热环境对大鼠纹状体神经元细胞膜的损伤作用

采用原代培养的大鼠纹状体神经元,施予43℃热环境处理1h。用气,质联用的方法测定细胞膜和细胞中的脂肪酸水平,主要是花生四烯酸的水平;荧光偏振法测细胞膜流动性,用[^3H]花生四烯酸大肠杆菌膜检测细胞内磷脂酶A2活性。发现细胞内大量存在的脂肪酸水平在热环境处理前后没有明显差别,而花生四烯酸的水平明显升高。热处理造成细胞膜的流动性明显降低,同时明显增加了神经元内磷脂酶A2的活性。表明热处理明显影响神经元细胞膜的流动性和磷脂代谢,进而影响细胞膜的功能,而热对细胞膜的损伤作用可能就是热致神经元损伤的重要事件。     

注射热损伤大鼠纹状体mRNA的爪蟾卵母细胞对多巴胺的反应

将从正常大鼠和热损伤大鼠的中枢纹状体提取的poly(A) mRNA ,注入非洲爪蟾卵母细胞表达。用电生理方法检测多巴胺诱发的膜电位和电流的变化 ,分析热损伤对中枢多巴胺受体表达的影响。结果表明 ,注射大鼠纹状体mRNA后 ,卵母细胞的静息电位与注射前没有变化 ,但多巴胺能诱发膜电流。经验证 ,此受体电流的主要载流离子是Cl-。注射热损伤大鼠纹状体mRNA的卵母细胞对多巴胺反应的敏感性降低 ,与正常大鼠组相比有显著性差异。因此可以断定 ,热损伤对大鼠纹状体中多巴胺受体的基因表达产生了明显的影响 ,并可能有离子通道的参与。     

无血清原代培养大鼠海马神经元的形态与膜电位

分离新生Wistar鼠海马,采用添加B27的无血清培养液进行海马神经元原代培养,动态观察海马神经元形态学变化;通过免疫荧光细胞化学法检测神经纤丝(NF)的表达,进行神经元鉴定及纯度计算;采用电位敏感的荧光探针标记神经元,在激光扫描共聚焦显微镜上动态监测去极化剂KCl作用前后膜电位的变化,观察神经元电生理反应。结果表明:此方法培养的大鼠海马神经元可在体外存活20天以上,9～14天为发育最成熟阶段,培养7天神经元纯度达90%。KCl作用于细胞后胞内荧光强度增强,细胞迅速去极化。本培养方法在体外获得高纯度的海马神经元并延长体外存活时间,且显示出神经元的电生理反应特性。     

高频刺激丘脑底核对帕金森病大鼠模型纹状体NOS阳性神经元的影响

目的观察高频刺激丘脑底核对帕金森病(PD)大鼠纹状体中NOS阳性神经元的影响,以探求其作用机制。方法应用6OHDA制备偏侧PD大鼠模型,丘脑底核区埋入刺激电极进行电刺激,采用组织化学方法观察纹状体中NOS阳性神经元的变化。结果PD大鼠纹状体中NOS阳性神经元数与正常大鼠相比明显增加(P<0.01),经电刺激后PD大鼠纹状体NOS阳性神经元数量明显减少,且与正常大鼠相比无显著性差异。结论高频电刺激丘脑底核治疗PD的机制之一可能是与其抑制纹状体NO的过度释放有关。     

电磁辐射对原代培养海马神经元的损伤效应及其机制探讨

研究X带高功率微波、S带高功率微波及电磁脉冲辐射对原代培养海马神经元的损伤效应并探讨其机制。通过体外培养原代海马神经元,建立电磁波辐照细胞模型。采用Annexin V-PI双标记、流式细胞术检测细胞凋亡与坏死,原子力显微镜检测细胞膜表面形态,Fluo-3-AM荧光探针负载、激光扫描共聚焦显微镜测定胞内[Ca2 ]i。结果表明,辐射后海马神经元凋亡与坏死均增加,其中坏死增加明显;细胞膜表面粗糙度加大,膜穿孔增多;胞内[Ca2 ]i明显升高。且以上变化均以X带高功率微波组最重,S带高功率微波组次之,电磁脉冲组最轻。提示细胞膜穿孔增多,膜通透性增加,导致胞外Ca2 内流增加,甚至胞内钙超载是辐射致海马神经元凋亡与坏死的机制之一;三种电磁辐射对海马神经元的损伤程度与照射频率呈正相关。     

