共查询到20条相似文献,搜索用时 31 毫秒
1.
Ablin J Verbovetski I Trahtemberg U Metzger S Mevorach D 《Apoptosis : an international journal on programmed cell death》2005,10(5):1009-1018
A number of mechanisms have been proposed to explain the etiology of drug-induced lupus (DIL) but the effect of apoptotic and necrotic cell handling has not been previously examined.Objective. To evaluate the effect of quinidine and procainamide at therapeutic range concentrations, on the uptake of apoptotic and necrotic thymocytes by murine peritoneal macrophages and on macrophage survival, as a novel mechanism for DIL.Methods. Thymocytes were stained and induced to undergo apoptosis by serum withdrawal. Apoptosis was evaluated using annexin V and propidum iodide (PI) and PI staining. Necrosis was induced by heating. Peritoneal macrophages were treated with quinidine or procainamide at a range of therapeutic concentrations and incubated with stained apoptotic and necrotic thymocytes. Apoptotic and necrotic cell uptake was evaluated by flow cytometry using double staining of thymocytes and macrophages and by confocal microscopy. Green fluorescent latex beads were used as controls for phagocytosis.Results. Significantly decreased uptake of apoptotic and necrotic cells was seen in the presence of quinidine and procainamide. The documented effect was mainly on the number of apoptotic/necrotic cells per macrophage. Uptake of fluorescent latex beads offered to resident macrophages was not significantly affected by quinidine or procainamide. No pro-apoptotic effect of quinidine or procainamide on macrophages was seen.Conclusion. Quinidine and procainamide at therapeutic range concentrations specifically inhibit clearance of apoptotic and necrotic cells by peritoneal macrophages. Altered handling of apoptotic and necrotic cells may represent a contributing mechanism for DIL. 相似文献
2.
Chow-Chin Tong Yew-Keong Choong Nor-Aini-B Umar Mohamed-Mustapha Noordin Suhaila Mohamed 《World journal of microbiology & biotechnology》2009,25(4):687-695
Ganoderma lucidum powder using hot water and methanol extraction methods indicated a twofold more active cytotoxic activity with IC50 of 44 ± 3.8 μg/ml in the latter method. The representative dose-response curves of the G. lucidum crude extracts on J558 cell-lines revealed that there were great similarities between the curves which reflected rapid killing
activities. The percentage viability of the J558 cell exposed to these crude extracts was dose dependent only up to 150 μg/ml.
After which, there was no significant reduction when the dose was increased to 200 or 400 μg/ml. The morphological alterations
induced by the crude extract were examined under the phase contrast, fluorescent and electron microscopy. When J558 cells
were treated with doses higher than 50 μg/ml of the crude extract, obvious morphological changes and apoptosis occurred after
72 h. At 400 μg/ml, most of the cells showed necrosis characterized as small fragments with uniformly stained red nuclei.
The apoptotic and necrotic cells increased by 16.5 and 29.1%, respectively whereas the viable cells decreased by as much as
45.6. The mode of cell death via apoptosis was 3.6% higher than necrosis. However, these morphological changes were not observed
in the case of 3T3 cells. Results obtained from scanning electron microscopy and transmission electron microscopy further
confirmed the occurrence of various apoptotic and necrotic features. 相似文献
3.
The blastogenic cycle of the colonial ascidian Botryllus schlosseri concludes in a phase of selective cell and zooid death called takeover. Every week, all asexually derived parental zooids synchronously regress over a 30-h period and are replaced by a new generation. Here we document the sequential ultrastructural changes which accompany cell death during zooid degeneration. The principal mode of visceral cell death during takeover occurred by apoptosis, the majority of cells condensing and fragmenting into multiple membrane-bounded apoptotic bodies. Cytoplasmic organelles (mitochondria, basal bodies, striated rootlets) within apoptotic bodies retained ultrastructural integrity. Dying cells and fragments were then swiftly ingested by specialized blood macrophages or intraepithelial phagocytes and subsequently underwent secondary necrotic lysis. Certain organs (stomach, intestine) displayed a combination of necrotic and apoptotic changes. Lastly, the stomach, which demonstrated some of the earliest regressive changes, exhibited intense cytoplasmic immunostaining with a monoclonal antibody to ubiquitin at the onset of takeover. Affinity-purified rabbit antiserum against sodium dodecyl sulfate-denatured ubiquitin detected a characteristic 8.6-kDa mono-ubiquitin band by Western blot analysis. Collectively, these findings raise the possibility that cell death during takeover is a dynamic process which requires active participation of cells in their own destruction. 相似文献
4.
