紫外辐射诱发NIH3T3细胞凋亡时DNA断裂的特性

用常规琼脂糖凝胶电泳UVB照射后培养不同时间的NIH3T3细胞DNA,两个样本均未出现凋亡梯形带,而用相同条件处理的昆明小鼠胸腺细胞DNA出现了典型的梯形带.再用反转电场琼脂糖凝胶电泳(FIGE)UVB照射后培养不同时间的NIH3T3细胞DNA,发现DNA先断裂成低于23 kb的大分子片段,然后进一步断裂成小分子片段,但始终未出现梯形带,说明DNA并不总是从核小体之间断裂的.     

紫外辐射诱发NIH3T3细胞调亡时DNA断裂的特性

用常规琼脂糖凝胶电泳ＵＶＢ照射后培养不同时间的ＮＩＨ３Ｔ３细胞ＤＮＡ,两个样本均未出现凋亡梯形带,而用相同条件处理拽明小鼠胸腺细胞ＤＮＡ出现的典型的梯形带。再用反转电场琼脂糖凝胶电泳（ＦＩＧＥ）ＵＶＢ照射后培养不同时间的ＮＩＨ３Ｔ３细胞ＤＮＡ,发现ＤＮＡ先断裂成低于２３ｋｂ的大分子片段,然后进一步断裂成小分子片段,但始终未出现梯形带,说明ＤＮＡ并不总是从核小体之间断裂的。     

斜纹夜蛾核型多角体病毒DNA诱导同源昆虫细胞的凋亡

发现野生型斜纹夜蛾核型多角体病毒（Spodoptera litura nuclear polyhedrosis virus,SpltNPV）DNA转染SL-1细胞能诱导细胞凋亡.SpltNPV-DNA转染其同源细胞系斜纹夜蛾核SL-1细胞6 h后,光镜下即可见细胞膜表面突出或形成小泡,细胞碎裂成凋亡小体,18 h后,细胞100%碎裂成凋亡小体.DAPI荧光染色显示感染细胞核渐呈半月形,直至碎裂被凋亡小体包裹.被转染的SL-1细胞DNA琼脂糖凝胶电泳呈典型梯形谱带.野生型SpltNPV病毒粒子感染的SL-1细胞既无多角体的出现,也无凋亡现象的发生.     

510.6 nm铜蒸汽激光诱导兔血管平滑肌细胞凋亡的形态学观察

目的：透射电镜下观察激光诱导的血管平滑肌细胞（VSMC）凋亡的形态学改变。方法：组织贴块法培养兔主动脉平滑肌细胞,予激光照射（能量密度200J/cm^2、功率密度200mW/cm^2)后4小时、8小时、12小时、16小时、24小时取材,制作电镜标本,于透射电镜下观察,照相并记录实验结果。结果：透射电子显微镜上可观察到自照光后8小时起VSMC依次出现细胞体积缩小,胞质浓缩,细胞核染色质边集,细胞核固缩,凋亡小体形成等改变。结论：经激光照射,VSMC可呈现凋亡细胞典型形态学改变。     

鸭呼肠孤病毒强毒株致鸭胚原代成纤维细胞凋亡的研究

通过光镜、电镜、DNA Ladder法、流式细胞术、荧光染色对鸭呼肠孤病毒(DRV)诱导鸭胚原代成纤维细胞(DEF)凋亡情况进行检测.结果显示,光镜可见细胞形态学上出现细胞皱缩,染色质浓染边移;电镜观察到细胞胞浆浓缩,细胞核染色质凝聚、部分形成凋亡小体;荧光染色结果显示,在感染后24h有激发绿色荧光的凋亡细胞出现,随着时间的推移,激发红色荧光的死亡细胞数量增多;DNA Ladder检测到感染后24～144h的DNA样品呈梯形条带;流式细胞术于感染后24h检测到凋亡细胞,其数量在72～96h达到高峰,144h开始下降.研究结果表明,DRV在DEF增殖的过程中具有诱导宿主细胞凋亡的作用.     

