DOI10.1007/s00702-004-0109-z
J Neural Transm(2004)111:667–681
Neurotoxic potential of haloperidol in comparison
with risperidone:implication of Akt-mediated
signal changes by haloperidol
https://www.doczj.com/doc/7c2946209.html,ai1,H.Ozawa2,M.Tateno1,E.Hashimoto1,and T.Saito1
1Department of Neuropsychiatry,School of Medicine,
Sapporo Medical University,Sapporo,and
2Division of Neuropsychiatry,Department of Neurosensory Medicine,
Nagasaki University School of Medicine,Nagasaki,Japan
Received November24,2003;accepted January9,2004
Published online April2,2004;#Springer-Verlag2004
Summary.The neurotoxicity of conventional antipsychotic drugs has emerged as a potential pathogenic event in extrapyramidal side effects(EPS)and in their limited ef?cacy for negative-cognitive symptoms in schizophrenic patients.The atypical antipsychotics,recently developed,have superior therapeutic ef?cacy to treat not only positive symptoms but negative symptoms and cognitive dysfunc-tions with much lower potentials of side effects,although the in?uence of atypical antipsychotics on the regulation of neuronal survival has been less investigated.It is important to clarify the effects of typical and atypical antipsychotics on neuronal survival and their contributions to the therapeutic development and understanding of the pathophysiology of schizophrenia.We measured the neurotoxicity of two antipsychotic drug treatments,haloperidol and risperidone,in primary cultured rat cortical neurons.Immunoblotting and pharmacological agent analyses were used to determine the signal transduction changes implicated in the mechanisms of the neurotoxicity.Haloperidol induced apoptotic injury in cultured cortical neurons, but risperidone showed weak potential to injure the neurons.Treatment with halo-peridol also led the reduction of phosphorylation levels of Akt,and activated caspase-3.The D2agonist bromocriptine and5-HT2A antagonist,ketanserin atten-uated the haloperidol-induced neuronal toxicity.Moreover,brain-derived neuro-trophic factor(BDNF)reduced the caspase-3activity and protected neurons from haloperidol-induced apoptosis.BDNF also reversed the reduced levels of phos-phorylation of Akt caused by treatment with haloperidol.Haloperidol but not risperidone induces caspase-dependent apoptosis by reducing cellular survival signaling,which possibly contributes to the differential clinical therapeutic ef?-cacy and expression of side effects in schizophrenia.
Keywords:Schizophrenia,extrapyramidal side effects,negative symptoms, haloperidol,risperidone,Akt,BDNF.
https://www.doczj.com/doc/7c2946209.html,ai et al.
Introduction
Conventional antipsychotics such as haloperidol have been widely used to treat schizophrenic patients,but it is well known that the typical antipsychotics often produce extrapyramidal side effects(EPS),including parkinsonism,akathisia and tardive dyskinesia(TD),and are less effective for negative-cognitive symp-toms rather than positive symptoms in such patients.The production of EPS is the major limitation of the use of this class of drugs(Diederich et al.,1998).
Recently developed atypical antipsychotics such as serotonin-dopamine antagonists(SDA)have superior therapeutic ef?cacy for negative symptoms, cognitive dysfunctions and treatment-resistant schizophrenia with lower poten-tials of side effects especially in EPS(Peuskens,1995;Kinon et al.,1996;Song, 1997).It is known that typical antipsychotics have high af?nity for dopamine D2 receptors in the brain(Farde et al.,1992).However,atypical antipsychotics have relatively low af?nity for D2receptors but higher af?nity for5-HT2receptors (Kapur et al.,1999).These pharmacological differences are basically thought to be responsible for the differences of therapeutic effects and side effects pro?les of typical and atypical antipsychotics(Meltzer et al.,1989),but the neurochem-ical mechanisms of the differences have not yet been understood.
Recently,as a potential pathogenic event in EPS,the neurotoxicity of antipsy-chotics has attracted attention.Treatment with haloperidol has been reported to cause necrotic and apoptotic cell death(Behl et al.,1995;Noh et al.,2000).It was demonstrated that haloperidol caused oxidative stress,resulting from alternations of mitochondrial function,and that vitamin E could protect neurons from halo peridol-induced toxicity(Cadet et al.,1994;Galili et al.,2000).However,the pre-cise mechanism of the neuronal toxicity is poorly understood,and little has been done to investigate the changes of intracellular signaling related to the toxicity.
