模拟失重与骨组织的细胞凋亡

背景：空间骨丢失主要表现为成骨细胞、骨细胞活性减弱,而包括Fas蛋白在内的诸多因素可调节成骨细胞、骨细胞的增殖和分化并最终调控骨形成。目的：观察失重对骨组织中细胞凋亡的影响。方法：5周龄SPF级雄性SD大鼠36只,随机分为尾吊模拟失重组及对照组。建立大鼠尾吊模拟失重模型,建模后7,14,21 d,使用自动生化仪检测大鼠血清中钙、磷、碱性磷酸酶含量,放射免疫分析法测定骨钙素含量,原位细胞凋亡检测技术检测骨组织中细胞凋亡,免疫组化方法检测骨组织中Fas蛋白含量。结果与结论：尾吊模拟失重组大鼠血清中碱性磷酸酶、骨钙素含量均显著低于对照组(P < 0.05),但血钙、磷浓度均较相应对照组升高,骨组织中成骨细胞、骨细胞及骨髓间充质细胞凋亡指数、骨组织中Fas抗原含量均高于相应对照组(P < 0.01)。说明尾部悬吊模拟失重增加骨组织中Fas蛋白表达,从而增加大鼠骨组织中成骨细胞、骨细胞及骨髓间充质细胞凋亡。关键词：尾吊;模拟失重;生化;骨钙素;细胞凋亡;Fas蛋白     

模拟失重雌性大鼠L5椎骨的应力松弛特点

背景：航天医学有必要了解模拟失重对雌性大鼠承重骨应力与时间的变化规律。 目的：观察模拟失重对雌性大鼠承重骨应力松弛的影响,为航天医学提供模拟失重动物椎骨应力松弛数据。 设计、时间及地点：随机对照动物实验,于2005-07/09在吉林大学力学实验中心完成。 材料：6月龄雌性大鼠30只,体质量275~296 g,随机分为正常对照组、失重组各15只。 方法：大鼠复制失重骨质疏松动物模型。在日本岛津电子万能试验机上对正常和失重组各10个试样进行应力松弛实验,应力松弛实验的应变增加速度为50%/min,设定时间为7 200 s,采集100个数据以一元线性回归分析的方法处理实验数据。 主要观察指标：①应力松弛实验数据和曲线。②归一化应力松弛函数计算。 结果：正常和失重组应力松弛最初600 s变化较快,之后应力缓慢下降,对照组7 200 s应力松弛量为0.88 MPa,失重组7 200 s应力松弛量为0.62 MPa。 结论：应力松弛曲线是以对数关系变化的,失重骨质疏松对应力松弛具有一定影响。     

吸烟大鼠脑细胞凋亡与Fas蛋白表达

目的 观察吸烟诱导大鼠脑组织细胞凋亡与Fas蛋白表达及两者之间的关系，探讨吸烟的时间和数量对细胞凋亡及Fas蛋白表达的影响。方法 利用脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记(TUNEL)技术及免疫组织化学法检测细胞凋亡和Fas蛋白表达。结果 除戒烟组外各吸烟组凋亡细胞数量及Fas蛋白表达比对照组有显著性增加。凋亡细胞数量随吸烟时间及数量的增加而增加，Fas蛋白表达强度随吸烟时间延长而增加。结论 吸烟可诱导大鼠脑组织细胞凋亡及Fas蛋白过度表达。Fas介导吸烟诱导的细胞凋亡，细胞凋亡与Fas蛋白表达强度与吸烟的时间及数量相关。     

