慢性缺氧/再氧合小鼠脑皮质Nip3表达及其与神经细胞凋亡的关系

目的研究慢性缺氧／再氧合对小鼠脑皮质Nip3的表达及神经细胞凋亡的影响．探索睡眠呼吸暂停综合征（SAS）脑损害的可能机制。方法自制慢性缺氧／再氧合小鼠模型：通过控制程序调控箱内氧浓度，使得每一间断性缺氧循环时间为2min，氧浓度循环于（21．72±0．551％与（6．84±0．47）％之间，每天缺氧／再氧合8h。30只ICR小鼠随机分为4组：模拟对照组10只．缺氧／再氧合二周组及四周组各5只，缺氧／再氧合八周组10只。免疫组织化学方法检测小鼠额叶脑皮质Nip3的表达，TUNEL法检测脑神经细胞凋亡。结果与模拟对照组比较，缺氧／再氧合各组脑皮质Nip3的表达和凋亡神经细胞数均显著增加（P〈0．05），八周组与四周组Nip3的表达和凋亡神经细胞数较二周组亦显著增加（P〈0．05），八周组与四周组比较差异无统计学意义（P〉0．05）；脑皮质Nip3的表达与脑神经细胞凋亡之间存在正相关关系（r=0．901，P〈0．05）。结论慢性缺氧／再氧合可增加脑Nip3的表达，引起皮质脑神经细胞凋亡，可能是其神经系统损害的机制之一。     

急性低氧条件下复氧后大鼠脑HIF-1α、nNOS蛋白的表达与人参皂甙Rd干预

目的观察大鼠脑缺氧状态下缺氧诱导因子1α(HIF-1α)、神经元性一氧化氮合酶(nNOS)蛋白的表达规律及低氧状态下人参皂甙Rd(Rd)干预对其影响并探讨机理。方法将成年Wistar大鼠120只随机分为3组:①急性低氧组(对照组);②低氧预处理组(低氧干预组);③人参皂甙Rd预处理组(药物干预组)。每组按照缺氧后不同时间点再分为4个亚组,各亚组10只大鼠,分别观察各组大鼠海马锥体细胞层神经元形态的变化及HIF-1α、nNOS蛋白的表达规律。结果在缺氧后复氧即刻,我们可以发现HIF-1α、nNOS蛋白的少量表达,主要存在于海马细胞,多在细胞质中表达;随着时间的推移,在复氧后4 h,HIF-1α、nNOS表达渐达高峰,至复氧后9h HIF-1α表达明显减少,而nNOS表达至复氧后24 h明显减少。低氧预处理组及人参皂甙Rd预处理组的实验结果发现HIF-1α、nNOS表达较对照组减少,与急性低氧组各时间点相比,均有统计学意义(P=0.009)。两个干预组之间各时间点比较无统计学差异(P>0.05)。结论急性低氧可以促使HIF-1α、nNOS蛋白在大鼠海马神经细胞的表达,具有时间依赖性;同时低氧预处理及人参皂甙Rd预处理均可促使大鼠脑组织HIF-1α、nNOS表达减少,可能对急性缺氧性脑损伤产生保护作用,其具体机理可能与其抗自由基、抑制Ca2+内流有关     