In the history of psychiatric research,the neurodevelopmental hypothesis has been presented as an etiologic and pathophysiologic theory(Weinberger et al.,1987).In addition to this hypothesis,the recent development of molecular neurobiology and neuroimaging and postmortem?ndings has given rise to the neurodegenerative hypothesis as a new pathophysiological model of schizo-phrenia(Lieberman et al.,1999).Both hypotheses regard morphological and structural abnormalities to be important in the pathophysiology of this illness. On the other hand,it is also reported that the volume and morphology of the prefrontal cortex and superior temporal gyrus are changed by pretreatment with antipsychotics and that their changes differ between typical and atypical anti-psychotics(Matsumoto et al.,2001).Postmortem study of TD brains showed ventricular dilatation and atrophy in several brain areas,as well as neurodegen-eration and gliosis(Mion et al.,1991;Dalgalarrondo et al.,1994).Furthermore, chronic treatment with haloperidol caused brain damage and TD in psychotic patients(Sunderland et al.,1987).All things considered,it is possible that typical and atypical antipsychotic differently affects neuronal survive and death,and that these effects considerably contribute to the differences in EPS and improvements of negative-cognitive symptoms in schizophrenic patients.
We thus,in the present work,carried out a direct comparison study of two kinds of antipsychotics and their potentials for neuronal toxicity in the cultured
Implication of Akt-mediated signal changes by haloperidol669 cells to clarify how the neurotoxic potentials of antipsychotics are related to the expression of EPS and the pathophysiology of schizophrenia.We used cortical neurons to investigate the involving cholinergic activity changes,which are related to the cognitive de?cits and production of TD.We especially analyzed the effects of antipsychotics on the regulation of the cellular survival signaling pathways,including the PI3-K=Akt pathway,on the basis of the apoptosis-related cascade.
Material and methods
Neuronal cell cultures
The animals used in this study were housed and treated according to the guidelines for care and use of experimental animals of the Ethics Committee of Sapporo Medical University.Primary cultures of cortical neurons were prepared from embryonic day18(E18)fetal rats.Jcl:Wistar rats were purchased from CLEAR Japan(Tokyo,Japan).Cortices were dissected from embryonic brains microscopically,and dissociated by incubation in trypsin(Invitrogen,Carlsbad,CA USA) for20min at37 C.Then the trypsin was inactivated by suspending the cells in serum-containing medium(DMEM=10%FBS),and triturated with a glass pipette followed by?ltration.DMEM was obtained from Nissui Pharmaceutical(Tokyo,Japan),and added20mM glucose,2mM L-glutamine1mM sodium bicarbonate and100U=ml gentamycin reagent(Invitrogen).Fetal bovine serum(FBS)was obtained from Sigma(St.Louis,MO USA).The cells were pelleted by centrifugation(400?g for5min at4 C)and washed once in DMEM=10%FBS.Then the cells were resuspended in a chemically de?ned plating medium(DMEM=B27[Invitrogen])and viable cells were determined by trypan blue exclusion.The cells were plated at0.5?105cells=cm2on poly-L-lysine(Sigma)coated tissue culture dishes.For the assessment of neurotoxicity,the cells were plated on24-well dishes(IW AKI,Japan)in0.5ml of plating medium.For the western blot analysis,the cells were plated on35mm dishes(IW AKI,Japan)in3ml of plating medium,and for the measurement of caspase-3activity,the cells were plated on100mm dishes(IW AKI, Japan)in10ml of plating medium.Cultures were maintained in a humidi?ed incubator with an atmosphere of5%CO2and95%air at37 C.Serum free culture with B27supplementation yields nearly pure neuronal cultures(90%>),as judged by the immunocytochemistry for glial ?brillary acidic protein(GFAP)and microtubule-associated-protein2(MAP2).Experiments were performed on culture days8–12in vitro(DIV).
Drug treatment
On the day of the experiment,the medium was removed and replaced by DMEM=B27containing 0.1–100m M haloperidol(Wako,Osaka Japan),0.1–100m M risperidone(gift from Janssen Phar-maceutical,Beerse Belgium),3–10m M bromocriptine(Sigma),1–3m M domperidone(Sigma), 0.1–1m M ketanserin(BIOMOL,Plymouth Meeting,PA USA)or10–30m M BDNF(PeproTech, London UK).Drugs were dissolved in dimethyl sulfoxide(DMSO),except for the BDNF.The?nal concentration of DMSO was below0.4%.BDNF was dissolved in PBS containing0.1%bovine serum albumin.The duration of each drug treatment is described in detail in the?gure legends.
DNA ladder assay
To examine DNA cleavage,soluble cytoplasmic DNA was isolated from5?106cells and sub-jected to1.8%agarose gel electrophoresis for DNA ladder analysis(Hockenbery et al.,1990) Quantitation of apoptosis by MTT and EIA
using antibodies against MAP2
The mitochondrial dehydrogenase activity that cleaves3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)was used to determine cell survival in quantitative colorimetric
https://www.doczj.com/doc/7c2946209.html,ai et al.
assays.The cortical neurons were incubated with MTT(0.5mg=ml)for2hours at37 C,and blue-colored formazan cleaved by dehydrogenase enzymes in active mitochondria was dissolved in DMSO,and quanti?ed spectrophotometrically at570nm.The results are expressed as a percentage of control culture viability.ED50values and95%con?dence limits were determined by the method of Fieller with linear regression analysis of log-logit plots(logit conversion:log (p=(1-p));p?neuronal viability reduction(%))using Yukms Statistical Library(Y ukms,Tokyo Japan).Neuronal death was also evaluated by the enzymatic immunoassay(EIA)using an anti-body against microtubule-associated-protein2(MAP2).
