荧光显微镜观察银杏内酯B对大鼠心肌细胞凋亡的影响

目的：建立一种简便、快速的细胞凋亡检测方法，探讨银杏内酯B（GB）对心肌细胞凋亡的影响。方法：体外培养大鼠心肌细胞，利用荧光显微镜观察经Hoeehst 33258染色的心肌细胞，记录凋亡细胞核形态学改变并计算凋亡百分率。结果：H2O2可诱导心肌细胞发生凋亡，与H2O2组相比，GB组大鼠心肌细胞凋亡率明显降低，并有显著性差异（P〈0．05）。结论：银杏内酯B对大鼠心肌细胞凋亡具有保护作用。     

高糖诱导的大鼠雪旺细胞凋亡的流式细胞术

探讨流式细胞术在检测高糖诱导的早期雪旺细胞凋亡中应用。选用培养大鼠的雪旺细胞,将细胞分为正常对照组(5.6mmol/L葡萄糖),高糖组(50mmol/L葡萄糖),高渗出组(50mmg/L甘露醇),培养7天后,收集细胞。48h后应用流式细胞术采用Annexin-V/PI检测法和JC-1检测早期细胞的细胞膜凋亡率和线粒体膜电位变化。结果表明:高糖与正常糖浓度处理的雪旺细胞比较,高浓度葡萄糖诱导雪旺凋亡率明显高于正常葡萄组的凋亡率,线粒体膜电位明显高于正常对照组。高渗透组与正常对照组凋亡率及线粒体无明显升高。通过采用流式细胞细胞术结合单克隆抗体,能够快速、灵敏和准确检测雪旺细胞在细胞膜的通透性改变和线粒体膜电位变化。为研究雪旺细胞凋亡提供客观、直接、准确的方法。     

雌二醇诱导人正常肝细胞的凋亡以及对细胞周期的影响

目的：观察雌二醇对诱导人正常肝细胞的的凋亡,并且研究其对细胞周期的影响。方法：采用人肝细胞为实验对象,用he染色观察细胞形态学变化;流式细胞记数仪检测细胞周期,AnnexinⅤ/PI双重染色定量检测早期细胞凋亡及Bcl-2/Bax的表达改变。结果：雌二醇48h可以明显抑制正常肝细胞株的增生与活力,诱导细胞的凋亡,且对细胞周期的影响非常显著。结论：其机制可能与Bcl-2/Bax表达的下调有关。     

流式细胞仪检测甘草多糖对小鼠S-180肉瘤的凋亡诱导作用

随着单克隆抗体及标记技术的发展,流式细胞术(FlowCytometry,FCM)检测细胞凋亡技术已广为使用,为探讨中药甘草多糖抑瘤作用及其机制,本文应用流式细胞术(FCM)检测细胞凋亡,结果表明甘草多糖抑制肿瘤生长可能与诱导细胞凋亡有关。     

用激光扫描共聚焦显微镜原位检测细胞凋亡

用激光扫描共聚焦显微镜在原位检测细胞凋亡具有多方面的优势。本文介绍用该仪器原位检测Annexin—Ⅴ试验样品、TUNEL试验样品及进行细胞凋亡形态学观察的过程和方法。     

应用流式细胞术检测大鼠心肌细胞缺氧/复氧后内质网应急引起细胞凋亡

利用流式细胞仪观察西洋参茎叶总皂甘通诱导大鼠心肌细胞后内质网应急引起细胞凋亡。选用原代培养心肌细胞,将心肌细胞分为正常细胞对照组,缺氧/复氧组和缺氧/复氧后加入西洋参茎叶总皂苷组。按照40mg/L,80mg/L,160mg/L浓度培养24h后,应用流式细胞仪采用Annexin-V/PI诱导凋亡。结果显示应用流式细胞仪检测最终浓度为160mg/L西洋参茎叶总皂苷凋亡率明显低于缺氧/复氧组。流式细胞仪检测由内质网应急诱导细胞凋亡对研究细胞凋亡的机理提供一种快速、准确检测方法。     

应用多参数技术方法对脑缺血皮质区神经细胞凋亡的检测

随着生物科学研究技术的不断提高和对细胞凋亡认识的不断深入,在细胞凋亡确认过程中,若单独使用一种方法检测会出现一定概率的假阳性,往往需要采用多参数技术方法相互印证。本实验结合透射电镜、流式细胞仪和共聚焦显微镜的检测技术及原理,通过多技术相结合观察判断了急性脑缺血皮质神经元细胞凋亡,并进行了定性、定量分析。     