Hirofumi Fujita Masanao Yamamoto Tetsuya Ogino Hirotsugu Kobuchi Naoko Ohmoto Eriko Aoyama Takashi Oka Tohru Nakanishi Keiji Inoue Junzo Sasaki 《Cell biochemistry and function》2014,32(1):77-86
A close relationship between cell death and pathological calcification has recently been reported, such as vascular calcification in atherosclerosis. However, the roles of cell death in calcification by osteoblast lineage have not been elucidated in detail. In this study, we investigated whether cell death is involved in the calcification on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hMSC) under osteogenic culture in vitro. Apoptosis and necrosis occurred in an osteogenic culture of hMSC, and cell death preceded calcification. The generation of intracellular reactive oxygen species, chromatin condensation and fragmentation, and caspase‐3 activation increased in this culture. A pan‐caspase inhibitor (Z‐VAD‐FMK) and anti‐oxidants (Tiron and n ‐acetylcysteine) inhibited osteogenic culture‐induced cell death and calcification. Furthermore, calcification was significantly promoted by the addition of necrotic dead cells or its membrane fraction. Spontaneously dead cells by osteogenic culture and exogenously added necrotic cells were surrounded by calcium deposits. Induction of localized cell death by photodynamic treatment in the osteogenic culture resulted in co‐localized calcification. These findings show that necrotic and apoptotic cell deaths were induced in an osteogenic culture of hMSC and indicated that both necrotic and apoptotic cells of osteoblast lineage served as nuclei for calcification on osteoblastic differentiation of hMSC in vitro. Copyright © 2013 John Wiley & Sons, Ltd. 相似文献
5.
Patel VA Longacre A Hsiao K Fan H Meng F Mitchell JE Rauch J Ucker DS Levine JS 《The Journal of biological chemistry》2006,281(8):4663-4670
Current models of autoimmunity suggest that delayed clearance of apoptotic cells leads to the presentation of apoptotic antigens in the context of inflammatory signals, with resultant autoimmunity. These models implicitly assume that, in contrast to early apoptotic cells (that retain membrane integrity), late apoptotic cells (with compromised membranes) act like necrotic cells (which also lack intact membranes), possibly because of the release of proinflammatory intracellular contents. We showed previously that early apoptotic and necrotic cells induce distinct mitogen-activated protein kinase modules in macrophages with which they interact. Exposure to apoptotic cells led to nearly complete inhibition of both basal and macrophage colony-stimulating factor-induced ERK1/2 by macrophages. In contrast, necrotic cells induced ERK1/2. We show here that apoptotic cells also strongly induced both c-Jun N-terminal kinase and p38, whereas necrotic cells had no detectable effect on c-Jun N-terminal kinase and p38. We also compared the signaling events induced in macrophages by exposure to early apoptotic cells, late apoptotic cells, and necrotic cells. The signaling events induced by late apoptotic cells were identical to and just as potent as those induced by early apoptotic cells. Thus, apoptotic cells are functionally equivalent throughout the cell death process, irrespective of membrane integrity. Moreover, the effects of both early and late apoptotic cells on signaling were dominant over those of necrotic cells. These data show that apoptotic cells do not become proinflammatory upon the loss of membrane integrity and are inconsistent with the notion that delayed clearance alone can lead to autoimmunity. 相似文献
6.
