凋亡调控因子在凝血酶诱导海马神经元凋亡中的变化

目的探讨凝血酶（TM）对海马神经元凋亡调控凶子Bcl-2、Bax及c-jun氨基末端激酶（JNK）表达的影响，阐明TM诱导凋亡的机制。方法海马神经元经40U／mLTM作用0h、6h、12h、24h、48h、72h后终止培养，应用免疫细胞化学方法检测Bcl-2及Bax蛋白表达，Western blot检测磷酸化JNK（P—JNK）及JNK蛋白表达。结果TM作用6h后，Bcl-2蛋白表达增加，12h后开始下降，到48h达到最低值（P〈0．01），72h后Bcl-2蛋白表达开始回升。TM作用6h后，Bax蛋白表达增加，随作用时间的延长，Bax蛋白表达水平逐渐上升，到48h达到最高值（P〈0．01）。Bcl-2／Bax比值逐渐降低，在48h达最低。TM作用0h时P-JNK蛋白无表达，6h后开始表达，与0h组比较差异存在显著性（P〈0．011，12h时达高峰（P〈0．01），并持续到24h，48h后逐渐降低。0h组可见有少量JNK蛋白表达，6h后JNK蛋白表达显著增加（P〈0．01），随TM作用时间的延长，JNK蛋白表达无明显变化。结论TM可能是通过抑制Bcl-2的表达，增强Bax的表达，降低Bcl-2和Bax的比值，激活JNK信号传导通路导致海马神经元凋亡。     

局灶性脑缺血再灌注大鼠海马CA1区神经元凋亡研究

目的 观察大鼠局灶性脑缺血再灌注(ischemia reperfusion,I/R)损伤后海马CA1区神经元凋亡、TUNEL阳性细胞变化,以及凋亡相关蛋白Bcl-2与Bax蛋白的表达情况.方法 将健康雄性SD (Sprague-Dawley)大鼠随机分为假手术组和I/R组,每组再分为缺血再灌注后3、6、12、24、48、72 h亚组.应用免疫组化方法检测再灌注后不同时间点大鼠海马CA1区神经元凋亡基因Bcl-2和Bax蛋白的表达及Bcl-2/Bax比值变化,采用原位细胞凋亡检测(TUNEL)技术检测凋亡阳性细胞数.结果 各组非缺血侧相应区域神经元胞质中Bcl-2均有微弱表达.I/R组缺血侧海马CA1区于再灌注3 h开始出现Bcl-2和Bax蛋白微弱表达,随再灌注时间延长神经元内Bcl-2表达逐渐增强,再灌注24 h后Bcl-2表达达高峰,假手术组与I/R组比较差异有统计学意义(P<0.05).结论 I/R损伤后海马CA1区神经元不仅存在变性坏死,还存在明显的细胞凋亡且细胞凋亡在大鼠I/R损伤中发挥重要作用;I/R可诱导海马CA1区细胞凋亡基因Bcl-2和Bax蛋白表达,且其表达呈一定规律.     