D-柠檬烯对人胃癌MGC803细胞增殖和凋亡的影响

目的研究D-柠檬烯对人胃癌MGC803细胞生长抑制和凋亡的影响。方法噻唑蓝（MTT）法比色检测细胞生长情况;流式细胞仪检测细胞凋亡率;激光共聚焦检测细胞内活性氧（reactive oxygen species,ROS）的含量和caspase-3蛋白的表达。结果D-柠檬烯对人胃癌MGC803细胞生长抑制作用呈剂量依赖关系;流式细胞仪检测发现D-柠檬烯可引起MGC803细胞凋亡,且呈时间依赖性;激光共聚焦显微镜荧光检测发现D-柠檬烯处理的细胞内ROS明显升高（P〈0．05）,caspase-3蛋白表达明显增加（P〈0．05）。结论D-柠檬烯能够有效地抑制癌细胞的生长以及诱导凋亡,这种作用机制可能是使MGC803细胞内产生大量的活性氧,也可能与细胞内caspase-3基因的表达变化有关。     

基质金属蛋白酶9在糖尿病视网膜病变发展中的凋亡作用

目的为了探索基质金属蛋白酶9(MMP9)在糖尿病视网膜病变(DR)进展中的潜在调节机制。方法利用Lipofectamine 2000将pc DNA-MMP9质粒和pc DNA-Ang2质粒分别转染至原代大鼠视网膜Müller细胞(RMCs)中。分别利用MTT法与流式细胞仪检测细胞生存率与凋亡情况。同时探索了MMP9与血管紧张素2(Ang2)之间的交互作用。另外,RMCs分别在正常血糖水平与高血糖水平下用MMP9处理2天。此外,通过Western印迹测定细胞凋亡蛋白MMP9、Ang2、Bax2、Bcl2、聚腺苷二磷酸核糖聚合酶(PARP)降解产物、天门冬氨酸特异性半胱氨酸蛋白酶-3(caspase3)降解产物的表达水平。结果与对照组相比,siRNA-MMP9组细胞生存率显著增长而MMP9过表达组下降。与对照组相比,MMP9过表达组中细胞凋亡显著增加,而si RNA-MMP9组中则下降。MMP9表达由Ang2显着调节,而当MMP9表达改变时,Ang2表达无明显变化。再者,高血糖组中MMP9的表达显著性增加,而正常血糖组与对照组比较差异无统计学意义。另外,高血糖组中Bax2,Bcl2,PARP降解产物、caspase3降解产物的表达也显著增加,相应地对照组与正常血糖组则没有显著性改变。结论我们的研究结果表明,MMP9通过由Ang2调控或定位细胞凋亡相关蛋白(如Bax2,Bcl2,PARP降解产物、caspase3降解产物)诱导细胞凋亡,在DR进展中扮演着一个重要角色。     

一种基于毛细管进样的无鞘液流式细胞分析技术

流式细胞技术对荧光素着色细胞的形态和生物学性状进行定性或定量大群体快速检测,以往的细胞驱动方式是鞘液包裹带动式,近年来诞生了一种毛细管进样的无鞘液驱动流式细胞分析技术,可以对微量细胞进行绝对计数、CD3/CD4,CD 8淋巴细胞计数艾滋病检测、细胞表面抗原等蛋白表达分析、细胞凋亡早期Nexin,线粒体膜电位分析、凋亡中期多种caspase分析和凋亡晚期TUNEL分析、细胞周期、细胞毒性、细胞增值和细胞示踪分析,是一种灵活易用的、低消耗的、便于实验人员自己常规操作的分析系统.     

Inhibitory of EV-A71 virus-induced apoptosis by ZVAD through ROS mediated signaling pathways

Emerging evidence that Enterovirus A 71 (EV-A71) infection closely related to apoptosis. The ZVAD is a caspase inhibitor that can prevent apoptosis. The aims of this project were to evaluate the mechanism of the ZVAD inhibited EV-A71 virus and to provide experiment basis for finding new antiviral drugs. In this study, after treated with ZVAD in EV-A71 infected Vero cells, the viral replication was reduced, and the cell viability was higher than EV-A71 group. Additionally, ZVAD decreased the cell apoptosis and the level of inflammatory cytokines induced by EV-A71 in the infected Vero cells. ZVAD inhibited cell apoptosis by regulating ROS mediated signaling pathway and inflammation cytokines to achieve antiviral.     