Method of specific detection of apoptosis using formamide-induced DNA denaturation assay. 总被引:3,自引:0,他引:3
Yuko Ito Masa-Aki Shibata Ken Kusakabe Yoshinori Otsuki 《The journal of histochemistry and cytochemistry》2006,54(6):683-692
We compared the reliability between apoptosis detection methods, namely, the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) method and formamide-induced DNA denaturation assay using a monoclonal antibody (MAb) to single-stranded DNA (ssDNA) (formamide-MAb assay). Reaction targets in these methods are different: the TUNEL method recognizes free 3'-OH DNA ends, whereas the formamide-MAb assay detects ssDNA itself (25-30 bp). We found that the formamide-MAb assay immunohistochemically detected apoptotic cells, whereas the TUNEL method detected apoptotic cells as well as mitotic and necrotic cells. The TUNEL method recognized not only 3'-OH DNA ends cleaved by DNase during apoptosis but also constitutive physiological nicking that occurs in DNA duplication and histone posttranslational modifications during mitosis and random DNA breaks during necrotic execution. By electron microscopy, the mean labeling density (the number of 3'-OH DNA ends/nuclear area) obtained by the TUNEL method was determined to be consistently higher than that (the number of ssDNAs/nuclear area) obtained by the formamide-MAb assay. On the basis of these findings, we conclude that the formamide-MAb assay was more specific than the TUNEL method for the detection of apoptotic cells using electron microscopy; however, the labeling intensity of the formamide-MAb assay was slightly weaker than that of the TUNEL method. 相似文献
7.
Phagocytosis of necrotic cells by macrophages is phosphatidylserine dependent and does not induce inflammatory cytokine production 总被引:9,自引:0,他引:9
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Brouckaert G Kalai M Krysko DV Saelens X Vercammen D Ndlovu MN Ndlovu M Haegeman G D'Herde K Vandenabeele P 《Molecular biology of the cell》2004,15(3):1089-1100
Apoptotic cells are cleared by phagocytosis during development, homeostasis, and pathology. However, it is still unclear how necrotic cells are removed. We compared the phagocytic uptake by macrophages of variants of L929sA murine fibrosarcoma cells induced to die by tumor necrosis factor-induced necrosis or by Fas-mediated apoptosis. We show that apoptotic and necrotic cells are recognized and phagocytosed by macrophages, whereas living cells are not. In both cases, phagocytosis occurred through a phosphatidylserine-dependent mechanism, suggesting that externalization of phosphatidylserine is a general trigger for clearance by macrophages. However, uptake of apoptotic cells was more efficient both quantitatively and kinetically than phagocytosis of necrotic cells. Electron microscopy showed clear morphological differences in the mechanisms used by macrophages to engulf necrotic and apoptotic cells. Apoptotic cells were taken up as condensed membrane-bound particles of various sizes rather than as whole cells, whereas necrotic cells were internalized only as small cellular particles after loss of membrane integrity. Uptake of neither apoptotic nor necrotic L929 cells by macrophages modulated the expression of proinflammatory cytokines by the phagocytes. 相似文献
8.
Growing evidence suggests that two modes of cell death, known as apoptosis and necrosis, are involved in postanoxic injury. The current opinion on these two types of cell death is that apoptosis and necrosis are not always the uniform and distinct events. The aim of this study was to determine ultrastructural criteria of postanoxic neuronal changes in model of anoxia in vitro. The organotypic cultures of rat hippocampus exposed to 10‐ and 20‐min of anoxic insult revealed the morphological features classic for both necrotic and apoptotic neuronal cell injury. Some neurones exhibited the typical necrotic lysis whereas others clearly reflected an active apoptotic form of cell death consisting of nuclear condensation with early preservation of cell membranes. However, numerous damaged cells shared both apoptotic and necrotic ultrastructural characteristics. These results evidenced the morphological continuum between apoptosis and necrosis under anoxia in vitro. 相似文献
9.