Immunocytochemistry and EIA
Following?xation in4%paraformaldehyde,cultures were rinsed with phosphate-buffered saline (PBS),and initially treated with a solution of3%aqueous H2O2for3min.The cultures were then rinsed in buffer and preincubated in PBS containing5%normal horse serum for30min at room temperature.Next they were incubated with mouse monoclonal IgG against MAP2(1:1000)for 30min at room temperature and then rinsed in buffer and incubated with the biotinylated anti-mouse IgG for30min at room temperature.After a buffer rinse,cultures were incubated with avidin-biotin-horseradish peroxidase complex(V ector Laboratories,Burlingame,CA USA)for 30min at room temperature.Finally,rinsed cultures were incubated with0.05%diaminobenzi-dine tetrahydrochloride(DAB)(Vector Laboratories)and0.01%hydrogen peroxide for3–5min. Reactions were stopped by rinsing the cultures with PBS.Quantitative analysis was performed using0.2mg=ml o-phenylenediamine dihydrochloride(OPD)(Wako)in0.2M Na2HPO3and 0.1M citric acid containing0.1%H2O2as a horseradish peroxidase substrate.In this EIA method, cultures incubated with avidin-biotin-horseradish peroxidase complex,were reacted with OPD solution for3min and then stopped with1N H2S04solution and aliquots were read in a micro-plate at450nm within30min of adding the stop solution.
Analysis of caspase-3activity
The activity of caspase-3,which cleaves the substrate acetyl-Asp-Glu-V al-Asp p-nitroanilide (Ac-DEVD-pNA)in the cortical neurons,was analyzed using a caspase-3assay kit(Sigma), following the manufacturer’s instructions.In brief,after the appropriate drug treatment,cortical neurons were scraped off the dish,and the cells were pelleted by centrifugation at600?g for5min at4 C.After one wash with ice-cold PBS,the cell pellets were suspended in lysis buffer containing 50mM HEPES(pH7.4),5mM CHAPS and5mM DTT.The lysates were incubated on ice for 20min,and centrifuged at18,000?g for15min at4 C.The reaction was started by adding caspase-3substrate Ac-DEVD-pNA to the cell lysates in96-well plates with assaying buffer(20mM HEPES[pH7.4],0.1%CHAPS,5mM DTT and2mM EDTA).The plates were incubated at 37 C for1–2hours,and the concentrations of pNA released from the substrates by caspase-3 cleaving were calculated,using colorimetrical detection of the absorbance value at405nM.The speci?city of the enzyme activity measured was assessed using a selective inhibitor of caspase-3, Ac-DEVD-CHO.The ability to block staurosporin induced caspase-3activation was determined by treating the cells with Ac-DEVD-CHO(10m M)before the treatment with staurosporine(1m M).
Western blot analysis
For analysis of the levels of phosphorylated Akt and total Akt,cortical neurons treated with drugs for appropriate times were washed twice with ice-cold PBS and harvested in200m l of lysis buffer containing50mM Tris-HCl,pH7.5,100mM NaCl,1%SDS,20mM EDTA.After sonication for 30sec,the amount of protein in each sample was estimated by the bicinchoninic acid method (Pierce,Rockford,IL),followed by addition of1=5volume of sample buffer(200mM Tris-HCl, pH6.8,10%SDS,25%b-mercaptoethanol,25%glycerol and0.01%bromophenol blue).The samples were boiled for5min,and20m g aliquots were subjected to SDS-PAGE on10%poly-acrylamide gels and transferred to polyvinylidene di?uoride(PVDF)membranes(Millipore, Bedford,MA).After blocking with5%nonfat dry milk in Tris-buffered saline containing 0.05%Tween-20overnight at4 C,blots were probed with speci?c antibodies(anti-Akt,
1:5000;anti-phosphorylated Akt,1:1000)for 1h at room temperature,and washed,then incu-bated with Rabbit Ig,HRP-Linked F (ab’)2Fragment (Amersham Pharmacia Biotech,Buckin-ghamshire,England)diluted to 1:5000for 1h at room temperature.Immunoreactive bands were detected with the enhanced chemiluminescence system (ECL).