骨折愈合延迟大鼠在模拟失重状态下的变化

背景：失重状态是一种特殊物理环境，长期处于该环境易引起机体骨质疏松等改变，而此环境下可影响骨折愈合，但骨折愈合延迟的机制尚不清楚。 目的：通过电子显微镜、免疫组织化学等观察在模拟失重情况下大鼠后肢骨折的愈合过程，观察其组织形态和细胞因子的变化及其特点。 设计、时间及地点：随机对照动物实验，于2005-08/2007-12在解放军空军总医院中心实验室完成。 材料：动物分组：SD成年清洁级雄性大鼠64只，体质量(170±5) g，随机分为失重组和单纯骨折组，每组32只。 方法：制备64只大鼠后肢腓骨骨折模型。单纯骨折组骨折形成后动物自由活动，失重组采用头低位尾悬吊模拟失重状态。两组大鼠分别于处理7，14，21，28 d各取8只麻醉后处死取骨折侧腓骨。 主要观察指标：用二步法免疫组织化学检测骨折断端骨形态发生蛋白2表达，透射电镜观察模拟失重后肢骨折断端的愈合特点。 结果：64只大鼠全部进入结果分析。失重7 d与单纯骨折相比骨形态发生蛋白表达差异无显著性意义(χ2=1.640，P > 0.05)；失重14 d与单纯骨折相比其骨折愈合较慢和骨形态发生蛋白表达低下(χ2=3.000，P < 0.05)；失重21 d与单纯骨折相比骨折愈合形态学改变延迟，骨形态发生蛋白表达显著降低 (χ2=4.063，P < 0.05)；失重28 d与单纯骨折相比，病理学显示骨折愈合明显延迟、骨形态发生蛋白2表达降低(χ2=4.655，P < 0.05)。 结论：模拟失重状态对后肢骨折大鼠骨折愈合过程有延迟作用，模拟失重状态持续的时间越长，则对后肢骨折大鼠的延迟愈合越明显。     

大鼠局灶性脑缺血再灌注后Fas表达与细胞凋亡关系的研究

目的 :本实验通过观察缺血再灌注后大鼠脑组织中 Fas的表达及细胞凋亡的情况 ;初步探讨脑缺血后 Fas表达与细胞凋亡的关系 ,以及其发生的机理。方法 :采用 TUNEL和免疫组化方法观察细胞凋亡及 Fas蛋白在脑缺血再灌注后随时间延长动态变化情况 ,并利用图像分析测定二者的免疫强度。结果 :脑缺血再灌注后神经细胞过度地表达 Fas,以缺血半暗带明显 ,且在缺血再灌注后 6小时达高峰 ,于 2 4小时开始下降。而细胞的凋亡于缺血再灌注后 6小时开始增加 ,于 2 4小时达高峰 ,于 36小时略有下降 ,凋亡细胞分布也以缺血半暗带明显。结论 :脑缺血再灌注可引起Fas的过度表达和神经细胞凋亡增加 ,而缺血后 Fas的过度表达是诱导细胞凋亡的重要的因素之一     

癫痫与新近发现的细胞凋亡相关基因研究动态

癫痫(Epilepsy)是神经内科常见病之一, 其持续状态(Status Epilepsy, SE)发作后, 各种酶、神经递质、 氨基酸等化学物质会迅速发生变化, 导致脑水肿, 造成选择性神经元损害, 甚至死亡.近来国内外大量研究认为SE后神经细胞死亡的方式除坏死外, 还可以细胞凋亡(apoptosis)的形式发生[1～3].本文主要就新近发现的有关调控细胞凋亡的基因Bcl-w、 ICE基因家族、 CystainB、 Fas、 GADD45、 PPTA与癫痫的关系作一综述.     

神经生长因子对大鼠前脑缺血再灌注后细胞凋亡及FaS蛋白表达的影响

目的：研究神经生长因子（NGF）对大鼠前脑缺血再灌注后海马CA1区Fas蛋白表达和细胞凋亡的影响。方法：夹闭大鼠双侧颈总动脉造成前脑缺血，30min后松夹再灌注，NGF组和生理盐水组于再灌注开始时分别肌肉注射NGF30μg·mL^-1和生理盐水0．1mL，应用免疫组化法和TUNEL法检测各组大鼠海马CA1区Fas蛋白表达和细胞凋亡。结果：再灌注后6和24h，NGF组Fas蛋白平均吸光度值小于生理盐水组（P〈0．001和P〈0．05）。再灌注后48h两组比较差异无统计学意义（P〉0．5）。再灌注后6、24和48h，NGF组TUNEL阳性细胞率均低于生理盐水组，差异有统计学意义（P〈0．005）。结论：NGF可以减少大鼠脑缺血再灌注后海马CA1区Fas蛋白表达，抑制细胞凋亡，从而发挥其神经保护作用。     