NIM811对Na2S2O4引起小鼠HT22细胞缺氧/复氧损伤的保护作用的研究

目的探讨环孢菌素衍生物NIM811对连二亚硫酸钠(Na₂S₂O₄)引起的小鼠海马神经元细胞(HT22)的缺氧/复氧损伤的保护作用及其机制。方法以小鼠HT22培养细胞制备缺氧/复氧细胞模型,实验分组为正常对照组、Na₂S₂O₄组、Na₂S₂O₄+NIM811组、NIM811组。CCK-8检测细胞生存率、流式细胞术检测细胞凋亡、JC-1试剂检测线粒体膜电位、用钙离子指示剂Rhod-2 AM观察线粒体内钙离子水平、DCFH-DA法检测细胞活性氧(ROS)水平。结果与Na₂S₂O₄组比较,给予NIM811处理后:(1)细胞活性增高38%(P<0.01);(2)细胞凋亡减少27%(P<0.01);(3)线粒体膜电位上升(P<0.01);(4)线粒体内钙离子水平下降(P<0.01);(5)活性氧(ROS)水平降低(P<0.01)。结论NIM811对Na₂S₂O₄引起小鼠海马神经元细胞缺氧/复氧损伤有保护作用,其机制可能为NIM811维持线粒体动态平衡和抑制细胞凋亡有关,NIM811对未来临床治疗缺血性脑卒中具有潜力。     

急性低氧条件下复氧后大鼠脑HIF—1α蛋白的表达与人参皂甙Rd干预研究

摘要：目的观察大鼠脑缺氧状态下缺氧诱导因子1α（HIF-1α）蛋白的表达及低氧状态下人参皂甙Rd干预对其影响。方法将成年Wistar大鼠90只随机分为急性低氧对照组、低氧预处理干预组、人参皂甙Rd预处理干预组,每组按缺氧后不同时间点（复氧后0h、4h、9h）再分为3亚组,每组10只大鼠。采用免疫组化法检测HIF-1α蛋白的表达。结果在缺氧后复氧即刻,HIF-1α蛋白少量表达,主要存在于海马细胞;随着时间的推移,在复氧后4h,HIF-1α表达逐渐达高峰,至9h时HIF-1α表达又明显减少。低氧预处理干预组及人参皂甙Rd干预组的实验结果发现HIF-1α表达较急性低氧对照组减少,与急性低氧对照组各时间点相比,差异均有显著性（P〈0．05）。两个干预组之间比较无统计学差异。结论急性低氧可以促使HIF-1α在大鼠海马神经细胞的表达,具有时间依赖性,低氧预处理干预及人参皂甙Rd干预均可促使大鼠脑组织HIF-1α表达减少。     

RNAi抑制星形胶质细胞Cx43对缺氧/再复氧后细胞凋亡的影响

目的探讨RNA干扰技术对星形胶质细胞Cx43蛋白的抑制作用以及对细胞缺氧/再复氧后凋亡的影响。方法化学合成针对大鼠Cx43基因的小干扰RNA(Small interfering RNA,siRNA)及其阴性RNA,通过脂质体转染体外培养原代星形胶质细胞,Western blot检测Cx43蛋白表达情况,MTT法检测siRNA转染对细胞的毒性作用;通过流式细胞术观察抑制Cx43蛋白对星形胶质细胞缺氧/再复氧后细胞凋亡情况的影响。结果 Western blot结果可见,转染siRNA后12h,Cx43蛋白表达开始抑制,48h抑制最明显,MTT结果显示siRNA组和脂质体组的细胞活性明显低于未转染组(P0.05),siRNA组与脂质体组未见明显区别(P0.05);流式细胞术示缺氧12h复氧不同时间,凋亡率逐渐增加,复氧48h凋亡率最高,siRNA干扰组明显低于正常对照组(P0.05)。结论 RNAi技术是研究Cx43功能的一种有效方法,siRNA转染能有效抑制星形胶质细胞Cx43蛋白表达,siRNA转染时,细胞毒性来自脂质体;siRNA抑制Cx43蛋白能够明显减轻星形胶质细胞缺氧/再复氧后的细胞凋亡。     

