玉米赤霉烯酮对Leydig细胞凋亡及caspase-9、caspase-3蛋白表达的影响

以不同浓度的玉米赤霉烯酮(Zearalenone,ZEA)对大鼠睾丸间质细胞(Leydig)细胞进行染毒,采用噻唑蓝比色法观察了ZEA对Leydig细胞活力的影响;流式细胞术分析检测了ZEA对细胞的凋亡率、线粒体膜电位变化的影响;Western blotting等技术检测了Bax、Bcl-2、caspase-3、caspase-9、PARP蛋白的表达情况。结果显示,ZEA可明显抑制睾丸Leydig细胞的活力(染毒各组与对照组比较P<0.05),40mg/L染毒组相对活力为40.67%;与对照组比较,5、10、20mg/L ZEA染毒组睾丸Leydig细胞凋亡率均极显著升高(P<0.05),呈明显剂量关系;线粒体膜电位变化测定显示,各染毒组与对照组比,线粒体膜电位均下降(P<0.05),且有明显的剂量关系;Western blotting分析显示,与对照组比较,5、10、20mg/L ZEA染毒组Bax、caspase-9、caspase-3、PARP蛋白表达均上调,bcl-2蛋白表达均下调。结果表明,ZEA能诱导大鼠睾丸间质细胞发生凋亡,Bax、Bcl-2、caspase-9、caspase-3等基因参与ZEA诱导Leydig细胞凋亡的调控。     

玉米赤霉烯酮对Leydig细胞凋亡及caspase-9、caspase-3蛋白表达的影响

以不同浓度的玉米赤霉烯酮（Zearalenone,zEA）对大鼠睾丸间质细胞（Leydig）细胞进行染毒,采用噻唑蓝比色法观察了ZEA对Leydig细胞活力的影响;流式细胞术分析检测了ZEA对细胞的凋亡率、线粒体膜电位变化的影响;Westernblotting等技术检测了Bax、Bcl-2、caspase-3、caspase-9、PARP蛋白的表达情况。结果显示,ZEA可明显抑制睾丸Leydig细胞的活力（染毒各组与对照组比较P〈0．05）,40mg／L染毒组相对活力为40．67％;与对照组比较,5、10、20mg／LZEA染毒组睾丸Leydig细胞凋亡率均极显著升高（P〈0．05）,呈明显剂量关系;线粒体膜电位变化测定显示,各染毒组与对照组比,线粒体膜电位均下降（P〈0．05）,且有明显的剂量关系;Westernblotting分析显示,与对照组比较,5、10、20mg／LZEA染毒组Bax、caspase-9、caspase-3、PARP蛋白表达均上调,bel-2蛋白表达均下调。结果表明,ZEA能诱导大鼠睾丸间质细胞发生凋亡,Bax、Bcl-2、caspase-9、caspase-3等基因参与ZEA诱导Leydig细胞凋亡的调控。     

呫吨酮衍生物XP-15对BEL-7402细胞线粒体膜电位的影响

目的 :探讨呫吨酮并吡啶衍生物9-(2-吗啡啉乙酰胺基)-7H-吡啶并[4,3-c]呫吨-7-酮(XP-15)对BEL-7402细胞线粒体膜电位的影响。方法:通过MTT法、细胞形态学和克隆实验观察XP-15对BEL-7402细胞增殖的影响;用荧光分光光度计检测线粒体膜电位的影响。结果:高剂量的XP-15能明显抑制BEL-7402细胞增殖(P<0.05);对BEL-7402细胞克隆的抑制作用,呈剂量和时间依赖性。XP-15作用后,BEL-7402细胞的线粒体膜电位降低(P<0.05)。结论:XP-15诱导BEL-7402细胞凋亡的作用,可能与其降低线粒体膜电位有关。     