Hypoxia induced apoptosis of rat gastric mucosal cells by activating autophagy through HIF-1α/TERT/mTORC1 pathway

The pathogenesis of high altitude-related gastric mucosal injury remains poorly understood, this study aimed to investigate the role of autophagy in hypoxia-induced apoptosis of rat gastric mucosal cells. Rats were randomized into four groups which were maintained at an altitude of 400 m (P) or received no treatment (H), autophagy inducer rapamycin (H+AI) or autophagy inhibitor 3-MA (H+AB) at an altitude of 4,300 m for 1, 7, 14 and 21 days, respectively, and the morphology, ultrastructure, autophagy, and apoptosis of gastric mucosal tissues were examined. Gastric mucosal epithelial cells CC-R039 were cultured under conditions of normoxia, 2% O2 (hypoxia), or 2% O2+anti-mTORC1 for 0, 24, 48, and 72 h, respectively, and the autophagy and apoptosis were analyzed. CC-R039 cells were transfected with siHIF-1α, siTERT, or siRNA and the autophagy was examined. The results showed that the exposure to hypoxia increased the autophagy and apoptosis of gastric mucosal cells in rats, and apoptosis was aggravated by rapamycin treatment but alleviated by 3-MA treatment. Increased duration of hypoxia from 0 to 72 h could increase the autophagy and apoptosis but decrease the proliferation of gastric mucosal cells. Inhibition of mTORC1 with rapamycin led to further increase in apoptosis and even substantial cell death, and inhibition of HIF- 1α and TERT increased mTORC1 expression and reduced autophagy. Moreover, the inhibition of HIF-1α reduced TERT expression. In conclusion, hypoxia could induce apoptosis of rat gastric mucosal cells by activating autophagy through HIF-1α/TERT/mTORC1 pathway     

Human umbilical cord blood mesenchymal stem cells conditioned media inhibits hypoxia-induced apoptosis in H9c2 cells by activation of the survival protein Akt

This work aimed to study the beneficial role of human umbilical cord blood-derived mesenchymal stem cellconditioned medium (MSC-CM) in hypoxia-induced apoptosis in H9c2 cardiomyoblasts, in which the serine/heroine kinases (Akt) pathway would be involved. For this, CM was collected by culturing MSCs in serum-free DMEM medium for 24 h, and paracrine factors were analyzed by protein chip. H9c2 cells were divided into the following groups: control group, hypoxia group, MSC-CM intervention group (CM group), MSC-CM + Akt phosphorylation inhibitor (LY294002) group (LY group). Apoptosis of the H9c2 cells was tested with chromatin dye Hoechst 33342 and FITC-conjugated Annexin V apoptosis detection kit by flow cytometer after a hypoxia/serum deprivation (H/SD) for 24 h. The apoptosis-related proteins were evaluated by Western blot. MSC-CM displayed significantly elevated levels of growth factors, anti-inflammatory, and anti-apoptosis cytokines. On Hoechst 33342 apoptosis staining, the H9c2 cell morphology displayed a lower proportion of apoptosis in the CM group than those in the hypoxia group, while apoptosis was increased in LY group. Flow cytometer analysis revealed the apoptosis ratio in the CM group was lower than the hypoxia group (12.34 ± 2.00% vs. 21.73 ± 2.58%; p < 0.05), while the LY group was significantly higher (22.54 ± 3.89%). Active caspase-3 expression was increased in hypoxia group than control group (p < 0.05), but decreased in CM group (p < 0.01). Umbilical cord blood-derived mesenchymal stem cell-conditioned media secrete multiple paracrine factors that are able to inhibit hypoxia-induced H9c2 cardiomyoblasts apoptosis, and in which the activation of Akt phosphorylation is involved to achieve the protective effect.     

Vitamin B3 inhibits apoptosis and promotes autophagy of islet β cells under high glucose stress

Background: Hyperglycemia is a typical symptom of diabetes. High glucose induces apoptosis of islet β cells. While autophagy functions in cytoprotection and autophagic cell death. The interaction between autophagy and apoptosis is important in the modulation of the function of islet β cells. Vitamin B3 can induce autophagy and inhibit islet β apoptosis.Method: The mechanism of vitamin B3-mediated protective effect on the function of islet β cells was explored by the method of western blot, immunofluorescence and flow cytometry.Results: In the present study, high glucose stress increased the apoptosis rate, while vitamin B3 reduced the apoptosis rate. The effect of vitamin B3 on autophagy flux under normal and high glucose stress was also investigated. Vitamin B3 increased the number of autophagosomes and increased the light chain (LC)3-II/LC3-I ratio. In contrast, vitamin B3 decreased sequestosome 1 (SQSTM1)/p62 protein expression and inhibited the phosphorylation of mammalian ribosomal protein S6 kinase β-1 (p70S6K/S6K1), which was a substrate of mammalian target of rapamycin (mTOR) under normal and high glucose stress. To further verify the protective effect of vitamin B3 on apoptosis, we treated islet β cell RIN-m5F with autophagy inhibitor 3-methyladenine (3-MA). Vitamin B3 decreased the apoptosis rate under high glucose stress, while the inhibition of apoptosis by vitamin B3 was blocked after adding 3-MA.Conclusion: Our data suggested that vitamin B3 reduced the apoptosis rate of β cells, possibly through inducing autophagy under high glucose stress.     