模拟失重大鼠延髓内脏带向下丘脑室旁核投射的儿茶酚胺能神经元表达Fos

确定延髓内脏带向下丘脑室旁核（PVN）投射通路是否参与对模拟失重的反应,用HRP逆行追踪结合抗Fos和抗栈氨酸羟化酶（TH）的免疫组织化学三重标记技术。观察4周模拟失重大鼠延髓内脏带向PVN投射的儿茶酚胺能神元Fos表达情况。发现有7种不同的标记细胞：HRP,Fos,TH单标细胞;Fos/HRP,Fos/TH,HRP/TH双标细胞;Fos/HRP/TH三标细胞,主要分布于延髓内脏带即延髓中尾段的孤束核和腹外侧区以及两者之间的网状结构。向PVN投射的神经元中有15.3%为Fos阳性细胞,即对失重起反应,而这些神经元中有62.6%为儿茶酚胺能神经元。结果显示,延髓内脏带投射至PVN的儿茶酚胺能神经元有些参与对失重的心血管反应。     

中药安胎养血合剂干预肾移植大鼠肾细胞凋亡及Fas/FasL基因的表达

背景：前期实验表明,作者自制的中药复方安胎养血合剂能够抑制肾移植大鼠急性排斥反应,延长受体存活时间。 目的：观察安胎养血合剂对异基因肾移植大鼠移植肾肾小管上皮细胞凋亡及Fas/FasL基因表达的影响,并与同基因肾移植大鼠进行对比。 设计、时间及地点：随机对照动物实验,于2005-07/2006-06在辽宁医学院附属第一医院外科实验室完成。 材料：选用雄性SD大鼠24只,雄性Wistar大鼠48只。 方法：按体质量相近配对后,按随机数字表法分为3组,每组12对。对照组大鼠同基因肾移植(Wistar→Wistar大鼠),生理盐水灌胃;生理盐水组大鼠异种基因肾移植(SD→Wistar大鼠),肾移植后不用任何免疫抑制剂,生理盐水灌胃;安胎养血合剂组大鼠异种基因肾移植(SD→Wistar大鼠),肾移植后给安胎养血合剂灌胃,灌胃剂量均为10 mL/(kg•d),移植后1次/d。 主要观察指标：于移植后第5天处死动物取肾脏组织,通过原位末端标记法检测凋亡指数,采用反转录-聚合酶链反应检测Fas/ FasL mRNA表达。 结果：生理盐水组移植肾组织凋亡细胞最多,对照组凋亡细胞极少见。生理盐水组和安胎养血合剂组凋亡指数显著高于对照组(P < 0.05),安胎养血合剂组凋亡指数显著低于生理盐水组(P < 0.05)。各组中均有Fas mRNA的表达,FasL mRNA的表达水平在安胎养血合剂组中明显低于生理盐水组(P < 0.05)。 结论：安胎养血合剂可以通过下调大鼠移植肾肾小管上皮细胞Fas L的mRNA表达,从而减少移植肾的细胞凋亡,起到免疫抑制剂的作用。     

缺血预处理后Fas蛋白表达与迟发性神经元凋亡的关系初步探讨

目的动态观察缺血预处理后大鼠大脑皮层和海马CA1区神经元凋亡与Fas蛋白表达变化情况,初步探讨缺血预处理后Fas蛋白表达与迟发性神经元凋亡的关系。方法四血管阻断法复制全脑缺血模型,动物随机分为非缺血对照组、预处理对照组、缺血预处理组和缺血组。采用尼氏和TUNEL染色法观察皮层及海马CA1区神经元存活数和凋亡细胞数,免疫组化方法检测Fas蛋白在缺血预处理后表达变化情况。结果缺血组缺血6h在皮质及海马CA1区Fas阳性表达细胞计数升高,12h达高峰;缺血预处理组缺血12h阳性细胞计数升高,24h达高峰。缺血组缺血6h出现凋亡细胞,48h凋亡细胞数达到高峰;缺血预处理组凋亡细胞数较缺血组明显减少。缺血组缺血7d神经元数明显减少,12周时神经元大量减少;缺血预处理组缺血7d时神经元数无明显变化,但12周时神经元同样大量减少。结论全脑缺血可能通过诱导Fas蛋白的表达增多,启动细胞凋亡,导致缺血后神经元凋亡的发生;缺血预处理虽可延缓缺血后神经元的凋亡,但无法提供真正的长时期的神经元保护作用,其有限的保护作用可能是通过延缓Fas蛋白的表达而减缓了神经元凋亡的进程。     

