Caspase-dependent retinal ganglion cell apoptosis in the rat model of acute diabetes

Background Neural apoptosis is generally believed to be mediated by two distinct pathways, caspase-dependant and caspase-independent pathways. This study investigated the apoptotic pathways involved in retinal ganglion cells in acute diabetes in rats. Methods Diabetes was induced in male Wistar rats by a peritoneal injection of streptozotocin （STZ）. Expression and localization of caspase-3 and apoptosis-inducing factor （AIF） proteins in the retina of diabetic rats was examined by Western blotting and immunohistochemistry analyses. Terminal transferase dUTP nick end labeling （TUNEL） assay and immunofluorescent staining specific for caspase-3 and AIF were applied to analyze for apoptosis of retinal ganglion cells. In addition, a caspase-3 inhibitor DEVD-CHO was injected intravitreally to further determine the apoptotic pathways of retinal ganglion cells triggered in acute diabetes. Results Two weeks after induction of diabetes, a significant increase in caspase-3 protein expression and localization occurred in the nerve fiber layer, ganglion cell layer, and inner plexiform layer of the retina. Four weeks after the onset of diabetes, the increase in caspase-3 expression was profound eight weeks postinduction of diabetes （P 〈0.05）. Meanwhile, no AIF protein expression was detected in this study. In addition, intravitreal administration of the caspase-3 inhibitor DEVD-CHO reduced apoptosis of retinal ganglion cells by its direct inhibitory action on caspase-3. Conclusion Caspase-dependent apoptotic pathways may be the main stimulant of STZ-induced retinal ganglion cell apoptosis in acute diabetes.     

Arylamine N-acetyltransferases： a new inhibitor of apoptosis in HepG2 cells

Objective： To explore how arylamine N-acetyltransferases （NATs） is related to cell apoptosis. Methods： NAT activity in apoptotic HepG2 cells was measured using high performance liquid chromatography （HPLC）; the apoptosis rate of HepG2 cells acted upon by an NAT inhibitor was measured using flow cytometry. Results： NAT activity was lowered in apoptotic HepG2 cells; apoptosis rate induced by camptothecin （CAM） increased after inhibition of NAT activity in HepG2 cells. Conclusion： NAT can inhibit apoptosis in HepG2 cells.     

Effect of phenylacetate on apoptosis and level of intracellular free calcium in rat C6 glioma cells

Objective To observe the effect of phenylacetate （PA） on apoptosis and the level of intracellular free caldum in rat C6 glioma cells and to study its mechanism. Methods C6 ceils were cultured in vitro. After induction by PA, apoptosis was detected by transmission electron microscope （TEM） and flow cytometry （FCM） with AnnexinV/propidium iodide（PI）.     

Inhibition of tumor necrosis factor-α reduces alveolar septal cell apoptosis in passive smoking rats

Background Recent studies have revealed that lung cell apoptosis plays an important role in pathogenesis of cigarette-induced chronic obstructive pulmonary disease （COPD）. Tumor necrosis factor alpha （TNF-α） is one of the most important cytokines which are involved in COPD. This study aimed at investigating the influence of its inhibitor, recombinant human necrosis factor-alpha receptor II：lgG Fc fusion protein （rhTNFR：Fc） on alveolar septal cell apoptosis in passive smoking rats. Methods Forty-eight rats were randomly divided into a normal control group, a passive smoking group, an rhTNFR：Fc intervention group and a sham intervention group. The passive smoking rats were treated by exposure to cigarette smoking daily for 80 days. After smoking for one month the rhTNFR：Fc intervention group was treated with rhTNFR：Fc by subcutaneous injection, the sham intervention group injected subcutaneously with a neutral preparation （normal saline 0.1 ml, manicol 0.8 ml, cane sugar 0.2 mg, Tris 0.024 mg as a control. Lung function was determined and the levels of TNF-a in serum and broncho-alveolar lavage fluid （BALF） were measured with enzyme-linked immunosorbnent assay （ELISA）. Lung tissue sections stained by hematoxylin and eosin （HE） were observed for study of morphological alternations. Mean linear intercept （MLI） and mean alveolar numbers （MAN） were measured and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） method was carried out to determine the percentage of positive cells and distribution of apoptotic cells. Results Increased MLI and decreased MAN were found in the passive smoking group compared with both the normal control group and the rhTNFR：Fc intervention group （P〈0.05）. Forced expiratory volume in 0.3 second （FEVo.3）/forced vital capacity （FVC） and peak expiratory flow （PEF） were lower in the passive smoking group than that in the normal control group （P〈0.05）. Compared with the sham intervention group     

