人重组白细胞介素-6可减少缺氧-复氧后大鼠海马培养神经元的DNA损伤

用原位末端标记（ＴＵＮＥＬ）法观察人重组白细胞介素－６（ｒｈＩＬ－６）对缺氧－复氧后大鼠海马培养神经元的ＤＮＡ损伤的影响。结果显示,海马神经元缺氧－复氧后ＤＮＡ损伤神经元百分率明显增高,经ｒｈＩＬ－６ 预处理的海马神经元缺氧－复氧后ＤＮＡ损伤神经元百分率明显低于对照组。本结果表明,缺氧－复氧能使体外培养海马神经元发生ＤＮＡ损伤,ｒｈＩＬ－６ 可减少缺氧－复氧后海马神经元的ＤＮＡ损伤。提示ｒｈＩＬ－６ 对海马神经元缺氧－复氧损伤可能具有一定保护作用。     

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。     

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     

低氧预处理对大鼠海马神经元缺氧耐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。     

低氧预处理对大鼠海马神经元缺氧耐受性和IL-6免疫反应的影响

目的 观察低氧预处理对大鼠海马神经元缺氧耐受性和IL-6免疫反应的影响。方法 取培养12d的两组（对照组和低氧预处理组）培养神经元,同时置于缺氧环境（0.9L/LN2、0.1L/LCO2)中培养2、4、8和12两组（对照组和低氧预处理组）培养神经元,同时置于缺氧环境（0.9L/LN2、0.1L/LCO2)中培养2、4、8和12h,分别观察它们的形态变化和神经元存活数,并用抗rhIL-6单克隆抗体进行免疫组化染色,观察缺氧对大鼠海马培养神经元IL-6免疫反应的影响。结果 低氧预处理可增强海马神经元对rhIL-6的免疫反应,经低氧预处理的海马神经元缺氧后神经元存活数和对rhIL-6的免疫反应均明显高于对照组。结论 低氧预处理氧预处理的海马神经元缺氧后神经元存活数和对thIL-6的免疫反应均明显高于对照组。结论 低氧预处理可使体外培养的海马神经元对缺氧产生耐受,其中rhIL-6的免疫反应增加可能是海马神经元对缺氧的一种适应性变化,提示IL-6可能参与脑缺氧耐受性的形成,并在海马神经元缺氧损伤的调控中起重要作用。     

人重组白细胞介素—6可减少缺氧—复氧后大鼠海马培养神经？…

用原位末端标记（ＴＵＮＥＬ）法观察人重组白细胞介素－６（ｒｈＩＬ－６）对缺氧－复氧后大鼠海马培养神经元的ＤＮＡ损伤的影响。结果显示,海马神经元缺氧－复氧后ＤＮＡ损伤神经元百分率明显增高,经ｒｈＩＬ－６预处理的海马神经元缺氧－复氧后ＤＮＡ损伤神经元百分率明显低于对照组。本结果表明,缺氧－复氧能使体外培养海马神经元发生ＤＮＡ损伤,ｒｈＩＬ－６可减少缺氧－复氧后海马神经元的ＤＮＡ损伤。提示ｒｈＩＬ－６对     

低氧预适应小鼠脑匀浆液提取液对大鼠鼠胚海马神经元缺氧复氧后神经细胞的影响

目的 探讨低氧预适应小鼠脑匀浆液提取液对大鼠鼠胚海马神经元缺氧复氧后神经细胞活性和凋亡的影响. 方法在96孔培养板中将大鼠鼠胚海马神经细胞原代培养至8 d,将培养细胞按照处理的不同分为以下5组:(1)正常对照组(仅加入PBS)、(2)H<,4>R<,48>组(缺氧4 h/复氧48h后加PBS)、(3)H0组(缺氧4 h/复氧48 h前加正常小鼠脑匀浆提取液)、(4)H1组(缺氧4h/复氧48h前加急性低氧对照小鼠脑匀浆提取液)、(5)H4组(缺氧4 h/复氧48 h前加低氧预适应小鼠脑匀浆提取液),分别用酶标仪和流式细胞仪测定神经细胞活性和凋亡情况.结果 正常对照组细胞活性明显高于H<,4>R<,48>组,H0、H1和H4组分别与H<,4>R<,48>组相比,细胞活性明显增加,且H4组细胞活性又明显高于H0、H1组;正常对照组仅有极少量的凋亡细胞,而H<,4>R<,48>组凋亡细胞显著增多,H0、H1和H4组分别与H<,4>R<,48>组相比凋亡细胞明显减少,且H4组又分别明显少于H0、H1组.结论低氧预适应小鼠脑匀浆液提取液可能通过增加大鼠鼠胚海马神经元缺氧复氧后神经细胞活性和减少神经细胞凋亡起到抗缺氧性损伤作用.     