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。     

ERK通路对体外培养星形胶质细胞缺氧/复氧后TNF-α分泌的影响

目的 分析ERK通路对缺氧/复氧后反应性星形胶质细胞TNF-α分泌的影响,从而探讨ERK通路在星形胶质细胞反应性改变的可能作用机制,为临床研究提供理论支持.方法 参照McCarthy方法星形胶质细胞（AC）原代培养,传至第3代,细胞自然纯化.将AC分为正常对照组（C组）、缺氧/复氧组（H/R组）、缺氧/复氧阻滞剂组（H/R＋M组）.每组设缺氧4 h、缺氧8 h、复氧6 h、12 h、24 h、48 h 6个时间点,建立AC缺氧/复氧模型.Western-blot法半定量分析T-ERK,P-ERK的表达情况,TNF-α ELISA试剂盒测定细胞凋亡情况.采用SPSS18.0统计软件包进行数据分析.结果 （1）Western-blot：缺氧组较正常组相比ERK表达明显升高（P＜0.05）,阻滞剂组较无阻滞剂组p-ERK蛋白表达量显著下降（P＜0.05）.（2）TNF-α ELISA：缺氧后TNF逐渐升高,复氧48 h时达高峰（P＜0.05）,加入ERK阻滞剂后升高更明显（P＜0.05）.结论 星形胶质细胞缺氧复氧后存在ERK通路的激活,ERK通路在星形胶质细胞缺氧损伤后反应中发挥生物学作用与TNF-α有关.     

依达拉奉对培养乳鼠海马神经元缺氧复氧损伤的保护作用

目的:探讨依达拉奉对海马神经元缺氧复氧损伤的保护作用及其机制。方法:制备培养乳鼠海马神经元缺氧复氧损伤模型,分别用依达拉奉1、10、100、300μmol·L-1干预后研究依达拉奉对脂质过氧化及细胞凋亡的作用。结果:海马神经元在缺氧复氧损伤后,依达拉奉干预能降低脂质过氧化产物丙二醛(MDA)含量,增加细胞内超氧化物歧化酶(SOD)活性,减少神经元凋亡。结论:缺氧复氧损伤后,依达拉奉具有清除氧自由基、抑制脂质过氧化及降低细胞凋亡率的作用。     

缺氧缺血性脑损伤大鼠Caspase-9的表达及高压氧干预疗效

目的:探讨高压氧(HBO)对缺氧缺血新生大鼠模型神经细胞凋亡的影响,以及Caspase-9在该过程中的作用。方法:建立缺氧缺血(HIBD)大鼠模型,观察HBO对缺氧缺血性脑损伤的改善情况,并用免疫组化的方法检测Caspase-9表达在缺氧缺血脑组织中的动态变化。结果:HIBD模型组大鼠造模后18h、24h、48h、96h不同时间段患侧海马区及皮层区Caspase-9活性蛋白的表达均明显高于假手术组(P值均<0.05);HBO干预组大鼠海马区和皮层区各相应时间点Caspase-9活性蛋白表达均低于HIBD模型组大鼠(P值均<0.05),部分时间点接近假手术组基线水平。结论:HBO可以减轻新生大鼠HIBD模型神经细胞的凋亡;不同时程HBO干预治疗可降低HIBD模型大鼠皮层、海马神经细胞Caspase-9的表达活性。     

低氧条件下星形胶质细胞对神经干细胞增殖的影响

目的 探讨体外低氧条件下星形胶质细胞对海马神经干细胞( NSCs)增殖的影响.方法 将分离出的新生小鼠海马NSCs分别与低氧条件星形胶质细胞培养液(低氧组)、低氧条件星形胶质细胞培养液+磷脂酰肌醇-3激酶/蛋白激酶B( PI3K/Akt)通路拮抗剂LY294002(拮抗剂组)以及常氧条件星形胶质细胞培养液(对照组)共培养;分别于培养24 h、48 h、72 h应用四甲基偶氮唑盐(MTT)法检测各组NSCs活性;应用Western Blot法检测各组NSCs磷酸化Akt (p-Akt)和磷酸化糖原合成酶激酶(p-GSK)-3β含量.结果 (1)共培养24h、48 h及72 h,低氧组NSCs活性的光密度(OD)值分别为0.292±0.006、0.382 ±0.005、0.649±0.028,拮抗组分别为0.197±0.003、0.255±0.005、0.325±0.012,对照组分别为0.107±0.006、0.198±0.008、0.254±0.006;低氧组各时间点NSCs活性显著高于拮抗剂组和对照组(均P＜0.05);(2)共培养24h、48 h和72 h,低氧组NSCs p-Akt和p-GSK-3β的吸光度(A)值分别为0.52、0.71、0.86及0.49、0.65、0.82;拮抗剂组分别为0.39、0.42、0.61及0.31、0.35、0.40;对照组分别为0.34、0.38、0.39及0.28、0.31、0.35;低氧组各时间点p-Akt及p-GSK-3β含量显著高于拮抗剂组和对照组(均P＜0.05).结论 缺氧刺激可激活星形胶质细胞PI3K/Akt信号通路,促进NSCs增殖.     