Effects of NHE-1 ribozyme gene transfection on apoptosis of rat pul-monary artery smooth muscle cells in vitro

Objective: To investigate the effects of the transfection of NHE-1 ribozyme gene on the apoptosis of pulmonary artery smooth muscle cells (PASMC) in vitro. Methods：After NHE-1 ribozyme gene was de-signed, synthesized and then cloned into plasmid pLXSN, the recombined plasmid was tansfected into cul-tured rat PASMC. Expression of NHE-1 mRNA was detected with semi-quantitative RT-PCR. Intracellular pH (pHi) was measured by using fluorescence dye BCECF-AM. Cell cycle was measured with aid of flow cy-tometric DNA analysis. Cell apoptosis was observed with electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) respectively. Results. The NHE-1 mRNA expres-sion level and pHi value were significantly lower in PASMCs transfected with NHE-1 ribozyme gene than those transfected with pLXSN or without transfection. Meanwhile, the apoptosis rate of cells transfected with NHE-1 ribozyme gene was increased significantly. Morphology of cell apoptosis was observed in the cells transfected with NHE-1 ribozyme gene under an electron microscope. Conclusions The transfection of NHE-1 ribozyme gene induces the apoptosis of PASMCs by inhibiting NHE-1 expression and intracellular acidification.     

蛛网膜下腔出血后早期脑损伤的研究进展

动脉瘤性蛛网膜下腔出血(SAH)是一种严重的疾病,具有很高的病死率和致残率。然而,传统的针对迟发性脑血管痉挛的治疗却不能取得很好的效果。最新研究认为,早期脑损伤(EBI)可能才是决定SAH患者预后的主要原因。EBI的发病机制是一系列复杂的病理、生理变化,主要包括全脑缺血、血脑屏障的破坏、脑水肿、氧化应激反应、免疫炎症途径和细胞死亡途径等。     

Inhibition of NF-κB by mutant IκBα enhances TNF-α-induced apoptosis in HL-60 cells by controlling bcl-xL expression

Backgound The aim of this study was to explore whether the inhibition of nuclear factor-κB (NF-κB)activation by mutant IκBα (S32,36→A) can enhance TNF-α-induced apoptosis of leukemia cells and to investigate the possible mechanism. Methods The mutant IκBα gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IκBα were obtained by the limiting dilution method. TNF-α-induced NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-α. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM). Results Mutant IκBα protein was confirmed to exist by Western blot. The results of EMSA showed that NF-κB activation by TNF-α in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IκBα repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-α, but changed very little in transfected HL-60 cells. The inhibition of NF-κB activation by mutant IκBα enhanced TNF-α-induced apoptosis. Thecytotoxic effects of TNF-α were amplified in a time- and dose-dependent manner. Conclusions NF-κB activation plays an important role in the resistance to TNF-α-induced apoptosis. The inhibition of NF-κB by mutant IκBα could provide a new approach that may enhance the antileukemia effects of TNF-α or even of other cytotoxic agents.     

Immunomodulatory effects of mesenchymal stem cells from adipose tissue in a rat orthotopic liver transplantation model

Objective To investigate immunomodulatory effects of adipose-derived mesenchymal stem ceils in a rat orthotopic liver transplantation model. Methods Mesenchymal stem ceils were isolated from adipose tissue of SD rats. In in vitro mixed lymphocyte culture system by using Wistar T cells as responders and SD spleen cells as stimulators,[第一段]     

Induction of type Ⅱ alveolar epithelial cells apoptosis in mouse by lipopolysaccharide does not require TNF-α

