EPO对全脑缺血后低氧诱导因子1α及存活素表达的调节

[目的]研究促红细胞生成素对全脑缺血再灌注大鼠缺氧诱导因子1α和凋亡相关蛋白存活素的调节,探讨其抗凋亡的可能机制。[方法]将成年雄性SD大鼠75只按随机数字表法分为全脑缺血组（n=35）和全脑缺血EPO干预组（n=35）,然后按再灌注时间不同又分为6h、12h、24h、48h、72h、5d和7d七个亚组。采用改良的Pu刘lsineli 4-VO法制作全脑缺血大鼠模型。应用TUNEL染色检测再灌注后不同时间点海马CA1区的神经元凋亡水平,免疫组织化学方法检测再灌注后不同时间点海马CA1区缺氧诱导因子1α和存活素表达水平的变化。[结果]全脑缺血EPO干预组24h至7d亚组TUNEL阳性神经元计数分别为1.50±0.73、3.14±0.88、5.78±1.03、7.78±1.79、10.34±1.82.与全脑缺血组相应时间点亚组相比明显减少,差异均有统计学意义（P〈0.05）。全脑缺血EPO干预组48h至5d亚组HIF-1α表达阳性细胞计数分别为11.26±0.02、20.28±2.03、3.33±0.04,与全脑缺血组相应时间点亚组相比明显减少,差异均有统计学意义（P〈0.05）.全脑缺血EPO干预组24h至5d亚组survivin蛋白表达阳性细胞计数分别为12.21±1.04、16.34±4.02、24.33±3.03、30.52±5.04,与全脑缺血组相应时间点亚组相比明显增高,差异均有统计学意义（P〈0.05）。[结论]在全脑缺血急性期,EPO抑制HIF-1α的表达而使存活素表达升高,具有一定的神经保护作用.     

益肺温阳化浊汤对Aβ诱导的AD大鼠海马Aβ含量及凋亡因子的影响

目的:探讨中药复方益肺温阳化浊汤对Aβ诱导的AD大鼠模型海马Aβ异常沉积与神经元凋亡的关系及Caspase-9、Caspase-3、Bcl-2、Bax凋亡相关因子的影响。方法:采用MorriS水迷宫测试各组大鼠行为学,流式细胞仪检测神经细胞凋亡率,Western Blot检测各组Caspase-9、Caspase-3、Bcl-2、Bax的表达;观察益肺温阳化浊汤低、中、高剂量对Aβ诱导的AD大鼠海马区Aβ含量及凋亡因子的影响。结果:模型大鼠经益肺温阳化浊汤处理后,海马区Aβ含量明显降低,神经元凋亡减轻,抑制Caspase-9、Caspase-3、Bax表达,上调抗凋亡蛋白Bcl-2的表达,并呈一定的剂量依赖性。结论:益肺温阳化浊汤可能通过促进Aβ诱导的AD大鼠海马区神经细胞中Aβ的清除,减轻Aβ的神经毒性,从而抑制神经元的凋亡,起到保护神经元的作用。     

不同负荷运动对大鼠海马CA1区Bax、Bcl-2表达的影响

目的:采用跑台训练方式,建立大鼠运动模型,运用免疫组织化学SABC法研究不同负荷运动对大鼠海马CA1区Bax、Bcl-2表达的影响。方法:将大鼠分为对照组、中等强度运动组和大强度运动组,对大鼠进行为期5周的跑台训练,用免疫组化结合图像分析技术观察海马神经元中Bax、Bcl-2的免疫反应活性。结果:中等强度运动后,大鼠海马CA1区神经元Bcl-2蛋白的表达显著上升,Bax蛋白略微增加,Bax/Bcl-2显著下降。大强度运动后,大鼠海马CA1区神经元中Bcl-2蛋白的表达略微增加,Bax蛋白的表达显著增加,Bax/Bcl-2显著增高。结论:中等强度运动可促使Bcl-2蛋白的表达,使Bax/Bcl-2显著降低,抑制细胞凋亡;而大强度运动促进Bax蛋白的表达,使Bax/Bcl-2比例显著增加,促进细胞凋亡。     

癫痫持续状态后大鼠海马神经元凋亡的形态学观察

本实验以腹腔注射青霉素诱导SE,以HE染色观察SE后大鼠海马神经元凋亡的细胞形态学改变。成功造模后24小时,模型组有细胞核内染色质凝聚和边集等不同程度的细胞凋亡表现。验证了在SE后神经元死亡形成中有凋亡现象存在,并成功摸索到一种操作简易的适合癫痫后细胞凋亡研究采用的大鼠模型。     