Statistical analysis
All values are presented as mean ?SEM.Data were analyzed using one-way ANOBA between subjects,and post hoc comparisons were made using the Tukey’s HSD test.In all cases,statistical signi?cance was set at p <0.05.
Results
Cortical neurons undergo apoptosis after exposure to haloperidol
Cortical neuron apoptosis was induced by exposure to haloperidol.In the absence of haloperidol,the cultured neurons exhibited normal cellular morphology with extending neurites (Fig.1).Haloperidol caused morphological changes charac-teristic of apoptosis,including degeneration of neurites and shrinkage of cell bodies.The morphological changes of neurons were evident over 24hr treatment with 30m M haloperidol.To determine whether haloperidol-induced toxicity
was Fig.1.Haloperidol exposure induces apoptosis in cortical neurons.A Representative photo-micrographs of cortical neurons (8DIV)treated with vehicle (0.3%DMSO)or 30m M haloper-idol for 16h and 72hr.The top panels are phase-contrast photomicrographs and the bottom panels are neurons stained with MAP2to clearly visualize apoptotic neuron morphology,including shrunken cell bodies,and fragmented processes.B DNA fragmentation manifests as a DNA ladder.Cortical neurons were treated with vehicle (C)or 30m M haloperidol (H)for 24hr.Positions of molecular size markers (MW)are indicated on the left in base pairs Implication of Akt-mediated signal changes by haloperidol 671
apoptosis,a DNA laddering assay was conformed.DNA cleavage into oligonu-cleosome fragments was performed 24hr after 30m M haloperidol exposure in cortical neurons.
The quantity of neuronal toxicity was evaluated by the MTT metabolism assay (Fig.2).Haloperidol reduced MTT metabolism in a dose-and time-depen-dent manner.The haloperidol concentration dependence and kinetics for neuro-nal viability assay were comparable with those seen when the cells were assayed for neuronal death by the ELISA method using an antibody against MAP2.
Effects of haloperidol and risperidone on cortical neuron survival
The neurotoxic potentials of haloperidol and risperidone were compared by MTT survival assays.The severity of neuronal toxicity was demonstrated by the evaluation of IC 50values for each drug.The conventional antipsychotic drug haloperidol had the higher toxicity on cortical neurons,with an IC 50value of 5.7m M (Fig.3),serotonin-dopamine antagonist (SDA)risperidone,showed neurotoxicity only with large doses (IC 50>30m
M).
Fig.2.Quantitation of dose response and kinetics for neuronal death induced by haloperidol exposure.Cortical neurons (8DIV)were treated with 0.1–100m M haloperidol for 72hr,or 30m M haloperidol for 0–96hr.Neuronal viability was determined by MTT metabolism (A ),and immunoreactivity of MAP2(B ),at each point after the initial treatment.Data are the
averages of three independent experiments
https://www.doczj.com/doc/7c2946209.html,ai et al.
Implication of D2receptor function in the neuronal
apoptosis induced by haloperidol
We tested whether the D2receptor agonist bromocriptine could also block cortical neuron toxicity after haloperidol exposure.Cortical neurons were treat-ed with 30m M haloperidol to induce neuronal toxicity (Fig.4).
Approximately Fig.4.Suppression of neuronal death,which correlates with D2receptors in haloperidol exposured neurons.Cortical neurons were pretreated with vehicle (C)or 3,10m M bromocrip-tine (Bro 3,Bro 10)for 24hr and then exposure to 30m M haloperidol for 48hr to induce neuronal toxicity.Neuronal toxicity was evaluated by MTT assay.Addition of 1or 3m M domperidone (dom 1,dom 3)reversed the protective effect of bromocriptine.Data are the
average of three independent
experiments
Fig.3.Evaluation of neuronal toxicity induced by different antipsychotics.Cortical neurons were treated with the indicated concentrations of haloperidol or risperidone for each 72hr.ED 50values and 95%con?dence limits were determined by the method of Fieller with linear regres-
sion analysis as described in Materials and methods
Implication of Akt-mediated signal changes by haloperidol 673
50%of cells survived 48hr after the treatment.However,90%of the cells survived in the presence of 10m M bromocriptine.Moreover,the neural protec-tion afforded by bromocriptine was primarily preserved by cotreatment with another D2receptor antagonist,domperidone,suggesting a role for the D2receptors and their signal transduction in bromocriptine protection against neu-ronal toxicity induced by haloperidol.