神经营养因子-3对海马神经元缺氧损伤保护作用的研究

目的观察单纯缺氧损伤对体外培养海马神经元内源性神经营养因子-3(neurotrophin-3, NT-3)表达的影响及外源性NT-3转导对单纯缺氧所致神经元凋亡的保护作用。方法体外分散培养新生Wistar大鼠海马神经元,体外培养第7天通过充氮法建立单纯缺氧损伤模型;用重组腺病毒载体pAC- CMV-PLPA构建携带NT-3全长cDNA的重组腺病毒Ad-NT-3,并分别于损伤前后向体外培养的海马神经元转导外源性NT-3;采用Western Blot检测在缺氧损伤前后及有无外源性NT-3转导的情况下NT-3 及Bcl-2的表达水平;采用TUNEL法检测缺氧及外源性NT-3转导后神经元凋亡的情况。结果 (1)单纯缺氧损伤后海马神经元的凋亡细胞标记指数由15．2％上升至56．4％,内源性NT-3表达量下降至对照组水平的71％。(2)缺氧损伤前重组腺病毒转导可使损伤后NT-3表达量上升至对照组的1.88倍、损伤后重组腺病毒转导亦可使NT-3表达量上升至对照组的1．42倍,而Bcl-2的表达量相应地上升至对照组的 1．69倍和1．32倍,凋亡细胞标记指数降至32．8％和45．4％。(3)统计学分析显示,海马神经元NT-3与Bcl- 2表达量间呈显著性正相关,二者与凋亡细胞标记指数间均呈显著性负相关。结论单纯缺氧损伤可使体外培养的海马神经元内源性NT-3表达量下降;腺病毒转导的外源性NT-3可保护单纯缺氧损伤神经元免于凋亡;其保护作用部分可能是通过对Bcl-2表达的诱导实现的。     

促红细胞生成素对低氧培养的海马神经元凋亡的影响

目的 探讨促红细胞生成素(EPO)对低氧条件下原代培养的海马神经元凋亡的影响及其可能机制.方法 培养7 d的大鼠海马神经元随机分为常氧对照组、低氧对照组和重组人促红细胞生成素(rHuEPO)低氧处理组(简称rHuEPO处理组,又分100 IU/mL、150 IU/mL两亚组),以四唑盐(MTT)比色法测定培养12、24、36 h的细胞存活率,以Western blot蛋白印迹法测定上述时间点B细胞白血病-淋巴瘤-2(Bcl-2)和Bcl-2相关X蛋白(Bax)表达.结果 EPO可明显增加低氧培养的海马神经元存活能力(P<0.01),但两剂量组之间差异无统计学意义;rHuEPO处理组神经元Bcl-2表达比同时间点低氧对照组明显增多(P<0.05),而Bax表达比低氧对照组明显减少(均P<0.01).结论 EPO可明显增加低氧培养的海马神经元存活能力,其作用机制可能通过调控Bcl-2和Bax表达实现.     

粒细胞-巨噬细胞集落刺激因子对体外培养的大鼠海马神经元氧糖剥夺后凋亡的影响

目的 探讨粒细胞-巨噬细胞集落刺激因子(GM-CSF)对体外培养大鼠海马神经元氧糖剥夺(OGD)后凋亡的影响.方法 体外培养乳鼠海马神经元并分为正常对照组、OGD组、GM-CSF 1 ng/ml、10ng/ml、20 ng/ml和100 ng/ml组;制备OGD模型,并给与相应剂量的GM-CSF干预.应用流式细胞仪及Annexin V/PI双染色法检测神经元凋亡率,测定上清液乳酸脱氢酶(LDH)活性了解神经元细胞膜损伤程度,RT-PCR法检测凋亡相关基因Bcl-2、Bax mRNA表达.结果 与正常对照组相比,OGD组细胞凋亡率及LDH活性明显升高(均P＜0.01);与OGD组相比,除GM-CSF 1 ng/ml组外,GM-CSF各浓度组神经元凋亡率及LDH活性明显降低(均P＜0.01),其中GM-CSF 20 ng/ml组对神经元凋亡影响最显著.与正常对照组比较,OGD组Bcl-2 mRNA表达明显下降,Bax mRNA表达显著升高,Bcl-2/Bax降低(均P＜0.01);与OGD组比较,GM-CSF 20 ng/ml组Bcl-2 mRNA表达升高,Bax mRNA表达降低,Bcl-2/Bax增加(均P＜0.01).结论 GM-CSF能够有效地抑制OGD后海马神经元的凋亡,20 ng/ml抗凋亡的效果最佳.其神经保护作用机制可能与其调节凋亡蛋白Bcl-2、Bax表达有关.     