A laser scanning confocal microscopy method. Simultaneous detection of intracellular Ca2+ and apoptosis using Fluo-3 and Hoechst 33342 总被引:5,自引:0,他引:5
Zhang T Cao EH Li JF 《Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology》2000,22(2):93-97
OBJECTIVE: To develop a simple and direct method to simultaneously determine apoptotic cells from a treated population of cells and detect the changes of intracellular Ca2+ in these apoptotic cells, in particular single ones, by confocal microscopy. STUDY DESIGN: MGC-803 cells treated with As2O3 were used as the double-staining cell model with Hoechst 33342 as a DNA probe and Fluo-3AM as a Ca2+ indicator. MGC-803 cell apoptosis induced by As2O3 was first demonstrated by DNA ladder in gel electrophoresis. Based on the difference in DNA stainability with Hoechst 33342 and corresponding fluorescence intensity between live and apoptotic cells, apoptotic cells and the changes in intracellular Ca2+ were detected at the same time by confocal microscopy. No necrotic cells in the group treated with As2O3 were found by the trypan blue exclusion test. RESULTS: The results from confocal microscope detection showed that intact and apoptotic cells were successfully recognized and the changes of intracellular Ca2+ in apoptotic and intact cells were simultaneously detected in the same sample. CONCLUSION: We provided a useful method to exactly detect changes in intracellular Ca2+ in apoptotic cells, especially in single ones, by confocal microscopy and to exclude the artifact effect of necrotic and intact cells. 相似文献
10.
Macrophages use different internalization mechanisms to clear apoptotic and necrotic cells 总被引:1,自引:0,他引:1
Krysko DV Denecker G Festjens N Gabriels S Parthoens E D'Herde K Vandenabeele P 《Cell death and differentiation》2006,13(12):2011-2022
The present study characterized two different internalization mechanisms used by macrophages to engulf apoptotic and necrotic cells. Our in vitro phagocytosis assay used a mouse macrophage cell line, and murine L929sAhFas cells that are induced to die in a necrotic way by TNFR1 and heat shock or in an apoptotic way by Fas stimulation. Scanning electron microscopy (SEM) revealed that apoptotic bodies were taken up by macrophages with formation of tight fitting phagosomes, similar to the 'zipper'-like mechanism of phagocytosis, whereas necrotic cells were internalized by a macropinocytotic mechanism involving formation of multiple ruffles directed towards necrotic debris. Two macropinocytosis markers (Lucifer Yellow (LY) and horseradish peroxidase (HRP)) were excluded from the phagosomes containing apoptotic bodies, but they were present inside the macropinosomes containing necrotic material. Wortmannin (phosphatidylinositol 3'-kinase (PI3K) inhibitor) reduced the uptake of apoptotic cells, but the engulfment of necrotic cells remained unaffected. Our data demonstrate that apoptotic and necrotic cells are internalized differently by macrophages. 相似文献
11.
This protocol describes a rapid and simple method for the identification of apoptotic cells. Owing to changes in membrane permeability, early apoptotic cells show an increased uptake of the vital DNA dye Hoechst 33342 (HO342) compared with live cells. The nonvital DNA dye 7-amino-actinomycin D (7-AAD) is added to distinguish late apoptotic or necrotic cells that have lost membrane integrity from early apoptotic cells that still have intact membranes as assayed by dye exclusion. The method is suitable to be combined with cell surface staining using Abs of interest labeled with fluorochromes that are compatible with HO342 and 7-AAD emissions. Surface antigen staining is carried out according to standard methods before staining for apoptosis. The basic assay can be completed in 30 min, and extra time is needed for cell surface antigen staining. 相似文献
12.
Yanming Sun Ruofeng Yan Charles I. Muleke Guangwei Zhao lixin Xu Xiangrui Li 《International journal of peptide research and therapeutics》2007,13(3):387-392
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. 相似文献
13.