单细胞凝胶电泳联合原子力显微镜观测UVB诱导的细胞DNA损伤模式变化

目的：检测UVB诱导的真核细胞DNA损伤。方法：采用单细胞凝胶电泳与原子力显微镜。结果：不同照射剂量的UVB引起的真核细胞DNA损伤模式不同。在0～20J／m2照射剂量范围内DNA无损伤;在20--360J／m2照射剂量范围内DNA损伤程度加快;当照射剂量超过360J／m2时DNA损伤速度减慢,实验组之间无显著性差异,出现“平台”。原子力显微镜的观察结果表明随着UVB照射剂量的增加,DNA结构的变化经历了断裂、交联与断裂并存的损伤增强趋势。当照射能量达到280J／m2时细胞DNA大都形成断片,并相互交联在一起。这一结果表明彗星电泳检测到的UVB照射剂量达到一定剂量后,DNA损伤出现”平台”的原因可能是此时DNA发生了链内或链间交联。结论：不同照射剂量的UVB造成的细胞DNA损伤模式不同;原子力显微镜是一种比较直观的观测DNA损伤的方法。借助原子力显微镜我们可以深入了解单细胞凝胶电泳检测的原理,为DNA损伤检测提供更优良的检测手段。     

Sindbis病毒的繁殖与宿主细胞BHK—21的凋亡

详细报道了Sindbis病毒诱导BHK-21细胞凋亡的过程,病毒感染6h后即可观测到核染色质的断裂,病毒感染12h后染色质可见明显的凝集,感染后24h DNA电泳出现明显的DNA“阶梯”(DNA ladder)。电镜观察更清楚地显示了凋亡小体形成的某些细节:在染色质凝集处核外膜突起,最后与细胞核分离形成凋亡小体。在此基础上将一段病毒非结构蛋白nsP2基因克隆到真核表达载体pMAMneo中,并得到瞬间表达,在其中一些细胞中出现DNA断裂这一细胞凋亡的基本特征,通过对nsP2氨基酸序列的分析,结合以前的实验结果推测nsP2可能与诱导细胞凋亡直接相关。     

原花色素对HepS细胞增殖抑制诱导凋亡的研究

本文通过体外培养肝癌HepS细胞,以不同浓度原花色素处理12—72h后,MTT法测定细胞生长抑制作用,采用DNA片断分析、DNA琼脂糖凝胶电泳、荧光染色以及流式细胞技术等方法来探讨原花色素体外抑制肝癌HepS细胞及诱导其凋亡的作用。实验结果显示原花色素能抑制HepS细胞的生长,并且呈现出明显的时效和量效关系,DNA电泳出现典型的凋亡DNA梯形带,在荧光显微镜下,凋亡细胞呈亮绿色,H和AnnexinV．FIFC双染后,经流式细胞仪检测、分析显示凋亡细胞明显增多。因此原花色素能抑制肝癌HepS细胞株的生长,可能与诱导其细胞凋亡有关。     

原子力显微镜观测紫外线诱导的K562肿瘤细胞DNA损伤

利用彗星电泳检测出UVB、UVC短时间照射会使肿瘤细胞的DNA发生断裂,而长时间照射之后彗星电泳无法检测到碎片,推测可能是由于DNA分子交联的原因[1],国内外尚无定论.为了更直观的研究这种现象,提取了UVB,UVA照射后K562细胞的DNA,并调节到合适的浓度在原子力显微镜下观测.实验结果表明UVB对K562肿瘤细胞DNA损伤的影响呈现时间/剂量效应,较短时间照射主要产生DNA的链断裂,较长时间辐射则主要产生DNA链的交联.UVC对K562肿瘤细胞DNA的损伤大于UVB.UVC短时照射即可引起DNA的断裂和交联,较长时间辐射主要产生交联和一些断裂;长时间照射不但产生大量交联,同时有大量断裂产生,并发生凝缩和缠绕等结构破坏.     