氢气预处理对乳鼠海马神经元细胞缺氧/复氧损伤后细胞活力的影响

目的探讨氢气对乳鼠海马神经元细胞缺氧/复氧损伤后细胞活力的影响。方法原代培养的SD乳鼠海马神经元细胞,随机分成对照组,缺氧/复氧组和氢气预处理组。对照组常规培养;缺氧/复氧组在100%N2中培养15min后复氧30min;氢气预处理组在2%H2+98%N2中培养15min后复氧30min。采用MTT法检测乳鼠海马神经元细胞活力。结果氢气预处理能够增强细胞活力(P<0.05)。结论氢气预处理对海马神经元细胞缺氧/复氧损伤具有保护作用,可能与提高细胞活力有关。     

Hypoxia/reoxygenation induces apoptosis through biphasic induction of protein synthesis in cultured rat brain neurons

To investigate biochemical events accounting for the outcome of central neurons following hypoxia/reoxygenation, cultured neurons from fetal rat forebrain were exposed to hypoxia (95% N₂/5% CO₂) for 6 h, and then reoxygenated for up to 96 h. Time-dependent changes in macromolecular biosynthesis were analysed by incorporation of [³H]uridine and [³H]leucine and were coupled to cell viability and lactate dehydrogenase leakage. Morphological features of necrosis and apoptosis were scored following nuclear incorporation of the fluorescent dye 4,6-diamidino-2-phenylindole. Hypoxia led to a 36% reduction of cell viability at the end of the reoxygenation period, while 23% of the neurons exhibited apoptosis. A biphasic increase in the rates of protein synthesis was measured 1 h after the onset of hypoxia (77% above controls) and by 48-h postreoxygenation (72%). The presence of cycloheximide during hypoxia inhibited both peaks of synthesis and prevented the development of apoptosis. Protein electrophoresis outlined specific alterations in constitutive proteins, and immunohistochemistry revealed an overexpression of the pro-apoptotic gene products Bax and ICE. Therefore, hypoxia followed by reoxygenation would trigger sequential changes in synthesis of specific proteins, leading to delayed and mainly apoptotic neuronal death.     

Neurogenesis response to hypoxia-induced cell death: map kinase signal transduction mechanisms

Hypoxic stress induces apoptosis of hippocampal CA1 neurons while selectively sparing those in CA2-3. Proliferation and differentiation of local stem cells may potentially replace lost neurons. We examined MAP kinase signaling regulation of these dual responses. Rat organotypic hippocampal cultures were exposed to hypoxia for up to 6 h followed by reoxygenation. JNKs and ERKs were maximally activated by 4 h, returning approximately to basal levels by 6 h. Apoptosis of CA1 neurons was maximal by 6-h hypoxia, although JNK activation had returned to basal levels. A neuroprotective protein, JNK-interacting protein 1 (JIP1), an inhibitor of JNK-mediated apoptosis, was reduced by 6-h hypoxia and markedly decreased by 24-h reoxygenation in CA1 neurons as was DENN/MADD, which also modulates JNK-mediated cell death. A second peak of ERK1 activation occurred at 24-h reoxygenation and declined to control levels by 48 h. Stem cells were detected by antinestin and cell proliferation confirmed with anti-PCNA immunohistochemistry and BrdU incorporation. With U0126, an inhibitor of ERK activation, BrdU labeling was strikingly reduced implicating ERKs in the proliferation response. Antidoublecortin (DCX), which detects neural progenitor cells, colabeled a subset of BrdU-positive cells that extended from the dentate granule neurons into CA1. Astrocytes were colabeled with BrdU. Thus, hypoxia concurrently triggered both JNK and ERK signaling, and with reoxygenation, ERK1 activation and stem cell proliferation followed by neuronal progenitor cell differentiation and targeted migration to the site of pyramidal neuronal loss.     