糖皮质激素对大鼠海马神经元影响的体外研究

目的　探讨在体外条件下糖皮质激素 (glucocorticoids ,GCs)对海马神经元的影响及其机制。方法　取新生大鼠海马组织进行原代分离培养 ,通过细胞形态学观察、细胞酶学噻唑蓝 (methylthiazolyl,MTT)测定、DNA原位末端标记 (TdT mediateddUTPNickEndLablelingmethod ,TUNEL)法以及凋亡相关蛋白Bcl 2、Bax的表达观察 0、10 - 9、10 - 8、10 - 7、10 - 6 、10 - 5mol/L不同浓度的地塞米松 (dexam ethasone ,DEX)作用 2 4h对大鼠海马神经元的影响。结果　高浓度的DEX可以引起明显的海马神经元形态变化 ,降低海马神经元的活性 ,并通过上调Bax的表达 ,下调Bcl 2的表达促进海马神经元的凋亡。结论　高浓度糖皮质激素可促进海马神经元的凋亡从而降低海马神经元的活性。     

经鼻给予TGFβ1对氯化锂-匹罗卡品诱导的SE大鼠海马神经元凋亡调控因子Bcl-2、Bax蛋白表达的影响

目的探讨经鼻给予TGFβ1(Transforming growth factor-beta1,TGFβ1)对氯化锂-匹罗卡品诱导的癫痫持续状态(status epilepticus,SE)大鼠海马神经元凋亡调控因子Bcl-2、Bax蛋白表达的影响。方法健康雄性SD大鼠60只,随机分为TGF组、Pilo组和正常对照组(Control)。建立氯化锂-匹罗卡品癫痫持续状态模型。采用免疫组化方法检测凋亡相关基因Bcl-2、Bax的蛋白表达。结果 (1)SE后24h、48h、72h,TGF组大鼠海马Bax阳性细胞均较Pilo组显著减少(P<0.05);72h最为明显(P<0.01)。HE染色是对各组大鼠海马神经元的形态结构变化的大体观察。(2)SE后24h、48h、72h,TGF组大鼠海马Bcl-2阳性细胞均较Pilo组显著增加(P<0.05);24h最为明显(P<0.01)。结论经鼻(IN)给予TGFβ1可以显著抑制癫痫持续状态诱导的大鼠海马神经元Bax蛋白的表达,上调Bcl-2蛋白表达,从而发挥神经保护作用。     

一氧化氮在缺氧复氧性神经元凋亡中的作用

目的观察神经元缺氧复氧损伤时NO的动态变化及其与神经元凋亡的关系。方法取孕13~15d ICR小鼠,无菌条件下对胎鼠大脑皮质神经元进行原代分离培养,建立缺氧复氧诱导的皮质神经元凋亡模型。用流式细胞仪检测细胞凋亡率,用硝酸还原酶法测定细胞培养上清液中NO的含量,用MTT测定细胞活性,并在电镜下观察细胞形态学变化。结果经缺氧复氧处理的神经元,随着缺氧时间延长细胞存活率逐渐下降,神经元凋亡率呈时间依赖性升高,NO含量逐渐升高,至缺氧8h复氧24h达到高峰,与正常对照组比较有显著性差异(P<0.01)。细胞超微结构呈现凋亡样改变。结论NO介导了缺氧复氧性神经元凋亡过程。     

脑出血灶周组织Bax、Bcl-2的表达与神经元凋亡关系的实验研究

目的 检测脑出血灶周组织Bax、Bcl-2的表达,探讨与神经元凋亡的关系。方法 对健康家犬30只在DSA介入下刺破大脑中动脉制作脑出血模型,选择出血后6h、12h、24h、48h、72h及96h共6个时间点,每点5只,均于相应时间点处死后取脑．检测出血灶周Bax、Bcl-2及神经元凋亡的表达。结果 Bax及神经元凋亡的表达均于48h达高峰,至96h皆明显下降（与48h比较。P〈0．01）;Bcl-2出血后72h达高峰,至96h末见明显下降（与72h比较,P〉0．05）;Bax的表达与神经元凋亡呈正相关（P〈0．05）;Bcl-2与神经元凋亡的表达出血后48h前均呈正相关（P〈0．05）,48h后均呈负相关倾向,但无统计学差异（P〉0．05）。结论 Bax及Bel-2 参与了ICH后出血灶周神经元凋亡的过程,前者诱导和加重了神经元凋亡,后者有抑制凋亡作用,但显示其强度不足。     