Differential expression of CD95, Bcl-2, and Bax in rat gastric chief and parietal cells

Apoptotic cell death is common in the inflamed gastric mucosa, but its role in the regulation of cell homeostasis in normal gastric mucosa is unknown. We investigated the expression of CD95, Bcl-2, and Bax and their roles in the regulation of apoptosis in normal rat gastric mucosa and in cultures of highly enriched rat chief and parietal cells by immunostaining, Western blotting, and FACS. In intact tissue CD95, Bcl-2, and Bax were localized predominantly in the glandular base region in chief cells. In freshly isolated cells, expression of CD95, Bcl-2, and Bax was much more pronounced in chief cells than in parietal cells. A lower intracellular Bcl-2/Bax ratio suggesting a higher susceptibility to apoptosis was noticed in chief rather than in parietal cells. In extended cultures of parietal and chief cells, Bax expression was upregulated and Bcl-2 expression was downregulated. These regulatory changes, presumably caused by in vitro effects, were not associated with an increase in spontaneous apoptosis. Treatment of chief and parietal cells with Fas-ligand induced apoptosis of all CD95 expressing cells. Expression of CD95, Bcl-2, and Bax predominantly in chief cells suggests that in this cell type regulation of apoptosis may differ from that in parietal cells. Binding of FasL with functionally active CD95 receptors on chief and parietal cells may be relevant for induction of apoptosis in inflamed gastric mucosa.     

Proteome analysis of apoptotic cells

Apoptosis, a genetically determined form of cell death, is a central and complex process involved in the development of multicellular organisms in the maintenance of cell homeostasis. During apoptosis, a large number of proteins involved in transducing signals are posttranslationally modified. Classical proteomics, the combination of protein separation by two-dimensional gel electrophoresis (2DGE) and protein identification by mass spectrometry (MS), enabled the discovery of more than 100 proteins altered during apoptosis. Functional data about protein degradation, modification, translocation, and synthesis were obtained. In addition to classical proteomics, some specifically designed proteome studies were carried out to analyze specific apoptotic components such as the mitochondrial releasing factors, death-inducing signaling complex (DISC), inhibitor of apoptosis (IAP) interacting proteins, and caspases. The identification of main regulators significantly influenced the elucidation of the concept underlying apoptosis signaling. Thus, the application of detailed protein analytical methods in the young field of apoptosis research was particularly fruitful.     

Impact of aging and life-long calorie restriction on expression of apoptosis-related genes in male F344 rat liver

Aging enhances apoptosis of hepatocytes under normal physiological conditions and increases the susceptibility to apoptosis of hepatocytes, whereas chronic calorie restriction (CR) suppresses the age-enhanced susceptibility to apoptosis. To clarify the subcellular mechanisms of age-associated dysregulation of apoptosis and the effects of CR, we analyzed the expression of genes promoting apoptosis (p53, Fas receptor, Fas ligand, TNF receptor 1, TNFalpha, Bax, TGF beta 1) and genes preventing apoptosis (Bcl-2 and Bcl-XL) in the livers of 3-, 6-, 15-, and 24-month-old male F344 rats that were either fed ad libitum or subjected to a 30% reduction in food intake (CR). After the age of 6 months, expression of p53, Fas receptor, Fas ligand, and TNFalpha mRNAs was up-regulated with aging. CR suppressed this age-enhanced p53 and Fas receptor mRNA expression, but expression of the other genes was not altered significantly by aging or CR. Expression of Fas receptor in hepatocytes, as detected immunohistochemically, increased with age, but CR suppressed age-accelerated Fas receptor expression. Our findings suggest that TNF ligand/TNF receptor family signaling, particularly Fas receptor expression, is important in age- and CR-modulated apoptosis of hepatocytes. Hepatocytes that were immunoreactive for p53 had slightly increased with aging, suggesting that p53 may mediate the age-enhanced up-regulation of Fas receptor in hepatocytes.