Haloperidol but not Risperidone activates caspase-3
in cortical neurons
To investigate what mechanism is distinct in the neuronal toxicity induced by the haloperidol and risperidone,we evaluated the activity of caspase-3in the cortical neurons.We found that 24hr exposure of the cells to 30m M haloperidol signi?cantly increased caspase-3activity compared with control cells,whereas after treatment with risperidone,it remained at the control level (Fig.5A).The role of caspase-3,DEVD-sensitive activity in cortical neuron apoptosis was also supported by the ?nding of a signi?cant 6-fold elevation of caspase-3after treatment with the potent apoptosis inducer staurosporine,and its almost com-plete reduction by cotreatment with the speci?c caspase-3inhibitor DEVD-CHO,similar to a previous report (D’Mello et al.,1998).Haloperidol increased caspase-3activity in a dose-dependent manner (Fig.5B)
BDNF suppresses caspase-3activation and protects neurons
from apoptosis after exposure to haloperidol
To de?ne signaling pathways that reduce survival of cortical neurons,we tested the effectiveness of neurotrophin,BDNF,against haloperidol-induced caspase-3acti-vation and neuronal apoptosis.Cortical neurons were treated with 30m M haloper-idol for 24hr for caspase-3activity analysis,and for 48hr for survival assay,in the presence or absence of varying concentrations of BDNF,in respective examina-tions.BDNF decreased haloperidol-elevated caspase-3activity in cortical neurons in a dose-dependent manner.For example,after 24hr of treatment,30ng =
ml
Fig.5.Activation of caspase-3in cortical neurons after exposure to haloperidol.A Cortical neurons were treated with vehicle (C)or 30m M haloperidol (Hal),30m M risperidone (Ris),1m M Staurosporine (Sta)or 1m M Staurosporine tAc-DEVD CHO (Inh)for 24hr.Exposure to haloperidol and staurosporine signi?cantly activated caspase-3compared with control.B Dose-dependent activation of caspase-3was observed by haloperidol (24h)treatment.Results in
panels are averages of three different experiments.Error bars are SEM
https://www.doczj.com/doc/7c2946209.html,ai et al.
BDNF decreased haloperidol-elevated caspase-3activity by 65%(Fig.6A).Simi-lar levels of neuroprotection were also observed,for example,after 48hr of treat-ment with haloperidol alone 60%of neurons survived,and 90%of neurons survived after cotreatment with 30ng =ml BDNF (Fig.6B).
Inhibition of the PI 3-Kinase-Akt pathway is critical
for cortical neuron apoptosis induced by haloperidol
BDNF can activate several signal transduction systems including the PI 3-kinase pathway (Segal et al.,1996;Yamada et al.,1997).We assayed PI 3-kinase activity by western blot analysis using a phospho-Akt antibody that speci?cally recognizes activated Akt.The same samples were also probed with an anti-Akt antibody to ensure an equal amount of protein loading in each lane.Phosphorylation of Akt was detectable in cortical neurons maintained in regular culture condition;probably because of PI 3-kinae activation by growth factors in the B27supplement.Treatment with 30m M haloperidol at 4,8,and 18hr caused large decrease in Akt phosphorylation (Fig.7A).
To determine whether activation of the PI 3-kinase pathway contributes to BDNF protection against haloperidol,we added BDNF to block PI 3-kinase inhibition by haloperidol.BDNF at 30ng =ml completely reversed the Akt phos-phorylation after its reduction by haloperidol (Fig.7B).
5-HT 2A antagonist suppresses neuronal apoptosis
induced by haloperidol
The atypical antipsychotic drug risperidone is known for its high af?nity to 5-HT 2A receptors as an antagonist.To obtain further insights into the
limited Fig.6.BDNF protects neurons from haloperidol-induced apoptosis through caspase-3inhibi-tion.Cortical neurons were treated with 30m M haloperidol in the presence or absence of 10or 30ng =ml BDNF.BDNF signi?cantly inhibited caspase-3activation (A ),and protected cortical neurons (B )against 30m M haloperidol exposure.Results in panels are averages of three
different experiments.Error bars are SEM
Implication of Akt-mediated signal changes by haloperidol 675
neurotoxicity induced by risperidone,we tested whether 5-HT 2A antagonism in?uenced the neuronal toxicity after haloperidol exposure.Cortical neurons were treated with 30m M haloperidol to induce neuronal toxicity (Fig.8).Approximately 55%of cells survived 48hr after the treatment.However,80%of the cells survived in the presence of 1m M 5-HT 2A speci?c antagonist ketanserin.This suggested a role for 5-HT 2A receptor antagonism on the pro-tective effect against neuronal apoptosis induced by haloperidol.
