【作者】 钱巍；

【导师】 张传汉；

【作者基本信息】 华中科技大学 ， 麻醉学， 2010， 博士

【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。更多还原

【Abstract】 Background and objectiveAnaphase-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 Results1. Changes in the Expression of APC-Cdhl in Rats with focal cerebral ischemiaMethods: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 neuronsMethod: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 survivalMethod: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 AnalysisAll 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. Conclusions1. 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 SummaryThe 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.更多还原