Fas mediates apoptosis in steroid-induced myopathy of rats.

Recently, apoptotic cell death has been reported in differentiated skeletal muscle, where apoptosis was generally assumed not to occur. To investigate whether apoptosis may contribute to the steroid-induced myopathy, rats treated with triamcinolone acetonide (TA) for 9 days were sacrificed for detecting apoptosis by in situ end-labelling (ISEL) and DNA electrophoresis in soleus muscles. Immunohistochemical stainings of Fas antigen and p53 protein were performed to examine whether apoptosis-related proteins were present in the myopathy. Muscle fibre necrosis and apoptotic myonuclei appeared in soleus muscles following administration of TA, while control muscles showed no evidences for apoptosis. Fas antigen was not detected in control muscles, but expressed in soleus muscles of steroid-induced myopathy. Some of the Fas antigen-expressing muscle fibres were positive for ISEL. p53 Protein was not detected in any muscle fibres. These findings indicate that TA can induce apoptosis in differentiated skeletal muscles, and Fas antigen might be partly related to apoptotic muscle death in steroid-induced myopathy.     

Problems of sensorimotor coordination in weightlessness

Previous studies about human sensorimotor coordination in space are inconclusive: it was reported that subjects in weightlessness point too high or too low, too fast or at normal speed, with increased or with normal variability; and that their tracking performance is degraded or normal. A better understanding of human performance in space would be desirable not only from the basic science perspective, but also for operational reasons. We propose a conceptual framework to explain the reported diversity, and to point out avenues for future research. We argue that exposure to weightlessness produces sensorimotor discordance, to which subjects gradually adapt through processes similar to those involved in earthbound adaptation. These processes require substantial information-processing resources in the brain, which may not be easily available during the hectic pace of a space mission. Within this framework, it is not surprising that previous data on sensorimotor performance in space were incongruent, as demand and availability of resources may have differed between missions, or even between subjects. We therefore propose that future work should control resource demand and availability, and study their effects on sensorimotor performance before and during space missions, in order to deconfound their effects from the immediate effects of gravity. A suitable hardware for such research is presented.     

Expression of Fas antigen is not associated with apoptosis in human myopathies

To determine whether the Fas-Fas-ligand mediated apoptosis occurs in the pathologic process of human muscle diseases, we examined the expression of Fas antigen, Fas-ligand messenger RNA, and chromosomal DNA fragmentation in the muscle cells of patients with a variety of human muscle disorders. The present study demonstrated that the Fas antigen of a whole molecule was expressed on the muscle fibers of patients with muscle wasting diseases. However, the apoptotic process did not occur in the muscle cells, and there was no evidence of Fas-ligand synthesis in the diseased muscle tissue. Our data suggest that the expression of Fas antigen on fibers in diseased muscle is related to unknown biological functions other than “apoptosis” in the process of muscle fiber injury. © 1997 John Wiley & Sons, Inc. Muscle Nerve, 20, 702–709, 1997.     