Objective To examine whether lipopolysaccharide (LPS)-induced apoptosis correlates with TNF-α release by type Ⅱ alveolar epithelial cells (AEC Ⅱ), whether TNF-α knockout (TNF KO) abrogates the induction of apoptosis by LPS and whether TNF-α is sufficient to induce apoptosis in this cell type.Methods AEC Ⅱ were isolated from wild type mice and TNF KO mice. Cells were stimulated with LPS or recombinant murine TNF-α for 24 h. TNF-α in culture supernatant was determined by ELISA following LPS stimulation. Apoptosis was determined by the terminal deoxynucleotidyl transferase end-labeling (TUNEL) assay after treatment with either LPS or TNF-α. Results LPS induced apoptosis in wild type AEC Ⅱ in a concentration-dependent manner. LPS-induced AEC Ⅱ apoptosis was accompanied by an 11-fold increase (from 0.073±0.065 ng/ml in control to 0.94±0.14 ng/ml in 50 μg/ml of LPS, P<0.01) in TNF-α release. However, increasing concentrations (5 or 25 ng/ml) of recombinant murine TNF-α failed to induce AEC Ⅱ apoptosis. In addition, apoptosis did occur in AEC Ⅱ isolated from TNF KO mice following LPS stimulation.Conclusions This study confirms that LPS induces TNF-α release and apoptosis in murine AEC Ⅱ in vitro. Exogenous TNF-α failed to induce AEC Ⅱ apoptosis, and apoptosis occurred following LPS stimulation in cells lacking the ability to produce TNF-α. Taken together, these results suggest that LPS-induced AEC Ⅱ apoptosis occurs by a TNF-α-independent mechanism.     

血管壁ICAM-1和细胞凋亡在大鼠脑血管痉挛中作用的实验研究

目的探讨血管壁炎症反应和细胞凋亡在CVS发病机制中的作用。方法枕大池二次注血法建立大鼠SAH后CVS模型,60只大鼠分为SAH组及对照组,每组按建模后1d、3d、7d、11d、14d分为5个亚组,分别测量基底动脉直径、基底动脉ICAM-1的OD值及凋亡指数。结果 SAH后第1天血管壁ICAM-1升高,第3天达高峰,第7天后逐渐下降,第11天正常;SAH后第1天在血管壁凋亡细胞增多,第7天达高峰,第14天仍高于对照组。结论血管壁的炎症反应及细胞凋亡在SAH后CVS中均发挥着重要作用,其起始及进展过程还需要进一步的深入研究。     

经颅多普勒超声在蛛网膜下腔出血后脑血管痉挛中的诊断价值

目的 探讨经颅多普勒超声（ＴＣＤ）在诊断蛛网膜下腔出血（ＳＡＨ）后脑血管痉挛（ＣＶＳ）中的作用。方法 总结我科１９９２年以来３０例ＳＡＨ病人的ＴＣＤ检测结果。结果 颅内血管血流速度明显增高,迟发性缺血性神经功能缺损（ＤＩＤ）发生的比率增加。结论 ＴＣＤ测定颅内血管的血流速度和ＰＩ值对于早期诊断ＳＡＨ后ＣＶＳ,预防ＤＩＤ是很有帮助的。     

TRAIL及其受体与蛛网膜下腔出血后痉挛动脉内皮细胞的凋亡

目的: 研究肿瘤坏死因子相关凋亡诱导配体(TRAIL)及其受体DR4、DR5在实验性蛛网膜下腔出血后痉挛血管内皮细胞表达与凋亡的关系.方法: 54只家兔随机分为3组,对照组(6只),SAH组(24只)和假手术组(24只).SAH组和假手术组再以时间随机分为4组,0,2,4,7 d组,每组6只.手术前及处死前分别行脑血管造影,评价血管痉挛模型.应用原位缺口末端标记(TUNEL)法显示痉挛血管内皮细胞的凋亡,免疫组化ABC的方法显示TRAIL、DR4、DR5在凋亡的内皮细胞中的表达.结果: 正常对照组基底动脉直径为(0.70±0.03) mm,SAH组与正常对照组基底动脉管径比较差异显著(P<0.01).痉挛血管内皮细胞的凋亡与对照组相比有显著性差异(P<0.01),TRAIL及其受体DR4、DR5在内皮细胞中有大量的表达,与对照组有显著性差异(P<0.01).结论: TRAIL及其受体DR4、DR5在蛛网膜下腔出血后痉挛血管内皮细胞的凋亡中有重要意义.TRAIL及其受体DR4和DR5介导的内皮细胞的凋亡在SAH后脑血管痉挛(CVS)的形成机制可能有重要的意义.     