TLR3在poly（I：C）-LMW预处理后缺糖缺氧诱导的原代皮质神经元的表达意义

目的观察TRL3激动剂poly（I：C）-LMW预处理对原代皮质神经元Toll样受体3（TLR3）表达的影响,探讨TLR3在缺糖缺氧诱导的原代皮质神经元损伤中的作用。方法取体外培养7d的大鼠原代皮质神经元细胞,对细胞分别予以正常条件下培养（空白对照组）;缺糖缺氧2h,复糖复氧24h（OGD组）;TRL3激动剂预处理12h（激动剂组）;TRL3激动剂预处理12h后缺糖缺氧2h,复糖复氧24h（激动剂＋OGD组）。免疫荧光法观察各组细胞生长状态,分别以Western blot法、RT-PCR法测定TLR3蛋白及TLR3mRNA的表达情况。结果 与空白对照组相比,激动剂及缺氧复氧处理方法均诱导原代皮质神经元TLR3、TLR3mRNA表达增强（P〈0.05）;激动剂预处理后,与OGD组相比,激动剂＋OGD组神经元细胞状态较好,数目较多（P〈0.05）。结论 TLR3参与缺糖缺氧诱导的原代皮质神经元的损伤过程,激动剂可减轻神经元缺糖缺氧后损伤。     

两种方法提取乳鼠神经细胞建立缺氧/复氧损伤模型的研究

目的比较两种不同方法培养乳鼠神经细胞稳定性,并探讨H_2O_2对原代培养神经细胞造成缺氧/复氧损伤模型的最佳处理时间。方法两种方法培养原代神经细胞,加入600μmol/L H_2O_2培养50 min、2 h、3 h后换正常培养液继续培养18 h,造成神经细胞缺氧/复氧损伤。MTT法检测各组细胞生存率,检测LDH活性、SOD、MDA含量和RhoA活性。结果与空白组相比,缺氧50 min、2 h、3 h培养各组细胞存活率下降,LDH活性增加,SOD含量下降,MDA含量升高,RhoA含量增加P均0.05。与直接法组相比,种植法组细胞在缺氧50 min、2 h各组细胞存活率高,细胞LDH漏出少,SOD含量高,MDA生成少P均0.05。结论与直接法相比,种植法培养的神经细胞状态更稳定,缺氧50 min、复氧18 h时制作神经细胞损伤模型最接近体内缺氧损伤状态,可用于实验。     

星形胶质细胞AQP4蛋白在缺氧/复氧条件下表达变化的研究

目的观察缺氧/复氧条件下星形胶质细胞形态和AQP4蛋白的表达变化以及葛根素对其表达变化的影响,探讨脑缺血再灌注损伤与AQP4的关系以及葛根素的干预作用。方法原代培养星形胶质细胞,用5%CO2+95%N2混合气体造成缺氧,以LDH漏出率及MTT降解率作为细胞受损指标,应用Western blot技术检验星形胶质细胞缺氧/复氧各个时间点AQP4蛋白的表达变化及葛根素的干预效果。结果体外培养的星形胶质细胞在缺氧环境下损伤不明显,随着复氧时间的延长细胞损害加重。AQP4蛋白在缺氧时表达与正常对照组无明显差异,复氧后表达升高并随时间延长呈增高趋势(P0.05)。葛根素干预组AQP4蛋白表达丰度与缺氧/复氧组无明显差异(P0.05)。结论星形胶质细胞AQP4蛋白表达变化与细胞损伤有明显相关性,葛根素对星形胶质细胞损伤的保护作用不是通过改变AQP4的表达来实现的。     

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。     

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。     

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.     

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.     

Sevoflurane-induced preconditioning of rat brain in vitro and the role of KATP channels

In the present study we tested the ability of the inhalation anesthetic sevoflurane to induce preconditioning against hypoxia in vitro. Rat hippocampal slices were prepared using established procedures. After 90 min of incubation, slices were exposed for 30 min to 0, 1, 2 or 3 minimum alveolar concentration (MAC) of sevoflurane under normoxic conditions (95% O2/5% CO2). Fifteen minutes later, slices were exposed to 13-min hypoxia (95% N2/5% CO2) followed by 30-min reoxygenation. The amplitude of extracellularly recorded, orthodromically evoked, CA1 population spikes (neuronal function) at the end of the reoxygenation period was measured and used to quantify the degree of recovery of neuronal function posthypoxia. To assess the role that the mitochondrial KATP channel plays in preconditioning, its blocker, 5-hydroxydecanoic acid (5-HD), was added during sevoflurane exposure. Sevoflurane-preconditioning with 1, 2 and 3 MAC increased the degree of recovery of neuronal function after 13-min hypoxia and 30-min reoxygenation from 51 +/- 1% (0 MAC), to 55 +/- 3%, 63 +/- 3%, and 72 +/- 2%, respectively. The effect of 3 MAC sevoflurane was blocked by 5-HD (53 +/- 3%), whereas 5-HD alone had no effect (48 +/- 3%) on the recovery of neuronal function from hypoxia. It is concluded that sevoflurane is capable of inducing preconditioning in vitro in a dose-dependent fashion and involves activation of mitochondrial KATP channels.     

