉.Cdhl-APC在中枢神经系统中的作用.中风与神经疾病杂志.2006,23：751-752.
3. Gieffers C, Peters BH, Kramer ER, et al. Expression of the CDH1-associated form of the anaphase-promoting complex in postmitotic neurons. Proc Natl Acad Sci USA,1999,96:11317-11322.
4. Konishi Y, Stegmuller J, Matsuda T, et al. Cdhl-APC controls axonal growth and patterning in the mammalian brain. Science,2004,303:1026-1030.
5. Almeida A, Bolanos JP, Moreno S. Cdhl/Hctl-APC is essential for the survival of postmitotic neurons. J Neurosci,2005,25:8115-8121.
6. 姚文龙,张传汉,钱巍,邱瑾,柳璐,祝畅,桂伶俐.APC-Cdhl在中枢神经系统中的表达分布特点.第四军医大学学报.2009,30：1161-1165.
7. Yao W, Qian W, Zhu C, Gui L, Qiu J, Zhang C.Cdhl-APC is involved in the differentiation of neural stem cells into neurons.Neuroreport,2010,21:39-44.
8. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 —ΔΔCt method. Methods,2001, 25:402-408.
9. Busl KM, Greer DM. Hypoxic-ischemic brain injury:pathophysiology, neuropathology and mechanisms. NeuroRehabilitation.2010,26:5-13.
10.钱巍,姚文龙,祝畅,柳璐,桂伶俐,刘志恒,张传汉.永生化神经前体细胞治疗大鼠局灶性脑缺血的实验研究.中华医学杂志,2008,88：867-870.
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12. Vogel GNobel Prizes. Gold medal from cellular trash. Science,2004,306: 400-401.
13. Almeida A, Bolanos JP, Moreno S. Cdhl/Hctl-APC is essential for the survival of postmitotic neurons. J Neurosci,2005,25:8115-8121.
14. Kim AH, Bonni A. Thinking within the D box:Initial identification of Cdhl-APC substrates in the nervous system. Mol Cell Neurosci,2007, 34:281-287.
15. Cuende J, Moreno S, Bolanos JP, et al. Retinoic acid downregulates Rael leading to APC (Cdhl) activation and neuroblastoma SH-SY5Y differentiation. Oncogene,2008,27:3339-44.
16. Liu W, Wu G, Li W, et al. Cdhl-anaphase-promoting complex targets Skp2 for destruction in transforming growth factor beta-induced growth inhibition. Mol Cell Biol,2007,27:2967-2979.
17. Almeida A, Bolanos JP, Moreno S. Cdhl/Hctl-APC is essential for the survival of postmitotic neurons. J Neurosci,2005,25:8115-8121.
1. Vogel G.Nobel Prizes. Gold medal from cellular trash. Science,2004, 306:400-401.
2. Konishi Y, Stegmuller J, Matsuda T, et al.Cdhl-APC controls axonal growth and patterning in the mammalian brain. Science,2004,303: 1026-1030.
3.姚文龙,张传汉,钱巍,邱瑾,柳璐,祝畅,桂伶俐.APC-Cdhl在中枢神经系统中的表达分布特点.第四军医大学学报,2009,13：1161-1165.
4. Yao W, Qian W, Zhu C, Gui L, Qiu J, Zhang C. Cdhl-APC is involved in the differentiation of neural stem cells into neurons. Neuroreport,2010,21:39-44
5. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2—ΔΔCt method. Methods, 2001,25:402-408.
6. Tuschl T and Borkhardt A. Small interfering RNAs:a revolutionary tool for the analysis of gene function and gene therapy. Mol Interv, 2002,2:158-167.
7. Kim D and Rossi J. RNAi mechanisms and applications. Biotechniques, 2008,44:613-616.
8. Kosaka Y, Kobayashi N, Fukazawa T, et al. Lentivirus-based gene delivery in mouse embryonic stem cells. Artif Organs,2004,28: 271-277.
9. Geraerts M, Eggermont K, Hernandez-Acosta P, et al. Lentiviral vectors mediate efficient and stable gene transfer in adult neural stem cells in vivo. Hum Gene Ther,2006,17:635-650.
10.姚文龙,张传汉,柳璐,祝畅,邱瑾,桂伶俐,田玉科.大鼠永生化神经前体细胞CDH1小干扰RNA真核载体的构建.中华麻醉学杂志.2008,28：919-921.