蛛网膜下腔出血后脑血管痉挛治疗现状

脑血管痉挛是蛛网膜下腔出血的严重并发症，探索有效的治疗方法对改善患者的预后有着重要的意义。研究表明，钙离子拮抗剂、内皮素转化酶抑制剂、抗CD11／CD18单克隆抗体、脑脊液引流和基因治疗等是新的有效治疗措施。     

磁共振灌注成像评估动脉瘤性蛛网膜下腔出血后脑血管痉挛的临床研究

目的 探讨磁共振灌注成像在动脉瘤性蛛网膜下腔出血后脑血管痉挛中的诊断价值,并探讨脑血流动力学与脑血管痉挛之间的关系.方法 采用磁共振灌注加权成像和全脑血管造影的方法,对38例动脉瘤性蛛网膜下腔出血后(7±2)d的患者进行检查并动态观察.通过磁共振灌注成像检查采集大脑全动脉供血区,大脑中动脉供血区和基底节区所选感兴趣区的...     

两种蛛网膜下腔出血模型所致脑血管痉挛的比较性研究

目的探讨两种蛛网膜下腔出血模型所致脑血管痉挛的情况。方法选用健康Wistar大鼠36只,随机分为对照组、一次注血组、二次注血组。对照组注入生理盐水0.3 ml、一次注血组注入无抗凝自体血0.3 ml、二次注血组在1次注血后48 h再次注入0.3 ml。不同时间点在体观察基底动脉管径变化,动态检测大鼠顶叶皮层局部脑组织血流量（rCBF）。结果（1）注血1 h后,一次及二次注血组基底动脉痉挛明显,与对照组比较均有显著显著性差异（P〈0.01）,注血3 d后一次注血组基底动脉痉挛得到缓解,二次注血组基底动脉则持续痉挛,与一次注血组比较均有显著显著性差异（P〈0.01）。（2）注血1 h后,一次及二次注血组rCBF值均较对照组降低（P〈0.01）。注血3 d后,一次注血组rCBF逐渐恢复,二次注血组与一次注血组比较明显降低（P〈0.01）结论一次注血模型适宜研究早期脑血管痉挛,二次注血模型则可造成脑血管的持续性痉挛。     

尼莫地平在防治蛛网膜下腔出血后脑血管痉挛的Meta分析

目的:系统评价尼莫地平注射液治疗蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后脑血管痉挛(cerebral vasospasm,CVS)的临床疗效及安全性。方法:检索1994~2007年国内发表的尼莫地平注射液治疗SAH后CVS临床对照试验的相关文献,利用M eta分析方法,采用Review Manager 4.2软件对符合条件的文献进行荟萃分析。结果:对符合标准的16项临床研究结果进行同质性检验表明各试验具有同质性(P>0.05)。Meta分析结果表明尼莫地平注射液能够减少住院期间SAH后CVS的发生率,病死率明显低于对照组(P<0.000001);不会引起再出血发生率的增加(P=0.17)。结论:现有的临床研究证据显示尼莫地平能够有效防治SAH后CVS,降低病死率且未增加再出血的危险。     

蛛网膜下腔出血后脑脊液引流与脑血管痉挛的关系

目的：研究实验性蛛网膜下腔出血（SAH）后脑脊液中积血量的变化与脑血管痉挛（CVS）的关系。方法：经皮枕大池二次注血法建立犬SAH动物模型,设立早期引流组、晚期引流组和对照组,测定脑脊液中积血量的变化,脑血管造影确定血管痉挛程度（proportion reduction of basilar artery diamiter,%RBAD)。结果：早期引流组脑脊液中积血的清除最明显,晚期引流组其次,脑血管痉挛早期引流组例数最少,程度最轻;晚期引流组其次。结论：SAH后脑脊液中积血越多,持续时间越长,脑血管痉挛发生率越高,程度越高。SAH后腰池持续引流有预防和治疗CVS的作用,早期引流可以取得更好的效果。     