不同强度力竭运动模型大鼠肝细胞凋亡及相关变化

背景：国内外不少实验证明,不同运动方式容易造成肝损伤,导致不同程度的肝细胞凋亡,其具体机制尚不明确。 目的：建立不同强度力竭运动模型,观察运动后大鼠肝细胞凋亡和肝糖元、NO、钙浓度变化。 设计、时间及地点：随机对照动物实验,超微结构观察,于2004-01/2006-12在湖南师范大学体育学院运动人体科学实验室、中南大学组胚实验室完成。 材料：30只8周龄雄性SD大鼠,体质量(219.2±19.5) g,根据Berdford运动模型将大鼠以随机数字表法分为对照组,中等强度、大强度力竭运动组,每组10只。 方法：运动组先进行3 d的适应性跑台训练,速度为10 m/min,坡度为0°。休息3 d后,中等强度力竭运动组初始速度为10 m/min,持续12 min,逐渐增加运动负荷,达到速度为19.3 m/min,持续到力竭。大强度力竭运动组初始速度为26.8 m/min,持续到力竭。共30 d,1次/d。对照组不进行运动训练。 主要观察指标：运动后即刻取肝组织检测肝糖元、NO和Ca2+及肝细胞凋亡情况。 结果：30只大鼠全部进入结果分析。不同强度力竭运动组大鼠都完成了运动,整个运动过程中未出现拒跑现象,中等强度力竭运动组力竭运动时间为(234.60±60.05) min,大强度力竭运动组力竭运动时间为(92.40±34.61) min。与对照组比较,两种强度力竭运动后,大鼠肝组织肝糖元含量、NO浓度均下降,线粒体Ca2+浓度、肝细胞凋亡指数均升高(P < 0.05,P < 0.01),中等强度力竭运动组效果更明显(P < 0.05)。 结论：中、大强度力竭运动均可导致大鼠肝细胞凋亡,肝糖元含量、NO浓度下降,线粒体Ca2+浓度升高,中等强度力竭运动效果更为显著,可能与力竭运动时间长有关。     

一氧化氮合酶对脑损伤后神经细胞凋亡的影响

目的探讨脑损伤后神经型一氧化氮合酶（nNOS）和诱生型一氧化氮合酶（iNOS）对神经细胞凋亡的影响及其相关机制。方法320只SD大鼠随机分成以下4组：假手术组、脑创伤组、7-硝基吲唑组（nNOS抑制剂）和氨基胍组（iNOS抑制剂），此4组分别划分为伤后3h、6h、12h、24h、2d、3d、7d、14d8个时相组，每个时相组均为10只大鼠，采用Marmarou法制造大鼠重型弥漫性颅脑创伤模型，运用末端脱氧核苷酸转移酶介导的生物素脱氧尿嘧啶核苷酸缺口标记法（TUNEL）和免疫组化法，观察4组不同时相点海马CA1区的神经细胞凋亡情况和Bcl-2、Fas的表达情况。结果①假手术组偶见TUNEL阳性凋亡细胞及Bcl-2、Fas阳性细胞；②脑创伤组，伤后各时相点均出现TUNEL阳性凋亡细胞及Bcl-2阳性细胞，先上升后下降；③7-硝基吲唑（7-NI）组，伤后6h、12h、24h凋亡细胞明显下降而Bcl-2阳性细胞却明显上升（同脑创伤组比较P〈0．05）；④氨基胍（AG）组，伤后2～7d，凋亡细胞及Fas阳性细胞明显下降（同脑创伤组比较P〈0．01）。结论在脑损伤的早期，nNOS能够通过抑制Bcl-2的表达来促进神经细胞的凋亡；在脑损伤的晚期，iNOS能够通过诱导Fas的表达来促进神经细胞的凋亡。     

Effects of topiramate on hippocampal neuronal apoptosis in rats after kainic acid-evoked seizures