Neuroprotective mechanisms of DNA methyltransferase in a mouse hippocampal neuronal cell line after hypoxic preconditioning

Hypoxic preconditioning has been shown to improve hypoxic tolerance in mice, accompanied by the downregulation of DNA methyltransferases(DNMTs) in the brain. However, the roles played by DNMTs in the multiple neuroprotective mechanisms associated with hypoxic preconditioning remain poorly understood. This study aimed to establish an in vitro model of hypoxic preconditioning, using a cultured mouse hippocampal neuronal cell line(HT22 cells), to examine the effects of DNMTs on the endogenous neuroprotective mechanisms that occur during hypoxic preconditioning. HT22 cells were divided into a control group, which received no exposure to hypoxia, a hypoxia group, which was exposed to hypoxia once, and a hypoxic preconditioning group, which was exposed to four cycles of hypoxia. To test the ability of hypoxic preadaptation to induce hypoxic tolerance, cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium assay. Cell viability improved in the hypoxic preconditioning group compared with that in the hypoxia group. The effects of hypoxic preconditioning on the cell cycle and apoptosis in HT22 cells were examined by western blot assay and flow cytometry. Compared with the hypoxia group, the expression levels of caspase-3 and spectrin, which are markers of early apoptosis and S-phase arrest, respectively, noticeably reduced in the hypoxic preconditioning group. Finally, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and western blot assay were used to investigate the changes in DNMT expression and activity during hypoxic preconditioning. The results showed that compared with the control group, hypoxic preconditioning downregulated the expression levels of DNMT3A and DNMT3B mRNA and protein in HT22 cells and decreased the activities of total DNMTs and DNMT3B. In conclusion, hypoxic preconditioning may exert anti-hypoxic neuroprotective effects, maintaining HT22 cell viability and inhibiting cell apoptosis. These neuroprotective mechanisms may be associated with the inhibition of DNMT3A and DNMT3B.     

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。     

去铁敏预处理对神经元缺氧性损伤的保护作用

目的 探讨去铁敏预处理对体内外神经元缺氧损伤的保护作用及可能机制.方法 体外培养皮质神经元,建立神经元去铁敏糖氧剥夺模型,采用细胞活力测定、细胞凋亡比率、形态学改变评价去铁敏预处理后的神经元保护效应.去铁敏预处理后不同时间点制作大鼠大脑中动脉阻塞(MCAO)模型,采用神经功能评分和梗死体积评价去铁敏预处理后的脑保护效应.用免疫荧光染色检测去铁敏预处理后神经元的缺氧诱导因子-1α(hypoxia inducible factor 1α,HIF-1α)和促红细胞生成素(erythropoietin,EPO)蛋白表达情况,RT-PCR检测HIF-1α和EPO的mRNA变化情况.结果 去铁敏预处理后再给予缺氧损伤后神经元活力下降至49%(无预处理组25%,t=8.544,P<0.05),凋亡细胞百分比为38%(无预处理组30%,t=4.409,P<0.05),预处理后细胞形态保持良好.去铁敏30 ms/kg预处理后第3天行MCAO手术,大鼠神经功能评分降低,梗死体积缩小8.5%.去铁敏100 mg/kg预处理后第2、3、7天行MCAO手术,与生理盐水组(8.13±0.17)相比,神经功能评分分别下降为7.44±0.39(t=2.903,P<0.05)、5.60±0.47(t=10.143,P<0.01)、6.97±0.73(t=3.142,P<0.05),脑梗死体积分别缩小12.0%(t=5.056,P<0.05)、32.3%(t=10.993,P<0.01)、10.6%(t=4.385,P<0.05).免疫荧光染色显示:体外培养的皮质神经元在预处理后出现HIF-1α和EPO蛋白表达,大鼠预处理后脑内神经元也出现HIF-1α和EPO蛋白表达上调.RT-PCR显示去铁敏化学缺氧能上调体外培养的神经元HIF-1α及EPO mRNA表达.结论 去铁敏预处理有确切有效的抗缺氧保护效应,这种效应与保护神经元有关,其机制可能是去铁敏诱导了神经元HIF-1α和EPO的表达增加.