BACKGROUND: Methods widely used to detect apoptosis do not allow us to easily distinguish between nuclei from viable or necrotic cells. Even if apoptosis and necrosis seem to occur as alternatives at the single cell level, they could be present simultaneously in a cell population much more frequently than expected. For this reason, attention was focused on attempting to recognize, by multiparameter flow cytometry, the characteristics of viable cells and of apoptotic or necrotic dead cells. METHODS: Apoptosis and necrosis were induced in vitro in murine thymocytes and lymphocytes from adult peripheral blood by using dexamethasone or prostaglandin E2 treatment and heat shock at 60 degrees C or hydrogen peroxide, respectively. Traditional methods, such as DNA gel electrophoresis and propidium iodide staining followed by single-fluorescence analysis or annexin-V-fluorescein isothiocyanate plus propidium iodide staining by using flow cytometry, were compared with a new method. This method consisted of combined light-scatter and red fluorescence analysis by flow cytometry after isolation of nuclei by hypotonic solution as well as high-dose detergent treatment and DNA staining with propidium iodide. RESULTS: Results showed that, although traditional methods such as DNA-gel electrophoresis and single-parameter fluorescence flow cytometry analysis were unable, as expected, to discriminate among viability, apoptosis, and necrosis, our new method has enabled us to easily identify nuclei from viable, apoptotic, and necrotic cells. Results obtained by using our method were comparable to those obtained by using two-color analysis of cells after propidium iodide/annexin V staining. CONCLUSIONS: A highly reproducible, inexpensive, rapid, and easily accessible method of analysis has been developed for simultaneously detecting apoptosis and necro sis. 相似文献
14.
Juszczak K Kaszuba-Zwoińska J Chorobik P Ziomber A Thor PJ 《Cellular & molecular biology letters》2012,17(2):196-205
Highly concentrated urine may induce a harmful effect on the urinary bladder. Therefore, we considered osmolarity of the urine
as a basic pathomechanism of mucosal damage. The influence of both cyclophosphamide (CYP) and hyperosmolar stimuli (HS) on
the urothelium are not well described. The purpose was to evaluate the effect of CYP and HS on rat urothelial cultured cells
(RUCC). 15 Wistar rats were used for RUCC preparation. RUCC were exposed to HS (2080 and 3222 mOsm/l NaCl) for 15 min and
CYP (1 mg/ml) for 4 hrs. APC-labelled annexin V was used to quantitatively determine the percentage of apoptotic cells and
propidium iodide (PI) as a standard flow cytometric viability probe to distinguish necrotic cells from viable ones. Annexin
V-APC (+), annexin V-APC and PI (+), and PI (+) cells were analysed as apoptotic, dead, and necrotic cells, respectively.
The results were presented in percentage values. The flow cytometric analysis was done on a FACSCalibur Flow Cytometer using
Cell-Quest software. Treatment with 2080 and 3222 mOsm/l HS resulted in 23.7 ± 3.9% and 26.0 ± 1.5% apoptotic cells, respectively,
14.3 ± 1.4% and 19.4 ± 2.7% necrotic cells, respectively and 60.5 ± 1.4% and 48.6 ± 5.3% dead cells, respectively. The effect
of CYP on RUCC was similar to the effect of HS. After CYP the apoptotic and necrotic cells were 23.1 ± 0.3% and 17.9 ± 7.4%,
respectively. The percentage of dead cells was 57.7 ± 10.8%. CYP and HS induced apoptosis and necrosis in RUCC. 3222 mOsm/l
HS had the most harmful effect based on the percentage of necrotic and apoptotic cells. 相似文献
15.