BACKGROUND:Apoptosis plays an important role in brain injury after seizures and the formation of chronic epilepsy.It is important to investigate whether topiramate exhibits either antiepileptic and/or anti-apoptotic effects on hippocampal neurons.OBJECTIVE:To observe euronal apoptosis in hippocampus of rat seizure models,and to investigate the antagonizing effect of topiramate on neuronal apoptosis after seizures.DESIGN:An animal experiment of comparative observation.SETTING:First Affiliated Hospital of Guangxi Medical University.MATERIALS:Sixty healthy male Sprague Dawley(SD)rats,4-6 weeks old and weighing 160-220 g,were provided by the Experimental Animal Center of Guangxi Medical University.Main apparatus and reagents were as follows:Rat brain solid positioner(SR-6N,made in Japan); kainic acid by Sigma(USA);pathological image analyzer(DMR 550)by Leica(Germany); in situ apoptosis detection kit by Wuhan Boster Biological Technology Co.,Ltd; topiramate by Xi'an-Janssen Pharmaceutical,Ltd.The treatment on animals in the experiment was in accordance with the standards of animal ethics.METHODS:The experiments were performed at the Scientific Experimental Center of Guangxi Medical University from June to December 2006.The rats were randomly divided into a topiramate-treated group(n＝30)and a model group(n＝30).① After anesthesia,all rats were administered a kainic acid injection(0.2 μ L,2 g/L)into the right lateral ventricle.Grade Ⅲ and greater Racine standards were considered to be a successful model establishment.Thirty minutes after seizure ,rats in the topiramate-treated group were treated with an intraperitoneal(i.p.)injection of topiramate every day(40 mg/kg/d)for 2 weeks.The rats in the model group were treated with an equal volume of saline for 2 weeks.③Six rats in the topiramate-treated group were sacrificed at 1 day,and 1,2,3,and 4 weeks after treatment,respectively.The model group animals were sacrificed at corresponding time points.The brain tissues of hippocampal dentate gyrus,CA1,CA2,and CA3 region were removed and prepared into sections.Neuronal apoptosis was detected with in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling.MAIN OUTCOME MEASURES:Hippocampal neuronal apoptosis in various rat brain areas was detected in the two groups.RESULTS:All 60 rats were included in the final analysis of results.In the topiramate-treated group,the number of apoptotic cells in hippocampal dentate gyrus and CA3 region at 1 day,1,and 4 weeks after seizures were significantly lower than the model group(P＜0.054-0.01).The number of apoptotic cells in hippocampal CA1 and CA2 regions at 1 day and 4 weeks after seizures in the topiramate-treated group were significantly lower than the model group(P＜0.05).CONCLUSION:Hippocampal apoptosis is closely associated with kainic acid-evoked seizures,and topiramate can alleviate early(1 day and 1 week)and delayed(4 weeks)hippocampal neuronal injury induced by kainic acid.     

Effects of topiramate on hippocampal neuronal apoptosis in rats after kainic acid-evoked seizures*☆

BACKGROUND： Apoptosis plays an important role in brain injury after seizures and the formation of chronic epilepsy. It is important to investigate whether topiramate exhibits either antiepileptic and/or antiapoptotic effects on hippocampal neurons. OBJECTIVE： To observe neuronal apoptosis in hippocampus of rat seizure models, and to investigate the antagonizing effect of topiramate on neuronal apoptosis after seizures.
DESIGN： An animal experiment of comparative observation.
SETTING： First Affiliated Hospital of Guangxi Medical University. MATERIALS： Sixty healthy male Sprague Dawley （SD） rats, 4-6 weeks old and weighing 160-220 g, were provided by the Experimental Animal Center of Guangxi Medical University. Main apparatus and reagents were as follows： Rat brain solid positioner （SR-6N, made in Japan）; kainic acid by Sigma （USA）; pathological image analyzer （DMR＋550） by Leica （Germany）; in situ apoptosis detection kit by Wuhan Boster Biological Technology Co., Ltd; topiramate by Xi＇an-Janssen Pharmaceutical, Ltd. The treatment on animals in the experiment was in accordance with the standards of animal ethics. METHODS： The experiments were performed at the Scientific Experimental Center of Guangxi Medical University from June to December 2006. The rats were randomly divided into a topiramate-treated group （n = 30） and a model group （n = 30）. ① After anesthesia, all rats were administered a kainic acid injection （0.2 μL, 2 g/L） into the right lateral ventricle. Grade Ⅲ and greater Racine standards were considered to be a successful model establishment. Thirty minutes after seizure , rats in the topiramate-treated group were treated with an intraperitoneal （i.p.） injection of topiramate every day （40 mg/kg/d） for 2 weeks. The rats in the model group were treated with an equal volume of saline for 2 weeks. ③ Six rats in the topiramate-treated group were sacrificed at 1 day, and 1, 2, 3, and 4 weeks after treatment, respectively. The model group ani