Caspase-8和caspase-3在镉致NRK细胞凋亡中的表达变化

为研究镉致NRK细胞凋亡中caspase-8和caspase-3 mRNA表达的变化,分别将终浓度为0、10、20、40、60 μmol/L的CdCl-2添加到培养液中,共同培养6 h。应用TUNEL法检测NRK细胞凋亡形态改变;分光光度法检测caspase-8和caspase-3蛋白活性;半定量RT-PCR检测caspase-8、caspase-3 mRNA。结果表明,随着Cd暴露剂量的加大,NRK细胞凋亡指数升高,caspase-8和caspase-3蛋白活性增强,caspase-8、caspase-3 mRNA转录增加,并呈现剂量效应关系。说明,caspase-8和 caspase-3在镉引起的NRK细胞凋亡中起重要作用。     

MPTP在庆大霉素诱导MDCK细胞线粒体凋亡途径中的作用

为研究线粒体膜通透性转换孔(mitochondrial permeablity transition pore,MPTP)在庆大霉素(gentamicin,GM)诱导MDCK细胞线粒体凋亡中的作用,试验用4 mmol/L GM和2μmol/L抑制剂CsA(cyclosporin A,CsA)单独或联合处理MDCK细胞24 h,流式细胞术检测MPTP开放情况和细胞凋亡率;JC-1探针检测线粒体膜电位的变化;荧光检测线粒体变化;免疫印迹法检测Bax与Bcl-2表达量,Caspase-9、Caspase-3和PARP蛋白活化情况,Cyt C和AIF释放。结果显示,CsA能够显著或极显著抑制GM引起的MDCK细胞MPTP的开放,细胞凋亡率的上升,线粒体膜电位下降,Bcl-2/Bax比值下降,Caspase-9、Caspase-3和PARP蛋白的活化以及Cyt C与AIF的释放(P0.05或P0.01);说明MPTP正向调节GM引起的MDCK线粒体凋亡。     

二烯丙基二硫化物诱导MDCC-MSB-1细胞的凋亡及Fas/FasL信号通路调控作用

为探究二烯丙基二硫化物(DADS)对马立克氏病肿瘤细胞系(MDCC-MSB-1细胞)的增殖抑制作用及其信号通路的调控机制,以MDCC-MSB-1细胞作为研究对象,采用CCK-8法检测不同浓度DADS分别作用MDCC-MSB-1细胞24、48、72 h后对细胞增殖抑制的影响。用0、80、160、240μmol/L DADS预处理MDCC-MSB-1细胞24 h后,光学显微镜观察细胞形态变化;Hoechst 33342核酸染色法检测细胞形态变化;caspase-8活性试剂盒检测细胞中caspase-8蛋白酶活性;并用荧光定量PCR法检测Fas/FasL信号通路关键因子mRNA的转录水平。结果显示,40μmol/L～240μmol/L的DADS抑制MDCC-MSB-1细胞的增殖呈时间剂量依赖性;随着浓度的升高,细胞形态逐渐出现细胞核核膜消失,染色体降解为多个片段并且边缘化,形成凋亡小体;240μmol/L的DADS可诱导MDCC-MSB-1细胞caspase-8蛋白酶活性及Fas、FADD、caspase-8、Bid和caspase-3 mRNA的转录水平极显著升高(P0.01)。结果表明,DADS可诱导MDCC-MSB-1细胞的凋亡,并活化其Fas/FasL死亡受体凋亡信号通路,可为鸡马立克氏病的治疗提供参考。     

α-硫辛酸对镉致PC12细胞线粒体凋亡途径的保护作用

为研究α-硫辛酸(α-lipoid acid,α-LA)对镉致PC12细胞线粒体凋亡途径的保护作用。10μmol/L醋酸镉和100μmol/Lα-LA单独或联合作用PC12细胞24h,流式细胞术检测细胞凋亡率,Western blot法检测cleaved caspase-3、Bcl-2和Bax蛋白的表达量和细胞色素C(cytochrome c,Cyt C)的释放。结果表明,镉引起细胞凋亡率上升,cleaved caspase-3表达上调,Bcl-2/Bax比值极显著下降(P<0.01),Cyt C从线粒体释放进入胞浆中(P<0.01);α-LA联合Cd处理,细胞凋亡率下降,cleaved caspase-3表达量下降,Bcl-2/Bax比值升高,并且能抑制线粒体中Cyt C的释放。表明α-LA对于镉诱导的PC12细胞凋亡的线粒体途径具有一定的保护作用。     