低氧预处理对大鼠海马神经元缺氧耐受性和热休克蛋白表达的影响

采用免疫组织化学方法 ,观察低氧预处理对大鼠海马培养神经元缺氧耐受性和热休克蛋白(Hsp70 )表达的影响。结果显示 ,低氧预处理能诱导海马神经元对Hsp70的表达。经低氧预处理的海马神经元缺氧 复氧后Hsp70表达较对照组明显增强 ,神经元损伤程度减轻 ,神经元存活数明显高于对照组。本结果表明 ,低氧预处理可诱导海马培养神经元对Hsp70的表达 ,并使海马神经元对缺氧产生耐受 ,减少缺氧 复氧后神经元的死亡     

缺氧复氧大鼠海马星形胶质细胞凋亡及Bcl-2和Bax蛋白表达的时间进程变化及异丙酚干预效应

BACKGROUND： Cerebral hippocampal astrocytes are more sensitive.to ischemic injury than neurons. Hypoxic-ischemic brain injury induces profound astrocyte apoptosis, and propofol may protect against astrocyte apoptosis.
OBJECTIVE： To verify the protective effects of propofol against astrocyte apoptosis and to investigate anti-apoptotic Bcl-2 and pro-apoptotic Bax expression in primary cultures of rat hippocampal astrocytes exposed to hypoxia-reoxygenation for different periods of time following propofol treatment.
DESIGN, TIME, AND SETTING： In vitro neural immunocytochemistry was performed at the Central Laboratory of Yunyang Medical College between September 2007 and March 2008.
MATERIALS： A total of 30 Wistar rats, aged 1-3 days, wJth equal numbers of males and females, were included for isolation and culture of .hippocampal astrocytes.
METHODS： Hippocampal astrocytes were purified and cultured for 3 weeks and treated with four culture conditions： 50 μL Hank＇s solution （normal control）; 0.2 mL/L Intralipid; 50 μL Hank＇s solution for 10 minutes followed by hypoxic incubation for 4 hours and normoxic incubation for 12, 24, 36, 48, 60 or 72 hours; propofol （250 μmol/L final） for 10 minutes followed by hypoxic incubation for 4 hours and normoxic incubation for 12, 24, 36, 48, 60 and 72 hours.
MAIN OUTCOME MEASURES： （1） Morphologic changes in hippocampal astrocytes. （2） Levels of astrocyte apoptosis and Bcl-2 and Bax expression.
RESULTS： Hypoxia and reoxygenation increased apoptosis over time, with Bcl-2 expression peaking at 24 hours and decreasing gradually （P 〈 0.01 ）; Bax expression peaked at 72 hours （P 〈 0.01）; the ratio of Bcl-2/Bax was 1.4, 0.8, and 0.6, respectively, at 24, 48 and 72 hours. Non-apoptotic astrocytes showed significant proliferation and swelling. Propofol treatment decreased apoptosis after hypoxia-reoxygenation （P 〈 0.01）, as well as Bct-2 and Bax expression （P 〈 0.05, P 〈 0.01）, with Bcl-2/Bax ratios of 1.6-1.8. Propofol treatmentalso blocked astrocyte proliferation and swelling. No apoptotic cells or Bcl-2/Bax expression was detected in astrocytes cultured in Hank＇s or Intralipid solution.
CONCLUSION： Propofol protects astrocytes against injury caused by hypoxia and reoxygenation via a mechanism that involves maintaining high ratios of Bcl-2/Bax.     