p38MAPK在体外培养星形胶质细胞缺氧/复氧损伤中的表达

目的 建立SD大鼠星形胶质细胞缺氧复氧损伤模型,探讨p38MAPK活性变化与星形胶质细胞损伤的关系.方法 体外培养新生SD大鼠星形胶质细胞,实验设正常对照组（N）、SB203580组（SB组,10 μmol/L）、缺氧/复氧组（H/R组）和缺氧/复氧组＋SB203580阻断p38MAPK组（H/R＋SB组）.应用MTT法、WB法、ELISA法检测缺氧4 h、8 h、复氧6 h、12 h、24 h、48 h时细胞存活率,p38MAPK、p-p38（磷酸化p38MAPK）及TNF-α的变化.结果 培养星形胶质细胞GFAP阳性表达率大于97%.缺氧/复氧使星形胶质细胞活力降低,SB203580阻断p38MAPK细胞活力高于H/R组,各组星形胶质细胞总p38MAPK水平无显著变化,缺氧复氧干预后p-p38表达上调,TNF-α水平显著增高.用SB203580阻断p38MAPK通路后,SB＋H/R组较H/R组p-p38、TNF-α水平降低.SB组总p38MAPK、p-p38、TNF-α水平与N组比较无显著变化.结论 p38MAPK信号通路参与了星形胶质细胞缺氧复氧损伤过程.     

细胞周期蛋白D1在环加氧酶-2诱导的大鼠神经细胞死亡中的作用

目的探讨环加氧酶-2诱导的大鼠神经细胞死亡的分子发病机制。方法原代培养大鼠胎鼠皮质神经细胞,给予缺氧再加氧处理;实验随机分为对照组、缺氧再加氧组及选择性抑制剂 缺氧再加氧组;用噻唑蓝比色法测定神经细胞生存率,用Western印迹法检测蛋白质的表达。结果环加氧酶-2及细胞周期蛋白D1(Cy-clinD1)在缺氧再加氧后神经细胞表达呈平行性、一致性增高;COX-2选择性抑制剂NS398能明显降低CyclinD1的表达;而CyclinD1选择性抑制剂flavopiridol对COX-2的表达则没有影响;flavopiridol还能明显改善缺氧再加氧后神经细胞的生存率(P<0.001)。结论COX-2诱导的缺氧性神经细胞死亡的分子发病机制可能是通过CyclinD1表达的增加使成熟神经细胞重新进入细胞周期而实现的。     