单侧颈动脉结扎结合枕大池二次注血建立改良脑血管痉挛模型

目的 在枕大池二次注血模型基础上结扎单侧颈动脉,尝试建立一种适合脑血管痉挛后脑损害研究的动物模型。方法 30只新西兰兔随机分为假手术组、注血组和结扎注血组各10只。注血组和结扎注血组均行枕大池二次注血;结扎注血组加行单侧颈动脉结扎。于首次注血后5 d处死全部动物,脑组织切片后行H-E及Tunel染色,测量基底动脉直径并行海马神经元凋亡计数。结果 假手术组动物术后全部存活,注血组术后存活率90%,结扎注血组存活率70%。注血组及结扎注血组均出现明显基底动脉痉挛及海马神经元凋亡(P＜0.001),结扎注血组基底动脉直径与注血组差异无统计学意义(P=0.342),结扎注血组海马神经元凋亡细胞计数较注血组高,差异有统计学意义(P=0.005)。结论 单侧颈动脉结扎结合枕大池二次注血可建立脑缺血损伤症状更严重的脑血管痉挛模型。     

Research on the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage

目的 :进一步研究脑血管痉挛的发生机理 ,为临床治疗服务。方法 :采用 1 4只成年家犬 ,实验组 9只 ,对照组 5只 ,通过 2次枕大池注血法建立蛛网膜下腔出血 (SAH)模型。观察痉挛血管的自由基变化、血管活性、血管造影像、超微结构变化。另外 2 0只犬用来观察血管扩张剂的作用。结果 :实验组较对照组自由基含量高 ,血管活性降低 ,血管狭窄 ,管壁损害重。结论 :尼莫地平对急性痉挛有效 ,对慢性痉挛无效 ;慢性血管痉挛是以管壁结构性狭窄为特点。     

ET在SAH后继发CVS病理机制中的作用

采用单次注射１５０μｌ自体新鲜血液至大鼠蛛网膜下腔Ｗｉｌｌｉｓ环处，建立蛛网膜下腔出血（ＳＡＨ）后继发脑血管痉挛（ＣＶＳ）的动物模型。用激光多普勒血流仪滥测ＣＶＳ的过程，平行地对脑脊液（ＣＳＦ）和血浆作内皮素（ＥＴ）含量的放射免疫测定。在ＳＡＨ发生的同时静脉注射ＥＴ抗体（１∶１０００，５００μｌ），在ＳＡＨ前５ｍｉｎ及ＳＡＨ后不同时间脑池内注射ＥＴ抗体（１∶１０００，２０μｌ）。结果显示：（１）ＣＳＦ中ＥＴ升高在ＳＡＨ后１５ｍｉｎ和３０ｍｉｎ时有显著性意义；血浆中ＥＴ升高在ＳＡＨ后３０ｍｉｎ时有显著性意义。（２）ＳＡＨ前５ｍｉｎ向脑池内注射ＥＴ抗体能有效地预防ＣＶＳ，在ＳＡＨ后１５ｍｉｎ时同样的注射能大部分消除ＣＶＳ，但在ＳＡＨ后３０ｍｉｎ时只能部分消除ＣＶＳ，而在ＳＡＨ后２４ｈ同样的注射对ＣＶＳ无作用。（３）在ＳＡＨ发生的同时静脉注射ＥＴ抗体对ＣＶＳ无预防作用。上述结果提示：（１）ＣＳＦ中高水平的ＥＴ是ＳＡＨ后引起ＣＶＳ的重要因素；（２）在ＣＶＳ的发展过程中可分为早期ＥＴ依赖阶段和晚期非ＥＴ依赖阶段；（３）在ＳＡＨ后早期从蛛网膜下腔阻断ＥＴ是预防和逆转ＣＶＳ的重要途径和方法。