The preconditioning modified neuronal expression of apoptosis-related proteins of Bcl-2 superfamily following severe hypobaric hypoxia in rats

The patterns of expression of the Bcl-2, Bax, and Bcl-xL proteins were examined immunocytochemically in rat hippocampus and neocortex after severe hypobaric hypoxia (180 Torr for 3 h) and severe hypoxia preconditioned by intermittent mild hypoxia (360 Torr for 2 h daily, for 3 consecutive days, 24 h prior to severe hypoxia). As revealed by TUNEL assay, severe hypobaric hypoxia produced extensive apoptotic damage to the neurons of hippocampal CA1-CA4 and the neocortex but not the dentate gyrus granule cells. Remarkable posthypoxic up-regulation of Bax expression maximal at 24 h was detected in the CA1-CA4 areas of hippocampus and neocortex 3-72 h after severe hypoxia. The preconditioning to severe hypoxia protected neurons from the posthypoxic apoptotic transformations, the up-regulation of Bax expression, and resulted in persistent overexpression of Bcl-2 and Bcl-xL. We conclude that the protective action of hypoxic preconditioning is at least in part mediated by shifting of neuronal Bax/Bcl-2-Bcl-xL ratio to a favor of antiapoptotic proteins Bcl-2 and Bcl-xL.     

Bcl-2 and Bax proteins are increased in neocortical but not in thalamic apoptosis following devascularizing lesion of the cerebral cortex in the rat: an immunohistochemical study

The hypothesis that devascularization of somatosensory and motor cortex causes apoptosis in infarcted regions and in the linked thalamic nuclei was evaluated. To unravel whether Bcl-related proteins, known to regulate apoptosis, participate in neuronal and glial responses to devascularization, we analyzed immunohistochemically the distribution and intensity of staining of Bcl-2 and Bax proteins at different time points after lesion. Both early (up to 6 h) and late (1-7 days) responses were studied. Devascularization led to rapid (within hours) apoptosis in the cortex and to a delayed (within 3-7 days) apoptosis in thalamic nuclei. In control groups, Bcl-2 and Bax immunoreactivity (IR) was detected in neurons and oligodendrocytes but not in astrocytes or microglia. Following devascularization, Bcl-2 IR and Bax IR increased in neurons before the onset of the apoptosis. In the ischemic focus, the increase reached maximal values 3 h after the lesion. The increase was of slower onset in the penumbra zone (24 h and after), a region in which both proteins were induced in astrocytes also. The change of Bax IR intensity exceeded four times that of Bcl-2 at all time points investigated, indicating a diminution of Bcl-2/Bax ratio that may direct neurons to apoptotic pathway. In numerous neurons, an increase of IR in the cytoplasm was accompanied by induction of nuclear staining. No changes of Bcl-2 and Bax IR were found in thalamic nuclei. Our results point to different mechanisms underlying apoptosis of cortical and thalamic neurons. Nuclear appearance of Bcl-2 and Bax suggests they possess regulatory role of gene expression changes triggered by cortical infarct.     

大鼠全脑缺血再灌注损伤后调控基因Bcl-2、Bax的表达及与细胞凋亡的关系

目的:本研究旨在探讨Bcl-2及Bax蛋白在大鼠全脑缺血再灌注损伤中的变化及与细胞凋亡的关系。方法:雄性Wistar大鼠56只,随机分为假手术组、缺血15分钟再灌注1、6、12、24、48、72小时组。采用大鼠四条血管阻断方法制备大鼠全脑缺血再灌注模型。采用TUNEL法观察不同再灌注时间组海马CAl区细胞凋亡的变化。采用免疫组化法观察Bcl-2及Bax蛋白表达水平的变化。结果:脑缺血损伤后随再灌注时间延长凋亡细胞逐渐增多,至再灌注48小时达到高峰,72小时后减少。Bcl-2表达至再灌注12小时达高峰,再灌注24~72小时组逐渐减弱。Bax表达至48小时达高峰,再灌注72小时减少。结论:Bcl-2于再灌注早期表达增强,Bax于再灌注中期表达增强,Bcl-2/Bax比例失衡可能是大鼠全脑缺血再灌注后神经细胞凋亡的机制之一。     