蝙蝠葛碱对AD大鼠海马神经元凋亡的影响

目的:探讨蝙蝠葛碱(DAU)对AD大鼠海马神经元凋亡的影响。方法:透射电镜观察AD大鼠海马超微结构,Western Blot检测大鼠脑Bcl-2和Bax表达。结果:与模型组比较,治疗组、DAU高、中、低剂量组Bax表达明显降低(P0.05),而Bcl-2表达明显升高(P0.05)。结论:DAU可通过调节脑Bax和Bcl-2表达来抑制神经元凋亡。     

米诺环素对压力作用下体外培养神经细胞凋亡的抑制

目的在开放式压力控制培养系统作用下体外培养大鼠视网膜神经细胞（retinal neurons,RNs）,观察米诺环素对其活性及凋亡的影响．并进—步探讨其对受损RNs保护的可能机制．方法采用出生0-3d的Sprague Dawlev（SD）大鼠视网膜神经细胞体外混合培养。制备RNs加压培养模型．通过细胞形态学观察,四唑盐（MTT）比色法测定细胞活力,吖啶橙／溴化乙锭（AO／EB）染色法检测细胞的凋亡率来观察米诺环素对上述损伤细胞的保护及治疗作用．以及应用免疫细胞化学染色法。观察诱导型一氧化氮台酶（iNOS}和半胱天冬酶-3,（caspase-3）表达的改变、结果 加压培养后,在倒置显微镜下RNs与对照组相比形态改变较咀.细胞活力降低,大量细胞发生凋亡（占53.93％）.而米诺环素治疗组（20μmol／L）细胞则形态改善．活力显著增高：凋亡数目减少（占17．29％）．差异具有显著性（P〈0.0051）。免疫细胞化学染色法示米诺环素治疗组细胞内NOS和caspase-3表达较加压损伤组减少。结论 一定剂量的米潜环素在体外可有效抑制蓝力。引起的大鼠视网膜神经细胞损伤及凋亡。抑制NOS和capase-3的表达可能是其潜在作用机制。     

医药卫生

阻遏caspase-3基因表达延缓海马硬化及神经元凋亡治疗颞叶癫痫；预交感神经阻滞对放烧复合伤小鼠HPA轴的调节及相关机制研究；三氧化二坤对黑色素瘤B-16细胞增殖与凋亡的影响；慢性酒精中毒大鼠学习记忆及内侧隔核胆碱能神经元的变化；[编者按]     

胡桃醌对人乳腺癌MCF-7细胞增殖抑制作用研究

目的:研究胡桃醌诱导人乳腺癌MCF-7细胞凋亡及其形态学观察。方法:四甲基偶氮唑蓝(MTT)法检测胡桃醌对MCF-7细胞的增殖抑制作用;Hochest33258荧光染色法观察细胞凋亡形态;流式细胞术(FCM)检测细胞凋亡率。结果:胡桃醌对人乳腺癌MCF-7细胞的IC50为11.99μmol/L;胡桃醌作用24h后,细胞生长密度变疏,细胞皱缩,出现凋亡小体,且随着给药浓度升高,凋亡小体也逐渐增多;胡桃醌5、10、20μmol/L作用24h后MCF-7细胞凋亡率分别为6.35%、12.43%、28.55%。结论:胡桃醌对人乳腺癌MCF-7细胞具有增殖抑制作用。     

氟哌啶醇对N-甲基-D-门冬氨酸诱导的大鼠海马神经细胞损伤的保护作用

用原代培养大鼠海马神经细胞的方法，观察氟哌啶醇对N－甲基－D－门冬氨酸（NMDA）诱导的大鼠海马神经损伤的保护作用。结果表明，氟哌啶醇能剂量依赖地抑制NMDA诱导的大鼠海马神经细胞内乳酸脱氢酶（LDH）的释放，并改善受损细胞形态，四唑盐比色法（MT）显示存活率增高。提示氟哌啶醇对NMDA诱导的大鼠海马神经细胞损伤有保护作用。     