Fas/FasL在镉致PC12细胞凋亡线粒体通路中的作用

为探讨Fas/FasL在镉致PC12细胞凋亡线粒体通路中的作用,用10μmol/L CdAc_2和10 mg/L Anti-FasL单独或联合处理PC12细胞12 h,通过Hoechst 33258染色检测细胞的凋亡形态学变化,流式细胞术检测线粒体膜电位的变化情况,Western blot检测BID、caspase-9、caspase-3、PARP的活化情况,Bax和Bcl-2的表达情况。结果:与对照组相比,Cd处理组细胞凋亡率、tBID/BID比值、Bax/Bcl-2比值、cleaved caspase-9、cleaved caspase-3、cleaved PARP蛋白表达量均极显著升高(P0.01),线粒体膜电位极显著降低(P0.01);与Cd处理组相比,Anti-FasL与Cd共处理组细胞凋亡率、tBID/BID比值、Bax/Bcl-2比值、cleaved caspase-9、cleaved caspase-3、cleaved PARP蛋白表达量均极显著降低(P0.01),线粒体膜电位极显著升高(P0.01)。结果表明,Fas/FasL在镉致PC12细胞凋亡线粒体通路中具有重要作用。     

沙冬青生物碱对MDCC-MSB1细胞增殖及细胞线粒体膜电位的影响

采用体外细胞培养技术、噻唑蓝(MTT)还原法、流式细胞技术检测梯度浓度沙冬青总生物碱对体外培养鸡马立克氏病肿瘤HDCC-MSB1细胞株的增殖抑制及对线粒体膜电位的影响。结果显示,沙冬青生物碱可显著抑制HDCC-MSB1细胞增殖,且这种抑制具有剂量和时间效应关系。经沙冬青生物碱作用的HDCC-MSB1细胞培养样本中有明显的DNA低含量颗粒(亚G1期峰),细胞周期各时相分布发生改变,细胞增殖在G1被阻滞。细胞线粒体膜电位(△Ψm)明显下降,也呈现出剂量和时间效应关系。结果表明,沙冬青生物碱对HDCC-MSB1细胞增殖有显著抑制效应,且抑制依赖于诱导HDCC-MSB1细胞线粒体膜电位(△Ψm)的下降,并进一步诱导了该细胞的凋亡。     

DSS诱导血管内皮细胞ECV304凋亡及机制研究

为了研究葡聚糖硫酸钠(DSS)诱导血管内皮细胞:ECV304增殖和凋亡的影响。体外培养血管内皮细胞ECV304,采用不同浓度DSS诱导细胞,流式细胞术分析细胞凋亡、活性氧(ROS)、线粒体膜电位(MMP)、细胞周期;WST-1法检测细胞增殖抑制率。结果显示,随着DSS浓度的增加细胞凋亡率增加,并导致细胞GOG1期阻滞,细胞内活性氧含量减少,同时线粒体膜电位下降,细胞增殖抑制率明显升高。结果表明,DSS可以抑制血管内皮细胞ECV304增殖并诱导细胞凋亡。     

Characterization of canine caspase-3

The canine caspase-3 gene was cloned and sequenced. The canine caspase-3 cDNA clone was 1473 base pairs in length and encoded 277 amino acids. The predicted amino acid sequence showed 88.4%, 88.0%, 85.9%, 65.9% and 60.6% homology with that of human, pig, mouse, chicken and zebra fish caspase-3, respectively. The caspase-3 mRNA was confirmed to express in skin, lymph node, bone marrow, small intestine and lung from a healthy dog by RT-PCR analysis.     