地塞米松诱导红系细胞凋亡的观察

正常机体内,红细胞生成素（EPO）诱导红系细胞分化,并阻止其凋亡,使其维持机体所需足够的数量。本文采用诱导贫血病毒（FVA）诱导BALB／c小鼠脾细胞所形成的红细胞实验系统,研究了外界因子－地塞米松（Dex）对EPO作用的影响。取6－8周雌性BALB／c小鼠,尾部静脉注射含FVA的小鼠血清。15天后,断颈处死,取脾于IMDM中漂洗、剪碎、过滤、离心、制成单细胞悬液,在EPO存在下,于平皿中培养至不同的时期后,进行不同浓度、不同时间的Dex处理。取细胞进行：（1）台盼蓝染色计数死细胞数;（2）观察DNA电泳图谱;（3）电镜检察细胞学变化。Fig.1表明：随着Dex处理浓度和时间的增加,细胞死亡率也增加。Fig.2表明：10^-4浓度Dex处理,即可使早（12h）、中（24h）幼期红细胞凋亡,DNA断裂成多聚核小体,产生明显DNA梯形带。Fig.3表明：高浓度Dex可以使早期幼期红细胞凋亡。电镜观察表明：与对照（Fig.4)比较,Dex处理后,细胞核固缩,染色质沿核膜内面周边凝聚,核周间隙扩张,胞质内出现空泡（Fig.5);随后细胞破碎,出现大量凋亡小体（Fig.6),Dex拮抗EPO,诱导红系细胞凋亡的现象此前未见报导。这对深入认识Dex作用机理,指导临床具有重要意义。     

Plasma levels of cell-free apoptotic DNA ladders and gamma-glutamyltranspeptidase (GGT) in diabetic children

The plasma levels of apoptotic DNA ladders (i.e., apoptosemia) and gamma-glutamyltranspeptidase (GGT) in diabetic outpatients and rats were investigated. Apoptotic DNA ladders were detected in plasma from 26.8% of type 1 (T1) and 18.5% of type 2 (T2) diabetic children 1-20 years of age, 25.7% of hospitalized children and 35.7% of adult RA outpatients, but in only 3.5% of adult pre-op patients. Plasma from 7.7% of young streptozotocin-induced diabetic but not control rats contained apoptotic DNA ladders. Apoptosemia was detected more often in male T1 (31%) and T2 (30.8%) diabetic outpatients than in female T1 (20.8%) and T2 (15.4%) diabetic outpatients. GGT in apoptosemic plasma was significantly higher than in nonapoptosemic plasma from T1 (P = 0.001) but not T2 diabetic children. The highest amounts of apoptotic DNA were detected most often in diabetic children > or =14 years of age. In vitro study results suggest that cell-free apoptotic DNA ladders appear prior to an increase in GGT activity in serum from human blood incubated at 37 degrees C. The results suggest that 24.7% of plasma samples from diabetic children contained apoptotic DNA ladders, the incidence and amounts of apoptotic DNA ladders were higher in the older diabetic children, and GGT was elevated in apoptosemic T1 diabetic children (P = 0.01). The results indicate that "silent" apoptosemia occurs in T1 and T2 diabetic children and suggest elevated GGT in diabetic children could be due to release from apoptotic cells.     

In vivo time course of morphological changes and DNA degradation during the degeneration of castration-induced apoptotic prostate cells

The in vivo time course of the morphological changes and DNA degradation in castration-induced apoptotic prostate cells was studied from the earliest to the latest stage of the degeneration process. To study this problem, we first induced apoptotic prostate cells in rats by castration for 3 days and then promptly and continuously blocked the death of healthy prostatic cells in the castrated rats by in vivo testosterone replacement. Because testosterone replacement could not stop the irreversible lysis of already damaged prostate cells, apoptotic cells at different stages of the degeneration process were eliminated sequentially from the prostate after the healthy prostate cells had been protected. Prostate cells at the earliest stage of apoptosis at the time when the castrated rats received testosterone replacement disappeared last. By tracing the morphological and DNA degradation of apoptotic cells after hormone treatment, we estimated the time course of prostate cell death from the early to the final stage. In the morphological evolution of apoptotic prostate cells, the clumping of nuclear chromatin, the degeneration of cytoplasm and the involution of the cell surface occurred and progressed simultaneously, resulting in the rapid formation of apoptotic bodies that were gradually digested by other cells. The DNA ladders of apoptotic cells were progressively cleaved into a mononucleosomal subunit that was further degraded at an additional site, generating a heterogeneous population of small nucleotides. The final digestion of DNA fragments occurred within the apoptotic bodies. The whole course of prostate cell death after castration took about 44 h.     