11. Nakayama KI, Hatakeyama S, Nakayama K. Regulation of the cell cycle at the G1-S transition by proteolysis of cyclin E and p27Kip1. Biochem Biophys Res Commun,2001.282:853-860.
12. Peters JM. The anaphase-promoting complex:proteolysis in mitosis and beyond. Mol Cell,2002,9:931-943.
13. Bashir T, Dorrello NV, Amador V, et al. Control of the SCF (Skp2-Cks1) ubiquitin ligase by the APC/C (Cdh1) ubiquitin ligase. Nature,2004.428:190-193.
14. Liu W, Wu G, Li W, et al. Cdh1-anaphase-promoting complex targets Skp2 for destruction in transforming growth factor beta-induced growth inhibition. Mol Cell Biol,2007,27:2967-2979.
15. Kim AH and Bonni A. Thinking within the D box:initial identification of Cdh1-APC substrates in the nervous system. Mol Cell Neurosci,2007,34:281-287.
16. Tatton WG, Chalmers-Redman R, Brown D, et al. Apoptosis in Parkinson's disease:signals for neuronal degradation. Ann Neurol. 2003,53:61-72.
1. Vogel G. Nobel Prizes. Gold medal from cellular trash. Science,2004, 306:400-401.
2.姚文龙,柳璐,张传汉.Cdhl-APC在中枢神经系统中的作用.中风与神经疾病杂志.2006,23：751-752.
3. Konishi Y, Stegmuller J, Matsuda T, et al. Cdhl-APC controls axonal growth and patterning in the mammalian brain. Science,2004,303: 1026-1030.
4.姚文龙,张传汉,钱巍,邱瑾,柳璐,祝畅,桂伶俐.APC-Cdhl在中枢神经系统中的表达分布特点.第四军医大学学报.2009,30：1161-1165.
5. Pfleger CM, Kirschner MW. The KEN box:an APC recognition signal distinct from the D box targeted by Cdhl. Genes Dev,2000,14: 655-665.
6. Littlepage LE, Ruderman JV. Identification of a new APC/C recognition domain, the A box, which is required for the Cdh1-dependent destruction of the kinase Aurora-A during mitotic exit. Genes Dev,2002,16:2274-2285.
7. Reis A, Levasseur M, Chang HY, et al. The CRY box:a second APCcdhl-dependent degron in mammalian cdc20. EMBO Rep,2006,7: 1040-1045.
8. Yamashita YM, Nakaseko Y, Samejima I, et al.20S cyclosome complex formation and proteolytic activity inhibited by the Camp/PKA pathway. Nature,1996,384:276-279.
9. Kotani S, Tugendreich S, Fujii M, et al. PKA and MPF-activated polo-like kinase regulate anaphase-promoting complex activity and mitosis progression. Mol Cell,1998,1:371-380.
10. Rudner AD, Murray AW. Phosphorylation by Cdc28 activates the Cdc20-dependent activity of the anaphase-promoting complex. J Cell Biol,2000,149:1377-1390.
11. Kraft C, Herzog F, Gieffers C, et al. Mitotic regulation of the human anaphase-promoting complex by phosphorylation. EMBO J,2003, 22:6598-6609.
12. Lukas C, SΦrensen CS, Kramer E, et al. Accumulation of cyclin B1 requires E2F and cyclin-A-dependent rearrangement of the anaphase-promoting complex. Nature,1999,401:815-818.
13.Zachariae W, Schwab M, Nasmyth K, et al. Control of cyclin ubiquitination by CDK-regulated binding of Hctl to the anaphase promoting complex. Science,1998,282:1721-1724.
14. Jaspersen SL, Charles JF, Morgan DO. Inhibitory phosphorylation of the APC regulator Hctl is controlled by the kinase Cdc28 and the phosphatase Cdc14. Curr Biol,1999,9:227-36.
15. Hsu JY, Reimann JD, SΦrensen CS, et al. E2F-dependent accumulation of hEmil regulates S phase entry by inhibiting APC(Cdh1). Nat Cell Biol,2002,4:358-366.
16.Turnell AS, Stewart GS, Grand RJ, et al. The APC/C and CBP/p300 cooperate to regulate transcription and cell-cycle progression. Nature,2005,438:690-695.