Discussion
In the current work,we investigated neuronal toxicity caused by the two kinds of antipsychotics,haloperidol and risperidone,to clarify the molecular basis
of Fig.7.BDNF reversed the haloperidol-induced PI 3-K =Akt reduction.Cortical neurons were treated with 30m M haloperidol for 4,8or 18hr.Haloperidol reduced the level of Ser-473phosphorylated Akt at all exposure time points (A ).BDNF (10,30m M)increased levels of phosphorylated Akt reduced by exposure to 30m M haloperidol (B ).Akt phosphorylation (pAkt)was examined by Western blot analysis at the indicated times after haloperidol treat-
ments.The blots show that the total level of Akt (Akt)remained
constant
Fig.8.Suppression of neuronal death by treatment with a 5-HT2A antagonist.Cortical neurons were pretreated with vehicle (C),0.1or 1m M ketanserin (Ket 0.1,Ket 1)for 24hr and then exposed to 30m M haloperidol for 48hr to induce neuronal toxicity.Neuronal toxicity was evaluated by
MTT assay.Data are the average of three independent experiments.Error bars are SEM
https://www.doczj.com/doc/7c2946209.html,ai et al.
Implication of Akt-mediated signal changes by haloperidol677 the differential expression of motor side effects,and differential ef?cacy on negative-cognitive symptoms.We found that treatment with haloperidol pro-duced apoptosis of cortical neurons accompanied by shrinkage of the cell body, and prominent DNA ladders,hallmarks of apoptosis(Raff et al.,1993;Stefanis et al.,1997).Haloperidol caused MTT metabolism reduction in a dose and time-dependent manner.We con?rmed that neuronal death paralleled MTT reduction using an established MAP2-ELISA.We con?rmed the reliability of this method by comparison of the immunoreactivity of MAP2with the number of neurons in cultured cortical neurons previously(data not shown).Recently,neurotoxic effects of antipsychotic drugs have emerged as potential pathogenic events of TD.One of the objectives of this study was to test the hypothesis that neurotoxic properties of antipsychotics are closely linked to the occurrence of EPS in schizo-phrenic patients.Haloperidol,a representative typical antipsychotic drug,showed about6-times higher neurotoxicity than risperidone,one of the atypical antipsy-chotic drugs.These differences suggest that haloperidol may have a stronger inhibitory effect on neuronal survival mechanisms than risperidone.
If haloperidol-induced neuronal toxicity is mediated by inhibition of D2 receptor function and subsequent disruption of signaling pathways underlying D2receptors,then agonists of D2receptors might prevent haloperidol-induced neuronal toxicity.In our experiment on cortical neurons,the D2receptor ago-nist bromocriptine blocked haloperidol-induced neurotoxicity.In addition, another speci?c D2antagonist,domperidone(Sokoloff et al.,1980)prevented the protective effect of bromocriptine against haloperidol-induced neurotoxi-city.Since it is reported that bromocriptine has neuroprotective effect against6-hydoxydopamine mediated by its hydroxyl free radical scavenging activity (Ogawa et al.,1994),the effect of bromocriptine here may relate to the pre-ventions of mitochondrial dysfunction and increasing hydroxyl free radicals by haloperidol.These data suggest that the haloperidol-induced neuronal apoptosis in cortical neurons is mediated by D2receptors,and that the inhibition of D2 receptor regulating signaling pathways plays a key role in the neurotoxicity of haloperidol.
To clarify the differences of cellular molecular changes,we next evaluated the activity of caspase-3,in the cortical neurons,because it is known as promi-nent apoptosis-inducing molecule acting in the?nal stages in the cell apoptosis program(Armstrong et al.,1997;Marks et al.,1998).We found that haloperidol activated caspase-3in cortical neurons.In contrast to haloperidol,risperidone exposure had no signi?cant effect on control caspase-3activity.These data sug-gested that the regulation of cellular survival signaling underlying the D2recep-tor inhibition differed between these two kinds of antipsychotics,and that caspase-3activation might contribute to neuronal apoptosis in cortical neurons induced by the antipsychotics,or at least by haloperidol exposure.
The active tissue brain level of risperidone at clinical dosage was estimated 1–50nM(Aravagiri et al.,1988,1998).That was about1000-times lower than that concentration of risperidone resulted in neither signi?cant toxicity nor caspase-3activation tested in the present study.The haloperidol concentration in the brain tissue of patients was detected at25–600nM(Kornhuber et al., 1999),and reported maximally to be10m M(Korpi et al.,1984).The concentration
https://www.doczj.com/doc/7c2946209.html,ai et al.
of haloperidol mainly used here(30m M)was several-fold higher than those brain levels.However,since the caspase-3activation by haloperidol was in-duced in dose-and time-dependently,the apoptotic mechanisms of haloperidol indicated here is possibly involved in a neuronal toxicity,especially in TD patients,during long-term therapeutic treatment with haloperidol.