Oskar S. Frankfurt James A. Robb Everett V. Sugarbaker Luis Villa 《Experimental cell research》1996,226(2):387
The most widely used histochemical marker of apoptosis (in situend labeling, TUNEL) detects both apoptotic and necrotic cells and evaluates only late stages of apoptosis. Hence, a specific and sensitive cellular marker of apoptosis is needed to determine the role of apoptotic death in biology and pathology. The present study describes a novel immunohistochemical procedure for the staining of apoptotic cells using a monoclonal antibody (MAb) to single-stranded DNA. This MAb stained all cells with the morphology typical of apoptosis in etoposide-treated HL-60, MOLT-4, and R9 cell cultures, in which apoptosis was accompanied by high, moderate, and low levels of internucleosomal DNA fragmentation, respectively. TUNEL stained all apoptotic cells in HL-60 cultures, nearly 60% of apoptotic cells in MOLT-4 cultures, and only 14% of apoptotic cells in R9 cultures. Apoptotic R9 cells, which progressed into secondary necrosis, retained MAb staining and became TUNEL-positive. Necrotic cells in MOLT-4 cultures treated with sodium azide were stained by TUNEL, but were negative for MAb staining. All floating cells at a late stage of apoptosis in MDA-MB-468 cultures treated with cisplatin were stained by both MAb and TUNEL. However, among adherent cells in the early stages of apoptosis, MAb stained nearly 20 times more cells than TUNEL. In histological sections of human tumor xenografts, MAb detected clusters of apoptotic cells in viable tumor tissue, but did not stain cells in areas of central ischemic necrosis. In contrast, TUNEL stained nuclei in necrotic areas. Thus, MAb to single-stranded DNA is a specific and sensitive cellular marker of apoptosis, which differentiates between apoptosis and necrosis and detects cells in the early stages of apoptosis. 相似文献
16.
Background
Analyzing apoptosis has been an integral component of many biological studies. However, currently available methods for quantifying apoptosis have various limitations including multiple, sometimes cell-damaging steps, the inability to quantify live, necrotic and apoptotic cells at the same time, and non-specific detection (i.e. "false positive"). To overcome the shortcomings of current methods that quantify apoptosis in vitro and to take advantage of the 96-well plate format, we present here a modified ethidium bromide and acridine orange (EB/AO) staining assay, which may be performed entirely in a 96-well plate. Our method combines the advantages of the 96-well format and the conventional EB/AO method for apoptotic quantification. 相似文献17.
Krysko DV D'Herde K Vandenabeele P 《Apoptosis : an international journal on programmed cell death》2006,11(10):1709-1726
The ultimate and most favorable fate of almost all dying cells is engulfment by neighboring or specialized cells. Efficient
clearance of cells undergoing apoptotic death is crucial for normal tissue homeostasis and for the modulation of immune responses.
Engulfment of apoptotic cells is finely regulated by a highly redundant system of receptors and bridging molecules on phagocytic
cells that detect molecules specific for dying cells. Recognition of necrotic cells by phagocytes is less well understood
than recognition of apoptotic cells, but an increasing number of recent studies, which are discussed here, are highlighting
its importance. New observations indicate that the interaction of macrophages with dying cells initiates internalization of
the apoptotic or necrotic targets, and that internalization can be preceded by “zipper”-like and macropinocytotic mechanisms,
respectively. We emphasize that clearance of dying cells is an important fundamental process serving multiple functions in
the regulation of normal tissue turnover and homeostasis, and is not just simple anti- or pro-inflammatory responses. Here
we review recent findings on genetic pathways participating in apoptotic cell clearance, mechanisms of internalization, and
molecules involved in engulfment of apoptotic versus necrotic cells, as well as their immunological consequences and relationships
to disease pathogenesis.
Katharina D’Herde and Peter Vandenabeele share senior authorship.
This study was supported by Ghent University GOA grant No. 12050502, IUAP-V/12-12.0C14.02, FWO-Vlaanderen 3G.0218.06, and
Flanders Interuniversity Institute for Biotechnology (VIB). 相似文献
18.