ECH-931长程治疗对老年性痴呆的分子基因药效学研究

阿尔茨海默病（AD）是最常见的与年龄有关的神经退行性疾病。全世界约15％的65岁以上人群和30％的80岁以上老人患有AD。AD的病因仍未明确，目前尚无有效的预防和治疗方法。随着人类社会的老龄化，本病已成为一个世界性的重大医学和社会难题。 许多证据显示β-淀粉样肽在阿尔茨海默病（AD）的病因学和／或病程发展中起着关键作用。很多研究提示β-淀粉样肽的神经毒性与氧的负荷和自由基损伤密切相关。最近的研究表明，NF-κB在神经元存活和突触的可塑性方面发挥重要作用，CREB是长时程记忆（LTM）和长时程增强效应（LTP）的必要基因开关。 本研究观察了科研新药ECH-931对β-淀粉样肽1-40，β-淀粉样肽25-35和H2O2所诱导的B104中枢神经系神经元细胞株神经毒性的预防和治疗作用。ECH-931的低，中，高实验浓度分别为50μg／ml，100μg／ml和150／200μg／ml，用ECH-931处理的方案如下：细胞经ECH-931预处理3天后，用ECH-931和H2O2（100-200μM）共同处理3—12小时，以观察ECH-931对H2O2神经毒性的防治效果；细胞经ECH-931预处理3天后，用ECH-931和Abeta 1-40（10μM）处理48小时来预防性治疗Abetal-40的神经毒性；在细胞暴露于Abeta25-35（25μM）8小时后再用ECH-931处理48小时（经ECH-931和Abeta 25-35共同处理48小时）以观察ECH-931对Abeta神经毒性的治疗作用：在NF-κB和CREB基因转染后以ECH-931处理5天，以观察ECH-931对NF-κB和CREB基因表达的影响；在NF-κB基因转染并加Abeta 1-40（5uM）后以ECH-931处理3天，以观察ECH-931拮抗Abeta 1-40对NF-κB基因表达影响的效果。 结果表明：经ECH-931（50-200μg／ml）预处理和共同处理B104神经元能完全拮抗β-淀粉样肽1-40（10μM）诱导的神经毒性（P〈0．05-0．01）；用ECH-931（50-200μg／ml）治疗性处理能显著阻止由β-淀粉样肽25-35（25μM）诱导的B104神经元细胞死亡／凋亡（P〈0．05—0．01）；用ECH-931（50—200μg／ml）预处理和共同处理能显著保护由H2O2（100—200μM）诱导的B104神经元细胞死亡／凋亡；用ECH-931（50-150μg／ml）治疗性处理能显著上调在B104CNS神经元细胞中转染基因NF-κB和CREB的表达（P〈0．05-0．01）；ECH-93150-150μg／ml能拮抗由β-淀粉样肽1-40诱导的NF-κB表达抑制（P〈0．01）。并且，所有ECH-931的处理效应都呈现剂量依赖性（P〈0．05-0．01）。 基于以上研究结果，我们认为ECH-931能保护（预防和治疗）神经元免受由β-淀粉样肽诱导的神经毒性。其机制与拮抗活性氧／氢氧根自由基损伤和激活NF-κB细胞存活信号通路有关。ECH-931治疗AD的另一个重要机理可能是它能调节CREB的表达，而CREB是长期记忆的基因开关。ECH-931的神经元保护效应尤其是其阻止β-淀粉样肽诱导的毒性和细胞死亡的效力显示出它治疗神经退行性疾病（如AD）的潜力，具有重要的研究开发价值和广阔的应用前景。     

Enumeration of lymphocyte subsets using flow cytometry: Effect of storage before and after staining in a developing country setting

Lymphocyte subset estimations by flow cytometry in population-based studies require transportation of samples from the field site to the laboratory. As samples arrive late in the day they have to wait overnight before being processed. The effect of two possible approaches, sample storage for 24 h before staining and immediate staining with analysis after 24 h and 48 h were evaluated. Two sets of experiments were performed with EDTA (ethylenediamine tetra-acetate) anticoagulated peripheral blood. In the first experiment, after collection, each sample was divided into two portions. One portion was stained at the time of blood collection and the other 24 h later after keeping it at room temperature (38–45°C). In the second experiment, blood samples were stained within 1–2 h. Each sample was analyzed immediately upon completion of staining process and subsequently after 24 h and 48 h of storage at 4°C. Results suggest that blood collected in EDTA can be processed using whole blood lysis method, after storage at room temperature (38–45°C) for 24 h with some but not significant alteration in T-cell subsets. Storage at 4°C after staining for 24 h results in a lesser and insignificant loss of cells or alteration of T-cell subsets and may be the method of choice.     