Through stimulation of the D1and D2subfamilies of G-protein-coupled receptors,dopamine can activate cAMP response element-binding protein (CREB)phosphorylation and gene transcription via distinct mechanisms.By elevating intracellular cAMP levels and activating PKA,D1receptor stimula-tion leads to phosphorylation of CREB(Konradi et al.,1994).On the other hand,although D2receptors are classically linked to reduction of cAMP pro-duction,they can couple to phospholipase C b(PLC b)via Gq,mobilize intra-cellular calcium stores,and also phosphorylate CREB(Yan et al.,1999).The transcriptional factor CREB is known as a key molecule regulating neuronal survival.It targets important genes for cell survival,including BDNF.BDNF,a member of the NGF neurotrophic factor family,protects several types of CNS neurons from apoptosis caused by the survival-reducing stimulation such as trophic factor support withdrawal(Skaper et al.,1998;Gunn Moore et al., 1997;Hegarty et al.,1997;Ghosh et al.,1994).However,it was not known whether BDNF decreased the elevated activity of caspase-3or protected against cortical neuron apoptosis induced by haloperidol.For the?rst time,in our experiment,BDNF was demonstrated to block caspase-3activation and neuro-nal apoptosis induced by haloperidol in cortical neurons.These results may suggest that haloperidol-induced apoptosis is mediated by the activation of caspase-3,and activation of BDNF survival signal transduction can suppress the apoptosis through inhibition of caspase-3dependent mechanism.
BDNF has been reported to activate several cellular signal transduction systems,including the PI3-kinase pathway(Segal et al.,1996;Yamada et al.,1997).Stimulation of PI3-kinase can lead to the activation and phos-phorylation of protein kinase Akt,and phosphorylation of Akt on Ser-473is primarily dependent on PI3-kinase activity(Franke et al.,1997;Hetman et al., 1999).Therefore,we analyzed the activity changes of Akt caused by haloper-idol and BDNF treatments of cortical neurons.In these experiments,haloper-idol treatment alone reduced the level of phosphorylated Akt,and the reduction of activated Akt,was completely blocked by additional treatment with BDNF. Although activation of both ERK and the PI3-kinase signal activation by BDNF have been reported,multiple survival pathways are used in cortical neurons to counteract different forms of apoptotic signals(Hetman et al., 1999).Our present results indicate that BDNF is an effective activator of the PI3-kinase pathway under haloperidol-induced apoptotic conditions in cortical neurons,and that the PI3-kinase signaling pathway may play a large role in BDNF neuroprotection against haloperidol.
The balance of multiple receptor occupation,in particular,5-HT2A antag-onism to added to D2antagonism,is thought to be a key pharmacological mechanism by which atypical antipsychotics exert their clinical usefulness (Altar et al.,1986;Meltzer et al.,1989).We therefore investigated the effects of a5-HT2A antagonist on the neurotoxicity of haloperidol.We found that
Implication of Akt-mediated signal changes by haloperidol679 co-treatment with a selective5-HT2A antagonist,ketanserin,reduced haloperidol-induced toxicity of cortical neurons,suggesting that5-HT2A receptor antago-nistic action regulates neuronal survival signals changed by haloperidol. Although the mechanisms underlying the protection of the5-HT2A antagonist against haloperidol-induced neurotoxicity are unclear,it has been reported that ketanserin reverses haloperidol-induced catalepsy in rat models(Ninan et al., 1999),moreover,chronic treatment with atypical antipsychotic quetiapine and 5-HT2A antagonist,ritanserin(Xu et al.,2002),but not haloperidol(Chlan-Fourney,1998),signi?cantly up-regulates BDNF mRNA in the hippocampus. These observations possibly indicate that5-HT2A antagonistic actions have the potential to reverse neuronal-function disturbances and behavioral changes induced by D2antagonists,which relates to their neuroprotective action mediated by the BDNF expression changes.
In conclusion,the potentials for apoptotic neuronal death induction and caspase-3elevation are different between haloperidol and risperidone.Akt is a crucial molecule in haloperidol-induced apoptosis and BDNF has the ability to protect against apoptosis occurring through Akt regulated actions.Our data strongly suggest the possibility that reduced activity of Akt and elevation of caspase-3are critical in the cellular survival signal transduction changes result-ing from the dopamine D2inhibition by haloperidol in cortical neurons,and that5-HT2A antagonistic action attenuates their anti-survival signaling.In the atypical antipsychotics,risperidone is closer to the typical antipsychotic such as haloperidol.In fact,the structure of risperidone is similar to the haloperidol and the af?nity for D2receptors is relatively high compared with the other atypical antipsychotic.However,in the clinical study,it is also reported that risperidone has much lower potential of EPS than haloperidol,and it has effectiveness to the treatment-resistant schizophrenia.We believe the distinct activation of this signaling and subsequent neuronal survival change is important mechanisms that contribute to the difference of EPS incidence and effectiveness against negative-cognitive symptoms of the antipsychotic drug treatments for schizo-phrenic patients.
Acknowledgements
This work was supported by grants from the Japan Society for the Promotion of Science(JSPS), and by the National Center of Neurology and Psychiatry,Japan(NCNP).