Apoptotic DNA fragmentation is detected by a semi-quantitative ligation-mediated PCR of blunt DNA ends

Apoptosis is a form of programmed cell death (PCD) characterized by morphological changes and stereotypical DNA degradation described as a nucleosomal ;ladder'. However, nucleosomal ladders have only been clearly demonstrated in vertebrate tissues when large numbers of cells die in synchrony. Their absence may be explained by asynchronous death under physiological conditions, or by distinct molecular mechanisms. In this study, nucleosomal ladders were revealed by a ligation-mediated polymerase chain reaction (LMPCR), that amplifies DNA fragments with blunt, 5' phosphorylated ends. Numerous tissues from different organisms were examined which demonstrated that nucleosomal ladders (a) accompany physiological cell death in mammalian tissues where previously DNA fragmentation has not been detected; (b) are produced during invertebrate cell death; (c) are invariably generated via the production of blunt, 5' phosphorylated double strand breaks. These results suggest that PCD in multicellular organisms consistently involves apoptotic mechanisms and that the endonuclease activity is evolutionarily conserved.     

Recombinant Galectins of <Emphasis Type="Italic">Haemonchus contortus</Emphasis> Parasite Induces Apoptosis in the Peripheral Blood Lymphocytes of Goat

The effect of Haemonchus contortus galectin peptides rHco-gal-m/f to induce apoptosis in the peripheral blood lymphocytes (PBLCs) of goats was investigated. Analysis of apoptosis was carried out with agarose gel electrophoresis, flow cytometry and transmission electron microscopy. The results indicated that there were visible apoptosis bodies and typical DNA ladders by genomic DNA fragmentation. The quantitative analysis of apoptosis by flow cytometry indicated that rHco-gal-m/f peptides induced apoptosis was time and dose dependent. Ultrastructural studies of the PBLCs revealed that a large number of apoptotic cells were present in galectin-treated cells, which had the typical morphologic changes of apoptosis such as reduction of the cytoplasmic volume, loss of cell surface microvilli, chromatin condensation and fragmentation of the apoptotic cells into small apoptotic bodies.     

Novel evidence for apoptotic cell response and differential signals in chromatin condensation and DNA cleavage in victorin-treated oats

Histological and cytological evidence of where and when apoptotic cells occur in Pc-2/Vb oat cells treated with victorin was obtained by observing DNA strand breaks at both light (LM) and electron microscope (EM) levels using TUNEL techniques. DNA from leaf segments that had been floated on victorin solution with the abaxial epidermis removed showed typical ladders on agarose gels. Nuclear chromatin condensation, followed by cell collapse, started in the mesophyll cells closest to the victorin solution. LM-TUNEL was positive in the non-collapsed cells but not in the collapsed cells in the treated leaves. However, the EM-TUNEL assay was positive in the nuclei of the non-collapsed as well as the collapsed cells where nuclear fragments dispersed into the cytoplasm, and the immunogold density was much higher than that in the cells killed by a high concentration of H2O2, suggesting that the victorin-treated collapsed cells are in the last stage of apoptotic cell death. The immunogold labelling in the victorin-treated non-collapsed cells was restricted to condensed heterochromatin, indicating that chromatin condensation is associated with DNA cleavage. Pharmacological studies indicated that proteases and nucleases may play a role in the apoptotic response. However, the EM-TUNEL assay indicated that EGTA co-incubated with victorin blocked DNA cleavage, but failed to prevent chromatin condensation. Moreover, protein kinases were involved in chromatin condensation, but did not affect DNA digestion, suggesting that chromatin condensation and DNA cleavage are differentially regulated in the death process in oats.     

镉诱导HEK293细胞凋亡及其线粒体凋亡途径

本课题研究了氯化镉(CdCl_2)诱导HEK293细胞(人胚胎肾细胞系)的凋亡,初步探讨了凋亡过程中Caspase-3、Bcl-2的变化和凋亡诱导因子(AIF)的转移以及它们的意义。MTT法检测CdCl_2对HEK293细胞增殖的抑制作用;通过倒置显微镜、电镜、琼脂糖凝胶电泳、流式细胞术、激光共聚焦观察细胞凋亡;应用Western blot法和荧光免疫法测定Caspase-3酶原、Bcl-2蛋白的变化以及检测AIF蛋白在细胞中的定位。结果显示:CdCl_2对HEK293细胞具有显著的生长抑制作用,并呈明显的剂量和时间依赖性。在琼脂糖凝胶电泳中,显示有凋亡细胞特有的DNA梯状条带,其中30μmol/L作用6-9h梯状条带最为清晰,时间过长或浓度过高则梯状条带逐渐模糊,表明镉浓度过高或处理时间过长,细胞有坏死。流式细胞仪检测也印证了这一结果。形态学观察可见明显的细胞凋亡特征。同时线粒体膜电位明显下降,发现Caspase-3酶原蛋白、Bcl-2蛋白含量减少,并具有时间依赖性;另外检测到线粒体AIF向细胞核转移。而Bcl-2转染后有一定的抑制凋亡作用。实验结果提示,CdCl_2能够诱导HEK293细胞凋亡,线粒体损伤导致AIF转移与细胞色素c释放,从而引发的非Caspases与Caspases凋亡途径可能在镉引发的细胞凋亡过程中起重要作用,而Caspase-3, Bcl-2起着重要的调控作用。     