热应激对大鼠丘脑、纹状体PLA2活性的影响

热应激作为一种外部应激信号 ,可以引起机体信号系统的反应。近年来 ,磷脂酰肌醇 (ＰＩ)信号转导系统受到人们的重视 ,它包括ＰＩ、磷脂酶Ｃ(ＰＬＣ)、磷脂酶Ａ2 (ＰＬＡ2 )、三磷酸肌醇 (ＩＰ3)、细胞内钙离子浓度 ([Ｃａ2 ]ｉ)等 ,参与许多重要的生理生化过程。其中 ,ＰＬＡ2 作为ＰＩ信号系统活化的启动因子 ,它在热应激时的变化颇受关注。离体实验表明 ,热可致ＰＬＡ2 的活性增强 ,在体实验中发现 ,热可使肝、肺组织中ＰＬＡ2 活性增强 ,膜磷脂 (ＭＰＬ)降解增强 ,用ＰＬＡ2 拮抗剂和膜稳定剂阿的平 ,可调节ＭＰＬ代谢 ,防止中暑发生 ,…     

注射热损伤大鼠纹状mRNA的爪蟾卵母细胞对多巴胺的反应

将从下沉大鼠和热损伤大鼠的中枢纹状体提取的ｐｏｌｙ（Ａ）＾＋ｍＲＮＡ,注入非洲爪蟾卵母细胞表达。用电生理方法检测多巴胺诱发的膜电位和电流的变化,分析热损伤对中枢多巴胺受体表达的影响。结果表明,注射大鼠纹状体ｍＲＮＡ后,卵母细胞的静息电位与注射 前没有变化,但多巴胺能诱发膜电流。经验证,此受体电流的主要载流离子是Ｃ１＾－。注射热务大鼠纹状体ｍＲＮＡ的卵母细胞对多巴胺反应的敏感性降低,与正常大鼠组相比     

阿糖胞苷对大鼠海马神经元原代培养的影响

目的：探讨在大鼠海马神经元原代培养过程中,阿糖胞苷对培养神经元的影响。方法：将新生24 h大鼠,分离出海马组织,进行原代海马神经元培养,再将细胞分为阿糖胞苷组和对照组,阿糖胞苷组加入1μmol/L阿糖胞苷,通过检测神经元特异性标志物微管相关蛋白-2（Map-2）计算培养神经元的数量,通过台盼蓝染色法观察细胞的存活率。结果：培养第7天,阿糖胞苷组神经元数量为（11±3）个,对照组为（10±4）个,两组无明显差异;阿糖胞苷组神经元细胞在培养第14天时存活率为74%,培养第21天时存活率为49%,而对照组神经元14天时存活率为96%,21天存活率为88%,两组神经元存活率差异明显。结论：原代培养海马神经元时,阿糖胞苷对神经元产量及形态影响不明显,但是由于阿糖胞苷的毒性作用,明显缩短神经元的存活时间,影响长期培养神经元的存活率。     

GDAF对原代培养脊髓神经元的影响

The effect of GDNF on long-term cultured spinal cord neurons was studied. GDNF could promote spinal cord neurons survival after 7 d or 14 d culture by MTT assay. The effect of GDNF on growth cones, neuron soma magnitude, neurite length and spines formulation of spinal cord neurons in cell culture was observed by phase microscopy, Nissl stain and NSE immunocytochemistry stain. The results indicated that GDNF had significant trophic effects on long-term cultured spinal cord neurons.     