家蚕凋亡蛋白酶(caspase-1)基因在大肠杆菌中的表达

为了研究家蚕凋亡蛋白酶(caspase-1)的原核表达及剪切方式,用PCR法扩增出家蚕caspase-1基因的cD-NA,然后插入pET28a载体,得到阳性质粒pET28a-caspase-1,并转化感受态细胞BL21,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导了caspase-1蛋白的表达。应用SDS-PAGE和Western blot分析对其性质作检测,诱导3 h后caspase-1蛋白得到剪切,5 h开始降解。研究结果表明,caspase-1基因可在大肠杆菌BL21中诱导表达,并且能够进行自我剪切。     

Colocalization of DNA fragmentation and caspase-3 activation during atresia in pig antral follicles

Apoptosis is the cellular mechanism of ovarian follicular atresia. The major downstream effector of this phenomenon in many tissues is caspase-3 but little is known about its role in pig ovarian apoptosis. In the present study, we detected the localization of caspase-3 in parallel with nuclear fragmentation (TUNEL) on healthy and early atretic antral follicles. In healthy antral follicles caspase-3 and TUNEL positivity were occasionally recorded within theca layer. The incidence of DNA fragmentation, as indicated also by the biochemical detection, increased mainly in the granulosa layer of early atretic follicles. Quantitative analysis revealed, besides, that atresia was accompanied by a higher incidence of caspase-3 (57.20 +/- 20.05 versus 3.64 +/- 0.61 positive cells in atretic versus healthy follicles, respectively; P < 0.05), of TUNEL positivity (20.13 +/- 9.33 versus 0.42 +/- 0.12; P < 0.05) and simultaneous immunostaining for caspase-3 and TUNEL (15.02 +/- 6.95 versus 0.31 +/- 0.05; P < 0.05) in the granulosa layer. In detached granulosa cells isolated from the follicular fluid of early atretic follicles a further significantly increase was recorded in the percentage of TUNEL positivity and in the incidence of cells that showed colocalization of caspase-3 activity and DNA fragmentation. Granulosa cells of early atretic follicles exhibited a higher positivity for caspase-3 localized in the cytoplasm and occasionally in the nucleus area of granulosa cells. These results indicate that capsase-3 was involved and precociously activated during the process of atresia. Finally, the progressively higher incidence of TUNEL positivity and of double immunostaining in atretic cells collected within the follicular fluid seems to indicate that proteases activity leads only tardily in a detectable DNA fragmentation.     

The bovine viral diarrhea virus (BVDV) NS3 protein, when expressed alone in mammalian cells, induces apoptosis which correlates with caspase-8 and caspase-9 activation

The bovine viral diarrhea virus (BVDV) strains exist as two biotypes, cytopathic (cp) and noncytopathic (ncp), according to their effects on tissue culture cells. It has been previously reported that cell death associated to cp BVDV in vitro is mediated by apoptosis. Here, experiments were conducted to determine the involvement of the NS3 protein in the induction of apoptosis. The NS3- and NS3Delta50 (deleted from the NH2-terminal 50 amino acids)-cDNA encoding sequences of BVDV NADL cp reference strain were cloned into adenoviral vectors (AdV) from which the BVDV gene of interest could be expressed from a tetracycline-responsive promoter. A549tTA cells infected in vitro with NS3 or NS3Delta50-expressing AdV showed cytopathic changes characterized by cell rounding and detachment, and nucleus chromatin condensation. DNA fragmentation assays, cytochrome c release, and activation of cellular caspases performed on these infected cells clearly correlated with the observed cytopathic changes with apoptosis. The BVDV NS3Delta50-induced apoptotic process was inhibited by caspase-8- and -9-specific peptide inhibitors (Z-IETD-FMK and Z-LEHD-FMK). Furthermore, apoptosis was inhibited in cells expressing the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase (HSV2-R1) or hsp70, two proteins which are known to inhibit apoptosis associated with caspase-8 activation and cytochrome c release-dependent caspase-9 activation, respectively. Given that HSV2-R1, a specific inhibitor of the caspase-8 activation pathway, efficiently suppressed apoptosis and also prevented caspase-9 activation, the overall results indicate that the BVDV NS3/NS3Delta50 induces apoptosis initiated by caspase-8 activation and subsequent cytochrome c release-dependent caspase-9 activation.     