重度缺氧中海马神经元KATP通道的表达改变

目的 探讨重度缺氧状态中ATP敏感性钾通道(KATP)通道在海马神经元上的表达变化. 方法 取培养1周的新生大鼠海马神经元分为4组:第1组为正常对照组,在正常氧状态中(5%CO_2、95%空气)孵育8 h;第2组为处理组,在模拟的缺氧状态(5% CO_2、95%N_2)孵育8h(单纯缺氧组);第3组为二氮嗪+缺氧组,在缺氧处理的同时添加KATP通道激动剂二氮嗪(100μmo1/L1,处理时间为8 h;第4组为甲糖宁+缺氧组,在缺氧处理的同时添加KATP通道阻断剂甲糖宁(100 μmol/L),处理时间为8h.利用MTT、免疫印迹及RT-PCR技术,比较4组细胞存活情况以及缺氧状态中神经元上KATP通道的表达改变. 结果 缺氧8h后,二氮嗪能明显降低细胞的凋亡数量,甲糖宁使细胞的凋亡数量增加,与正常对照组比较,差异均有统计学意义(P<0.05).缺氧状态中KATP通道的SUR1亚基的表达明显增加,而Kir6.2亚基表达量则无明显改变,与正常对照组比较,差异均有统计学意义(P<0.05). 结论 KATP通道的活性及表达改变,对缺氧中的海马神经元的保护起到重要作用.     

Anti‐hypoxia effect of adenovirus‐mediated expression of heat shock protein 70 (HSP70) on primary cultured neurons

Heat shock protein 70 (HSP70) has attracted great attention recently in hypoxia injury because of its close link to the recovery after hypoxic–ischemic damage in organs. However, the cellular mechanism underlying its protective roles remains unclear. In this study, we developed a recombinant adenovirus containing HSP70‐GFP (vAd‐HSP70‐GFP) and studied the effect of virus‐mediated expression of exogenous HSP70 gene on neurons in response to hypoxia–reoxygenation injury. Virus‐mediated expression of HSP70 was detected as early as 24 hr and lasted until 10 days after infection. Neurons with 48 hr vAd‐HSP70‐GFP infection were exposed to 0, 0.5, 1, 2, 3, or 4 hr hypoxia followed by 1 hr reoxygenation. The mRNA and protein levels of HSP70 in neurons exposed to different lengths of hypoxia were compared by using RT‐PCR and Western blotting (WB). The 1‐hr hypoxia exposure showed the most significant increases in the HSP70 mRNA and protein level compared with other exposure durations. MTT assay showed that HSP70 overexpression significantly increased the neuronal viability, accompanied by decreased lactate dehydrogenase (LDH) activity in the culture medium after hypoxia–reoxygenation. Neurons with vAd‐HSP70‐GFP exhibited increased levels of mitochondrial cytochrome C (Cyt‐C) and decreased levels of cytoplasmic Cyt‐C compared with vAd‐GFP‐infected cells. These results suggest a neuroprotective role of exogenous HSP70 against hypoxia–reoxygenation injury, possibly via preventing initiation of mitochondrial apoptosis. © 2013 Wiley Periodicals, Inc.     

Mitogen-activated protein kinase signaling, oxygen sensors and hypoxic induction of neurogenesis

In the adult nervous system, neuronal subpopulations sustain a hierarchical pattern of selective vulnerability to hypoxia. Hypoxia also activates quiescent neural progenitor cells (NPCs) resulting in their amplification and subsequent differentiation into neurons and glia. Use of rat organotypic hippocampal cultures facilitates examination of early signaling events in response to hypoxia and reoxygenation that result in neurogenesis. Cultures were exposed to hypoxia for up to 6 h followed by reoxygenation. CA1 neurons showed focal nuclear condensation by 2 h of hypoxia, but CA2 and CA3 neurons were spared. JNKs and c-Jun reached peak activation by 4 h, returning to basal levels by 6 h. Expression of oxygen sensors, hemoxygenase 2 and HIF1, were elevated by 30 min and 2 h, respectively. By 24 h of reoxygenation, there was proliferation of nestin-positive NPCs. With U0126, an upstream inhibitor of ERK activation, BrdU labeling was markedly reduced immunohistochemically as well as PCNA protein expression, suggesting a role for ERKs in the proliferation response. Immunohistochemically, antinestin detected NPCs and on Western blots reached peak levels by 24-48 h of reoxygenation. Proliferation and differentiation of endogenous NPCs in the area of neuronal loss further suggests that mechanisms potentially exist in vitro for replacement with functional neurons.