Mitochondria are involved in apoptosis induced by ultraviolet radiation in lepidopteran Spodoptera litura cell line

Abstract Mitochondria are involved in apoptosis of mammalian cells and even single‐cell organisms, but mitochondria are not required in apoptosis in cultured Drosophila cells such as S2 and BG2 cell lines. It is not very clear whether mitochondria are involved in apoptosis in other insect cells such as lepidopteran cell lines. Thus, we determined to elucidate the role of mitochondria in apoptosis induced by ultraviolet radiation in Spodoptera litura (Lepidoptera: Noctuidae) cell line (SL‐ZSU‐1). The Western blot results suggested that cytochrome c in the ultraviolet‐treated SL‐1 cells was released from the mitochondria to cytosol as early as 4 h after the induction of ultraviolet radiation and increased in the cytosolic fractions in a time‐dependent manner. Flow cytometric analysis of mitochondrial membrane potential (ΔΨm) of SL‐ZSU‐1 cell treated with ultraviolet‐C (UV‐C) light indicated the decrease in mitochondrial membrane potential was dependent on the times of ultraviolet treatment. Both of them are different from apoptosis in cultured Drosophila melanogaster cell lines (S2 and BG2) and it appears evident mitochondria are involved in apoptosis of the studied lepidopteran cells.     

Dynamic analysis of apoptosis in primary cortical neurons by fixed- and real-time cytofluorometry

We describe here a cytofluorometric technology for the characterization of decision, execution, and degradation steps of neuronal apoptosis. Multiparametric flow cytometry was developed and combined to detailed fluorescence microscopy observations to establish the chronology and hierarchy of death-related events: neuron morphological changes, mitochondrial transmembrane potential (DeltaPsi(m)) collapse, caspase-3 and -9 activation, phosphatidyl-serine exposure, nuclear dismantling and final plasma membrane permeabilization. Moreover, we developed a reliable real-time flow cytometric monitoring of DeltaPsi(m) and plasma membrane integrity in response to neurotoxic insults including MPTP treatment. Taking advantage of recently developed specific fluorescent probes and a third generation pan-caspase inhibitor, this integrated approach will be pertinent to study the cell biology of neuronal apoptosis and to characterize new neuro-toxic/protective molecules.     

Novel piperazine induces apoptosis in U937 cells

The effect of 1,4-bis-(4-(1H-benzo[d]imidazol-2-yl-phenyl)) piperazine (BIPP), a newly synthesized piperazine derivative, on U937 leukemia cell viability was investigated. We show that BIPP induces dose-responsive apoptotic cell death in U937 cells by intrinsic mechanisms of apoptosis. Maximum apoptotic effect of BIPP on U937 cells was observed at 12.8μM. BIPP-induced apoptosis was evident by characteristics such as altered annexin-V binding, caspase activation, loss of mitochondrial membrane potential (MMP) and cytochrome c release. BIPP also differentially activates initiator and effector caspases combined with the loss of MMP strongly suggesting that BIPP causes an intrinsic apoptosis in U937 leukemia cells. Due to our observations that BIPP induces leukemia cell death without significantly affecting normal cells, our data suggests that it may be a potential therapeutic agent for human myeloid leukemia.     

颅脑创伤后大鼠脑组织脑红蛋白表达变化及其与神经元凋亡的关系研究

目的：研究大鼠弥漫性颅脑创伤后脑组织中脑红蛋白的表达变化情况,探究创伤后脑红蛋白表达变化及其与神经元凋亡的关系。方法：采用雄性SD大鼠50只,随机分为10组（n=5）空白对照组、伤后30min、1h、2h、6h、12h、24h、48h、72h和5d组。以Marmarou’s自由落体打击装置复制颅脑创伤模型,采用免疫组化技术检测伤后不同时间脑组织中脑红蛋白的表达情况及神经元凋亡相关基因Bax、Bcl-2表达情况,并对所得数据进行统计学分析。结果：致伤区皮层神经元脑红蛋白表达分别于伤后2h、72h呈现出两次高峰表达;伤后30min～1h、48～72h期间大脑皮层区脑红蛋白表达的上调均伴随着Bax/Bcl-2比值上升趋势减缓甚至呈现下降趋势。结论：弥漫性颅脑创伤后脑组织中脑红蛋白的高表达在一定程度上可以拮抗创伤应激及伤后继发缺血、缺氧性损伤所导致的神经元凋亡,在颅脑创伤的超早期（〈3h）、急性期（〈72h）可能具有一定的神经保护作用。     