草鱼细胞株（ZC—7901）乌苯苷抗性突变体的筛选

采用甲基磺酸乙酯（ＥＭＳ）诱变草鱼吻端组织细胞（ＺＣ－７９０１）．用乌苯苷（Ｏｕａｂａｉｎ）筛选抗药性突变体．结果表明，ＥＭＳ对草鱼细胞的诱变效果与该药物对哺乳类细胞的诱变效果有明显差异．草鱼细胞对乌苯苷的敏感性也大大低于哺乳类细胞．ＺＣ－７９０１细胞在传代后２４ｈ，用浓度为２．８ｍｇ／ｍｌ的ＥＭＳ处理２４ｈ，诱变效果较理想，细胞杀伤率约３０％，这比通常用ＥＭＳ诱变哺乳类细胞所使用的浓度（０．４ｍｇ／ｍｌ）增大了７倍，存留下来的细胞用浓度为２５ｍｍｏｌ／Ｌ乌苯苷筛选抗性突变体，有少数细胞存活，并具有分裂增殖能力．但在细胞瓶内不易形成单层     

Order of draw of blood samples affect potassium results without K-EDTA contamination during routine workflow

IntroductionA specific sequence is recommended for filling blood tubes during blood collection to prevent erroneous test results due to carryover of additives. However, requirement of this procedure is still debatable. This study was aimed to investigate the potassium ethylenediaminetetraacetic acid (K-EDTA) contamination in blood samples taken after a tube containing the additive during routine workflow. The study was also carried out to examine the effect of order of draw on potassium results, regardless of K-EDTA contamination.Materials and methodsIn 388 outpatients, to determine the probability of K-EDTA cross-contamination, blood was drawn sequentially into a serum tube, followed by a tube containing K-EDTA, and by another serum tube. In another 405 outpatients, to evaluate the effect of order of draw blood unrelated to K-EDTA contamination, two serum tube were successively collected. Potassium was measured on Cobas 6000 c501 analyser (Roche Diagnostic GmbH, Mannheim, Germany) by indirect ion selective electrode method.ResultsOf paired samples collected before and after a K-EDTA tube, 24% had a potassium difference of above 0.3 mmol/L. However, no EDTA contamination was detected in these samples as well as 95% confidence intervals (CI) of limits of agreement for calcium were within the allowable error limits based on reference change values. Interestingly, of blood samples drawn successively, 24% had also a difference greater than 0.3 mmol/L for potassium.ConclusionIncorrect order of draw using closed blood collection system does not cause K-EDTA contamination, even in routine workflow. However, regardless of K-EDTA contamination, order of draw has significant influence on the potassium results.     

Cytotoxicity and Apoptosis Inducing Activities of 2-Amino-4H-chromene-3-carbonitrile Derivatives Loaded on Gold Nanoparticles Against Human Breast Cancer Cell Line T47D

Chemotherapy drugs, used for prevention of uncontrolled cell proliferation in certain tissues as well as inducing apoptosis in tumor cells, are important candidates for treatment of cancer. The synthesized 2-amino-4H-chromene-3-carbonitrile derivatives effective on cancerous cells resistant to other drugs such as Paclitaxel were used due to their ability in induction of apoptosis. The growth inhibitory and inducing apoptosis activities were determined. In order to make it target-oriented, the best compound was conjugated with gold nanoparticles (NPs) by aspartic acid with chemical reduction method. Cytotoxicity effect of 2-amino-4H-chromene-3-carbonitrile derivatives against the T47D breast cancer cell line was determined by MTT assay. The synthesis of gold NPs was confirmed by transmission electron microscopy, UV–Vis and dynamic light scattering. To assess the effects of compounds on the process of apoptosis, staining methods with acridine orange–ethidium bromide and Hoechst staining by fluorescence microscopy and DNA fragmentation by the diphenylamine method were used. The synthesized compounds containing two NH₂ groups on benzene rings, demonstrated more cytotoxicity effect. The effect of conjugation with gold NPs and the induction of apoptosis were studied with the best compound. The cytotoxicity effects of the synthesized 2-amino-4H-chromene-3-carbonitrile compounds were changed by replacement of NO₂ group on thiol ring with different chemical groups on the benzene ring. Analyses of treated cell lines by conjugated and non-conjugated forms of compounds verified their ability in inducing apoptosis while conjugated form demonstrated higher apoptosis.     