硒对氟中毒鸡睾丸组织caspase-3 mRNA表达的影响

为探讨硒对氟中毒鸡睾丸组织caspase-3 mRNA表达的影响,将8日龄海兰褐雏鸡180只随机分为3组:Ⅰ组为正常对照组,饲喂全价日粮;Ⅱ组为高氟组,全价日粮中添加氟化钠(NaF),使每千克日粮中氟含量为1 000 mg;Ⅲ组为硒颉颃组,在高氟日粮中添加亚硒酸钠(Na2SeO3),使每千克日粮中硒含量为4 mg。试验期共90 d,分别于试验期第30、60、90天,空腹心脏采血后,剖杀试验鸡,取睾丸组织并检测caspase-3 mRNA表达水平。结果表明:在试验的各个阶段,Ⅲ组中caspase-3 mRNA转录水平均高于同期Ⅱ组。提示硒可以在一定程度上影响氟中毒鸡睾丸组织caspase-3 mRNA的表达水平。     

Induction of proinflammatory cytokines and caspase-1 by leptin in monocyte/macrophages from holstein cows

The proliferation of peripheral blood mononuclear cells (PBMC) containing both monocyte/macrophages and T lymphocytes increased after treatment with T-cell mitogen (concanavalin A: Con A). PBMC treated with either leptin alone or combination of leptin and ConA showed enhanced proliferative activity by 10-40%, compared with those treated with ConA alone. In contrast, isolated T lymphocytes treated with leptin and ConA showed lowered proliferative activity than the ConA-treated alone, indicating that leptin induced production of some cytokines from monocyte/macrophages, that subsequently resulted in enhancement of T lymphocytes proliferation in PBMC. Among the cytokines examined, monocyte/monocytes constitutively expressed interleukin (IL)-1beta, IL-12p35, IL-18 mRNA, and faintly expressed tumor necrosis factor (TNF)-alpha and IL-12p40 mRNA. Leptin treatment augmented the monocyte/macrophages mRNA expression of only TNF-alpha and IL-12p40 to comparable levels of cells treated with lipopolysaccharide (LPS). However, leptin treatment increased monocyte/macrophages production of IL-1beta as well as TNF-alpha, and induced the mRNA expression of caspase-1, which is shown to mediate the conversion of latent pro-IL-1beta and pro-IL-18 to active forms. These results suggest that leptin directly acts on monocyte/macrophages to produce factors that induce T lymphocytes proliferation such as IL-12p35/p40 complex through IL-12p40 induction and IL-1beta/IL-18 production through caspase-1 induction.     

茶皂素对PRRSV感染Marc-145细胞受体表达及Caspase-9活化的影响

以茶皂素(Teasaponin,TS)作为候选药物,研究茶皂素对Marc-145细胞受体CD163和波形蛋白(Vimentin)基因合成和蛋白表达的影响,以及茶皂素是否能通过细胞凋亡内源性通路影响PRRSV感染细胞,探究茶皂素抗PRRSV的作用机制。通过qRT-PCR和Western blot检测TS对细胞受体CD163和Vimentin的基因合成和蛋白表达的影响。运用Western blot技术检测TS对细胞内源性凋亡通路启动子caspase-9活化的影响,初探TS的抗PRRSV机制。qRT-PCR结果表明TS能显著抑制感染PRRSV的Marc-145细胞受体CD163和Vimentin基因的合成。Western blot结果表明TS能显著抑制细胞受体CD163和Vimentin的蛋白表达。TS能够引起细胞内源性凋亡通路启动子caspase-9的活化。研究表明,TS能抑制PRRSV在Marc-145细胞上的穿入过程,从而达到抗PRRSV的作用;亦可通过激活细胞凋亡内源性通路以早期促进细胞凋亡的方式产生抗PRRSV的作用。