Features of apoptotic cells measured by flow cytometry. 总被引:134,自引:0,他引:134
Z Darzynkiewicz S Bruno G Del Bino W Gorczyca M A Hotz P Lassota F Traganos 《Cytometry》1992,13(8):795-808
The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. Most of these methods were applied to studies of apoptosis triggered in the human leukemic HL-60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomerase inhibitors or prednisolone. In most cases, apoptosis was selective to cells in a particular phase of the cell cycle: only S-phase HL-60 cells and G0 thymocytes were mainly affected. Necrosis was induced by excessively high concentrations of these drugs. The following cell features were found useful to characterize the mode of cell death: a) Activation of an endonuclease in apoptocic cells resulted in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, led to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content made it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of the apoptotic process. b) Plasma membrane integrity, which is lost in necrotic but not apoptotic cells, was probed by the exclusion of propidium iodide (PI). The combination of PI followed by Hoechst 33342 proved to be an excellent probe to distinguish live, necrotic, early- and late-apoptotic cells. c) Mitochondrial transmembrane potential, assayed by retention of rhodamine 123 was preserved in apoptotic but not necrotic cells. d) The ATP-dependent lysosomal proton pump, tested by the supravital uptake of acridine orange (AO) was also preserved in apoptotic but not necrotic cells. e) Bivariate analysis of cells stained for DNA and protein revealed markedly diminished protein content in apoptotic cells, most likely due to activation of endogenous proteases. Necrotic cells, having leaky membranes, had minimal protein content. f) Staining of RNA allowed for the discrimination of G0 from G1 cells and thus made it possible to reveal that apoptosis was selective to G0 thymocytes. g) The decrease in forward light scatter, paralleled either by no change (HL-60 cells) or an increase (thymocytes) of right angle scatter, were early changes during apoptosis. h) The sensitivity of DNA in situ to denaturation, was increased in apoptotic and necrotic cells. This feature, probed by staining with AO at low pH, provided a sensitive and early assay to discriminate between live, apoptotic and necrotic cells, and to evaluate the cell cycle phase specificity of these processes. i) The in situ nick translation assay employing labeled triphosphonucleotides can be used to reveal DNA strand breaks, to detect the very early stages of apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
19.
Role of Oxidative Stress,Apoptosis, and Intracellular Homeostasis in Primary Cultures of Rat Proximal Tubular Cells Exposed to Cadmium 总被引:1,自引:0,他引:1
Cadmium (Cd) is a known nephrotoxic element. In this study, the primary cultures of rat proximal tubular (rPT) cells were
treated with low doses of cadmium acetate (2.5 and 5 μM) to investigate its cytotoxic mechanism. A progressive loss in cell
viability, together with a significant increase in the number of apoptotic and necrotic cells, were seen in the experiment.
Simultaneously, elevation of intracellular [Ca2+]i and reactive oxygen species (ROS) levels, significant depletion of mitochondrial membrane potential(Δ
Ψ) and cellular glutathione (GSH), intracellular acidification, and inhibition of Na+, K+-ATPase and Ca2+-ATPase activities were revealed in a dose-dependent manner during the exposure, while the cellular death and the apoptosis
could be markedly reversed by N-acetyl-l-cysteine (NAC). Also, the calcium overload and GSH depletion were significantly affected by NAC. In conclusion, exposure
of rPT cells to low-dose cadmium led to cellular death, mediated by an apoptotic and a necrotic mechanism. The apoptotic death
might be the chief mechanism, which may be mediated by oxidative stress. Also, a disorder of intracellular homeostasis induced
by oxidative stress and mitochondrial dysfunction is a trigger of apoptosis in rPT cells. 相似文献
20.
The in Vitro Growth of Human Chondrocytes 总被引:2,自引:0,他引:2
Harrison PE Ashton IK Johnson WE Turner SL Richardson JB Ashton BA 《Cell and tissue banking》2000,1(4):255-260
Autologous chondrocyte implantation (ACI) for the treatment of articular cartilage defects has been described by other workers, however, relatively few details of the in vitro growth of the cells have been published. Here we describe the release of cells from adult human articular cartilage and their growth characteristics in vitro.Cultures were successfully established from 29 of 30 biopsies taken from patients aged 20–72 year. No significant relationship was found between donor age and initial cell yield following cartilage digest, however, the time to primary confluence increased in direct proportion to age. Thereafter the kinetics of cell proliferation was independent of donor age.The proportion of apoptotic or necrotic cells in the cartilage digest was low and increased with time in culture only in those cells which remained non-adherent. Conversely, entry into cell cycle was restricted to those cells which had become adherent.These results illustrate that previously reported techniques for isolating and culturing chondrocytes are reproducible, that adherent chondrocytes have considerable proliferative potential, and that concern about cell growth and viability need not, in itself, limit the clinical application of ACI to younger patients. 相似文献