Withanolide induces apoptosis in HL-60 leukemia cells via mitochondria mediated cytochrome c release and caspase activation

The present study is on the growth inhibitory effect of Withania somnifera methanolic leaf extract and its active component, withanolide on HL-60 promyelocytic leukemia cells. The decrease in survival rate of HL-60 cells was noted to be associated with a time dependent decrease in the Bcl-2/Bax ratio, leading to up regulation of Bax. Both the crude leaf extract and the active component activated the apoptotic cascade through the cytochrome c release from mitochondria. The activation of caspase 9, caspase 8 and caspase 3 revealed that caspase was a key mediator in the apoptotic pathway. DNA fragmentation analysis revealed typical ladders as early as 12h indicative of caspase 3 role in the apoptotic pathway. Flow cytometry data demonstrated an increase of sub-G1 peak upon treatment by 51% at 24h, suggesting the induction of apoptotic cell death in HL-60 cells.     

Electrophoretic DNA analysis for the detection of apoptosis

There are many techniques available for the detection of apoptotic cells; some are based on morphological changes, others on biochemical events. However, electrophoretic detection of the systematic cleavage of DNA into oligonucleosomal multimers of 180–200 bp remains the “hallmark” of apoptosis. Conventional constant field agarose gel electrophoresis of DNA from apoptotic cells can be used to resolve the multimers into the characteristic DNA ladders indicative of apoptotic cell death. More recently, it has become clear that the generation of the lower molecular weight oligonucleosomal DNA is preceded by the generation of higher molecular weight fragments. In some cell types, DNA cleavage proceeds no further than the formation of 300 and/or 50 kbp cleavage products. DNA fragmentation of this size can only be resolved using a form of pulsed-field gel electrophoresis. Basic “starter” protocols for conventional and pulsed field electrophoresis for the detection of apoptotic cell DNA are described in this article.     

Apoptotic Cell Death and Cellular Surface Negative Charge Increase in Maize Roots Exposed to Cytotoxic Stresses

Maize root meristematic tissues were exposed to cytotoxic reagents,the RNA-synthesis inhibitor Actinomycin D (ActD), the protein-synthesisinhibitor cycloheximide (CHX) and the mitosis inhibitor colchicine(COL). Morphological and biochemical evidence of specific apoptoticnuclei and chromosomes in individual treated cells was identifiedusing a simple and highly efficient chromosome spreading-basedTUNEL assay, DNA laddering and DNA gel blotting. All of thesedrugs induced DNA cleavage, dose-dependent oligomeric ladders,and characteristic nuclear and chromosomal condensations. Resultsfrom DNA gel blotting showed that DNA ladders could be inducedby exposure to 0.1 mg l^-1ActD, 100 mg l^-1CHX and 500 mg l^-1COLfor 6 h, 6 h and 12 h respectively. The sequence of changesin single cells was studied in detail. DNA cleavage was foundto occur before condensation and disorganization of the nucleus,followed by deformation and condensation of metaphase chromosomes,and marginalization of chromatin. Finally, nucleoli disappearedand fragmentation of the nucleus occurred. Meanwhile, changesin the outer surface charge of apoptotic cells were assessedby electrophoresis. Results indicated quantitatively that thesurface negative charge increased during these apoptotic processes.Our results also showed that the apoptotic pathway induced byeach of these drugs could be reversed before serious cleavageof DNA into oligonucleosomal fragments and universal chromatincondensation. Copyright 2001 Annals of Botany Company Cytotoxin, chromosome spreading, apoptosis, cell electrophoresis