17. Listovsky T, Oren YS, Yudkovsky Y, et al. Mammalian Cdhl/Fzr mediates its own degradation. EMBO J,2004,23:1619-1626.
18. Rape M, Kirschner MW. Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry. Nature,2004,432:588-595.
19. Starborg M, Brundell E, Gell K, et al. A novel murine gene encoding a 216-kDa protein is related to a mitotic checkpoint regulator previously identified in Aspergillus nidulans. J Biol Chem,1994, 269:24133-7.
20. Zhao N, Lai F, Fernald AA, et al. Human CDC23:Cdna cloning, mapping to 5q31, genomic structure, and evaluation as a candidate tumor suppressor gene in myeloid leukemias. Genomics,1998,53:184-90.
21.Giefers C, Peters BH, Kramer ER, et al. Expression of the CDHl-associated form of the anaphase-promoting complex in postmitotic neurons. Proc Natl Acad Sci USA,1999,96:11317-11322.
22. Becker EB, Bonni A. Cell cycle regulation of neuronal apoptosis in development and disease. Prog Neurobiol,2004,72:1-25.
23. Greene LA, Biswas SC, Liu DX. Cell cycle molecules and vertebrate neuron death:E2F at the hub. Cell Death Differ,2004,11:49-60.
24. Peters JM. The anaphase promoting complex/cyclosome:a machine designed to destroy. Nat Rev Mol Cell Biol,2006,7:644-656.
25. Almeida A, Bolanos JP, Moreno S. Cdh1/Hctl-APC is essential for the survival of postmitotic neurons. J Neurosci,2005,25: 8115-8121.
26. Konishi Y, Bonni A. The E2F-Cdc2 cell-cycle pathway specifically mediates activity deprivation-induced apoptosis of postmitotic neurons. J Neurosci,2003,23:1649-1658.
27. DiAntonio A, Haghlghi AP, Portman SL, Et al. Ubiquitination-dependent mechanisms regulate synaptic growth and function. Nature,2001,412:449-452.
28. Zhao Y, Hegde AN, Martin KC. The ubiquitin proteasome systern functions as an inhibitory constraint on synaptic strengthening. Curr Biol,2003,13:887-898.
29. Speese SD, Trotta N。Rodeseh CK, et al. The ubiquitin proteasome system acutely regulates presynaptie protein turnover and synaptic efficacy. Curr Biol,2003,13:899-910.
30. Juo P, Kaplan JM. The anaphase-promoting complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of celegans. Curt Biol,2004,14:2057-2062.
31. van Roessel P, Elliott DA, Robinson IM, et al. Independent regulation of synaptic size and activity by the anaphase-promoting complex. Cell,2004,119:707-718.
32. Li M, Shin YH, Hou L, et al. The adaptor protein of the anaphase promoting complex Cdhl is essential in maintaining replicative lifespan and in learning and memory. Nat Cell Biol,2008,10: 1083-1089.
33. Moss A, Blackburn-Munro G, Garry EM, et al. A role of the ubiquitin-proteasome system in neuropathic pain. J Neurosci 2002, 22:1363-1372.
34. Ossipov MH, Bazov I, Gardell LR, et al. Control of chronic pain by the ubiquitin proteasome system in the spinal cord. J Neurosci 2007,27:8226-8237.
35. Stegmuller J, Konishi Y, Huynh MA, et al. Cell-intrinsic regulation of axonal morphogenesis by the Cdhl-APC target SnoN. Neuron,2006, 50:389-400.
36.祁月红,钱巍,李平,姚文龙,万里,张传汉.慢病毒介导RNA干扰Cdh1的表达对大鼠脊髓损伤的修复作用.第四军医大学学报,2009,30：1353-1356.
37. Wu J, Yoo S, Wilcock D, et al.Interaction of NG2(+) glial progenitors and microglia/macrophages from the injured spinal cord. Glia,2010,58:410-22.
38. Lasorella A, Stegmuller J, Guardavaccaro D, et al. Degradation of Id2 by the anaphase-promoting complex couples cell cycle exit and axonal growth. Nature,2006,442:471-4.
39. Kaplow ME, Korayem AH, Venkatesh TR. Regulation of glia number in Drosophila by Rap/Fzr, an activator of the anaphase-promoting complex, and Loco, an RGS protein. Genetics,2008,178:2003-16.