Effects of genistein on neuronal apoptosis, and expression of Bcl-2 and Bax proteins in the hippocampus of ovariectomized rats

Genistein is one of several isoflavones that has a structure similar to 17β-estradiol, has a strong antioxidant effect, and a high affinity to estrogen receptors. At 15 weeks after ovariectomy, the expression of Bcl-2 in the hippocampus of rats decreased and Bax expression increased, with an obvious upregulation of apoptosis. However, intraperitoneal injection of genistein or 17β-estradiol for 15 consecutive weeks from the second day after operation upregulated Bcl-2 protein expression downregulated Bax protein expression, and attenuated hippocampal neuron apoptosis. Our experimental findings indicate that long-term intervention with genistein can lead to a decrease in apoptosis in hippocampal neurons following ovariectomy, upregulate the expression of Bcl-2, and downregulate the expression of Bax. In addition, genistein and 17β-estradiol play equal anti-apoptotic and neuroprotective roles.     

Roles of p53, c-Myc, Bcl-2, Bax and caspases in glutamate-induced neuronal apoptosis and the possible neuroprotective mechanism of basic fibroblast growth factor.

By using flow-cytometric analysis, we examined the involvement of p53, c-Myc, Bcl-2 and Bax in the glutamate-induced cell death in cultured cortical neurons. The activities of caspase-1-like and caspase-3-like proteases were also measured after the glutamate treatment. The apoptosis rate of the cells increased after 12 h and 24 h treatment with glutamate. The temporal profile of p53, c-Myc, Bcl-2, Bax expression and caspases activation after glutamate treatment suggest that Bcl-2, c-Myc and caspase-3 play important roles in the excitotoxic neuronal cell death. The down-regulation of Bcl-2 may be an important early stage event, which may cause the activation of caspase-3. c-Myc is also involved in the process of apoptosis though its precise role remains elusive. bFGF exhibited the capability to antagonize the neuronal apoptosis caused by glutamate. The antiapoptotic potential of bFGF may result from its attenuating effect on the down-regulation of Bcl-2 induced by glutamate and, subsequently, blockade of apoptosis cascade. This may provide a possible explanation for its neuroprotective effect against ischemic cell death.     

缺血性脑卒中患者外周血CBX7的表达水平变化及其通过Nrf2/HO-1通路对神经元凋亡的影响

目的 探讨Polycomb chromobox7(CBX7)对氧糖剥夺/再灌注(Oxygen-glucose deprivation/reperfusion,OGD/R)神经元的影响及其可能的作用机制.方法 收集缺血性脑卒中(Ischemic stroke,IS)组患者和体检健康人群(对照组)外周血,分离外周血单个核细...     

Bcl-2、Bax蛋白产物在脑缺血耐受中的表达及前列腺素E1的干预作用

目的探讨大鼠脑缺血时凋亡相关基因Bcl-2、Bax蛋白的表达与脑缺血耐受的关系及前列腺素E1 (PGE1)对脑缺血耐受的影响。方法采用大鼠全脑-局灶脑缺血耐受模型。观察预缺血组、PGE1组在大脑中动脉梗死2h再灌注(MCAO)24h后神经行为评分、脑梗死体积、Bcl-2、Bax蛋白表达和TUNEL、流式细胞仪检测神经细胞凋亡。结果MCAO后24h,PGE1组的神经行为评分、脑梗死体积、Bax蛋白表达更少,Bcl-2蛋白表达较高,神经细胞凋亡降低。结论预缺血及PGE1预处理后,使Bax蛋白表达减少,Bcl-2蛋白表达增高能够诱导脑缺血耐受,且PGE1的作用优于预缺血。