钙-钙调素与小麦苗中热激蛋白的诱导

在 34℃热激条件下 ,种子经钙预处理的小麦苗中的钙调素含量随着热激时间的延长而增加 ,热激 90min时达最大值 ,而种子用钙离子螯合剂EGTA预处理的小麦苗中钙调素含量无明显增加。种子用EGTA及钙调素拮抗剂CPZ和TFP预处理的小麦幼苗在 34℃热激时 ,热激蛋白的合成量减少。 4d的小麦幼苗在34℃或 37℃热激条件下 ,能诱导耐热性的获得 ,分别用EGTA、钙离子通道阻断剂易博定、钙调素拮抗剂TFP或CPZ预处理种子后 ,所得幼苗热诱导的耐热性的提高程度有所下降     

Dissection of the multiple mechanisms of TNF-alpha-induced apoptosis in liver injury

Tumor necrosis factor (TNF)-alpha-induced hepatocyte apoptosis is implicated in a wide range of liver diseases including viral hepatitis, alcoholic hepatitis, ischemia/reperfusion liver injury, and fulminant hepatic failure. TNF-alpha exerts a variety of effects that are mediated mainly by TNF-receptor 1 (TNF-R1) in cell death. The activation of TNF-R1 leads to the activation of multiple apoptotic pathways involving the activation of the pro-death Bcl-2 family proteins, reactive oxygen species, C-Jun NH2-terminal kinase, cathepsin B, acidic sphingomyelinase and neutral sphingomyelinase. These pathways are closely interlinked and mainly act on mitochondria, which release the apoptogenic factors and other events, resulting in apoptosis. This article reviews the recent progress in the molecular mechanisms of TNF-alpha-induced apoptosis in hepatocytes, and discusses how these molecular findings are shaping our understanding of the pathogenesis of liver diseases and our strategy to develop novel therapeutics.     

Reactive oxygen and nitrogen species induce cell apoptosis via a mitochondria-dependent pathway in hyperoxia lung injury

Hyperoxia-induced lung injury limits the application of mechanical ventilation on rescuing the lives of premature infants and seriously ill and respiratory failure patients, and its mechanisms are not completely understood. In this article, we focused on the relationship between hyperoxia-induced lung injury and reactive oxygen species (ROS), reactive nitrogen species (RNS), mitochondria damage, as well as apoptosis in the pulmonary epithelial II cell line RLE-6TN. After exposure to hyperoxia, the cell viability was significantly decreased, accompanied by the increase in ROS, nitric oxide (NO), inflammatory cytokines, and cell death. Furthermore, hyperoxia triggered the loss of mitochondrial membrane potential (▵Ψm), thereby promoting cytochrome c to release from mitochondria to cytoplasm. Further studies conclusively showed that the Bax/Bcl-2 ratio was enlarged to activate the mitochondria-dependent apoptotic pathway after hyperoxia treatment. Intriguingly, the effects of hyperoxia on the level of ROS, NO and inflammation, mitochondrial damage, as well as cell death were reversed by free radical scavengers N-acetylcysteine and hemoglobin. In addition, a hyperoxia model of neonatal Sprague-Dawley (SD) rats presented the obvious characteristics of lung injury, such as a decrease in alveolar numbers, alveolar mass edema, and disorganized pulmonary structure. The effects of hyperoxia on ROS, RNS, inflammatory cytokines, and apoptosis-related proteins in lung injury tissues of neonatal SD rats were similar to that in RLE-6TN cells. In conclusion, mitochondria are a primary target of hyperoxia-induced free radical, whereas ROS and RNS are the key mediators of hyperoxia-induced cell apoptosis via the mitochondria-dependent pathway in RLE-6TN cells.