A microfluidic-based neurotoxin concentration gradient for the generation of an in vitro model of Parkinson's disease

In this study, we developed a miniaturized microfluidic-based high-throughput cell toxicity assay to create an in vitro model of Parkinson's disease (PD). In particular, we generated concentration gradients of 6-hydroxydopamine (6-OHDA) to trigger a process of neuronal apoptosis in pheochromocytoma PC12 neuronal cell line. PC12 cells were cultured in a microfluidic channel, and a concentration gradient of 6-OHDA was generated in the channel by using a back and forth movement of the fluid flow. Cellular apoptosis was then analyzed along the channel. The results indicate that at low concentrations of 6-OHDA along the gradient (i.e., approximately less than 260 μM), the neuronal death in the channel was mainly induced by apoptosis, while at higher concentrations, 6-OHDA induced neuronal death mainly through necrosis. Thus, this concentration appears to be useful for creating an in vitro model of PD by inducing the highest level of apoptosis in PC12 cells. As microfluidic systems are advantageous in a range of properties such as throughput and lower use of reagents, they may provide a useful approach for generating in vitro models of disease for drug discovery applications.     

Microfluidic culture platform for studying neuronal response to mild to very mild axonal stretch injury

A new model for studying localised axonal stretch injury is presented, using a microfluidic device to selectively culture axons on a thin, flexible poly (dimethylsiloxane) membrane which can be deflected upward to stretch the axons. A very mild (0.5% strain) or mild stretch injury (5% strain) was applied to primary cortical neurons after 7 days growth in vitro. The extent of distal degeneration was quantified using the degenerative index (DI, the ratio of fragmented axon area to total axon area) of axons fixed at 24 h and 72 h post injury (PI), and immunolabelled for the axon specific, microtubule associated protein-tau. At 24 h PI following very mild injuries (0.5%), the majority of the axons remained intact and healthy with no significant difference in DI when compared to the control, but at 72 h PI, the DI increased significantly (DI = 0.11 ± 0.03). Remarkably, dendritic beading in the somal compartment was observed at 24 h PI, indicative of dying back degeneration. When the injury level was increased (5% stretch, mild injury), microtubule fragmentation along the injured axons was observed, with a significant increase in DI at 24 h PI (DI = 0.17 ± 0.02) and 72 h PI (DI = 0.18 ± 0.01), relative to uninjured axons. The responses observed for both mild and very mild injuries are similar to those observed in the in vivo models of traumatic brain injury, suggesting that this model can be used to study neuronal trauma and will provide new insights into the cellular and molecular alterations characterizing the neuronal response to discrete axonal injury.     

CO_2、NH_3对YBa_2Cu_3O_(7-x)的结构及超导电性的影响

以热重分析(TGA)、电阻测量和X射线衍射分析等方法,研究了CO_2与NH_3对YBa_2Cu_3O_(7-x)超导体结构稳定性及超导,电性的影响。当温度低于373K时,CO_2对YBa_2Cu_3O(7-x)的结构和超导电性无影响;当温度为523K时,CO_2使YBa_2Cu_3O_(7-x)的Tc下降;温度高于703K时,CO_2将很快破坏YBa_2Cu_3O_(7-x)的结构,并产生化学反应,生成新的化合物,在CO_2中存在的O_2与N_2对YBa_2Cu_3O(7-x)与CO_2的反应及反应产物的影响有较大差异。NH_3对YBa_2Cu_3O(7-x)结构稳定性的影响不大。     

全植床深板层角膜移植术后神经重建实验研究

目的观察新西兰兔全植床深板层角膜移植术后不同时期的角膜神经重建过程。方法新西兰兔麻醉后行冰冻保存角膜全植床深板层角膜移植术。术后分别于第7、21天,第1、2、3、4、6个月摘取术眼角膜,行乙酰胆碱酯酶神经染色,光学显微镜下观察。结果术后7天,植片内神经溃变,同时植床远端未损伤神经发出分支,穿过创缘进入植片。术后21天,神经溃变完成。术后1个月,植片内较多细、长、直的神经纤维,创缘外侧植床基质神经发出较多分支平行于创缘,但未进入植片。术后3个月,植床近端基质神经同一部位同时发出2￣4个分支进入植片,植片内细、长、直的神经纤维达到最多。术后4￣6个月,植片内神经重新塑型。少数植床近端基质神经形态基本接近正常。结论全植床深板层角膜移植术后神经再生恢复较慢。     

图书消毒及其对纸质的影响

探讨了图书消毒的方法及其对纸质的影响，通过实验，紫外线消毒２０ｍｉｎ和甲醛薰蒸消毒（甲醛浓度１００ｍｌ／ｍ＾３）３ｈ可达到消毒目的，图书纸张张力随着紫外线照射时间和甲醛薰蒸时间延长而下降。