References
Altar CA,Wasley AM,Neale RF,Stone GA(1986)Typical and atypical antipsychotic occupancy of D2and S2receptors:an autoradiographic analysis in rat brain.Brain Res Bull16:517–525 Aravagiri M,Marder SR,Wirshing D,Wirshing WC(1988)Plasma concentrations of risperidone and its9-hydroxy metabolite and their relationship to dose in schizophrenic patients: simultaneous determination by high performance liquid chromatography with electroche-mical detection.Pharmacopsychiatry31:102–109
Aravagiri M,Yuwiler A,Marder SR(1998)Distribution after repeated oral administration of different dose levels of risperidone and9-hydroxy-risperidone in the brain and other tissues of rat.Psychopharmacology139:356–363
Armstrong RC,Aja TJ,Hoang KD,Gaur S,Bai X,Alnemri ES,Litwack G,Karanewsky DS, Fritz LC,Tomaselli KJ(1997)Activation of the CED3=ICE-related protease CPP32
https://www.doczj.com/doc/7c2946209.html,ai et al.
in cerebellar granule neurons undergoing apoptosis but not necrosis.J Neurosci17: 553–562
Behl C,Rupprecht R,Skutella T,Holsboer F(1995)Haloperidol-induced cell death-mechanism and protection with vitamin E in vitro.Neuroreport7:360–364
Cadet JL,Kahler LA(1994)Free radical mechanisms in schizophrenia and tardive dyskinesia.
Neurosci Behav Res18:457–467
Chlan-Fourney J,Ashe P,Nylen K,Juorio A V,Li XM(2002)Differential regulation of hippocampal BDNF mRNA by typical and atypical antipsychotic administration.Brain Res954:11–20
Dalgalarrondo P,Gattaz WF(1994)Basal ganglia abnormalities in tardive dyskinesia.Possible relationship with duration of neuroleptic treatment.Neur Arch Psychiatry Clin Neurosci244: 272–277
Diederich NJ,Goetz CG(1998)Drug-induced movement disorders.Neurol Clin16:125–139 D’Mello SR,Aglieco F,Roberts MR,Borodezt K,Haycock JW(1998)A DEVD-inhibited caspase other CPP32is involved in the commitment of cerebellar granule neurons to apoptosis induced by Ktdeprivation.J Neurochem70:1809–1818
Farde L,Nordstr€o m AL,Wiesel FA,Pauli S,Halldin C,Sedvall G(1992)Positron emission tomographic analysis of central D1and D2dopamine receptor occupancy in patients treated with classic neuroleptics and clozapine.Relation to extrapyramidal side effects.Arch Gen Psychiatry49:538–544
Franke TF,Kaplan DR,Cantley LC(1997)PI3K:downstream AKTion blocks apoptosis.Cell 88:435–437
Galili-Mosberg R,Gil-Ad I,Weizman A,Melamed E,Often D(2000)Haloperidol-induced neurotoxicity-possible implications for tardive dyskinesia.J Neural Transm107: 479–490
Ghosh A,Carnahan J,Greenberg ME(1994)Requirement for BDNF in activity-dependent survival of cortical neurons.Science263:1618–1623
Gunn-Moore FJ,Williams AG,Toms NJ,Tavare JM(1997)Activation of mitogen-activated protein kinase and p70S6kinase is not correlated with cerebellar granule cell survival.
Biochem J324:365–369
Hegarty JL,Kay AR,Green SH(1997)Trophic support of cultured spiral ganglion neurons by depolarization exceeds and is additive with that by neurotrophins or cAMP and requires elevation of[Ca2t]I within a set range.J Neurosci17:1959–1970
Hetman M,Kanning K,Smith-Cavanaugh JE,Xia Z(1999)Neuroprotection by brain derived neurotrophic factor is mediated by extracellular-signal-regulated kinase and phosphatidy-linositol-3kinase.J Biol Chem274:22569–22580
Hockenbery D,Nunez G,Millman C,Schreiber RD,Korsmeyer SJ(1990)Bcl-2is an inner mitochondrial membrane protein that blocks programmed cell death.Nature348: 334–336
Kapur S,Zipursky RB,Remington G(1999)Comparison of the5-HT2and D2receptor occupancy of clozapine,risperidone,and olanzapine in schizophrenia:clinical and theore-tical implications.Am J Psychiatry156:286–293
Kinon BJ,Lieberman JA(1996)Mechanisms of action of atypical antipsychotic drugs:a critical analysis.Psychopharmacology(Berl)124:2–34
Konradi C,Cole RL,Heckers S,Hyman SE(1994)Amphetamine regulates gene expression in rat striatum via transcription factor CREB.J Neurosci14:5623–5634
Kornhuber J,Schultz A,Wiltfang J,Meineke I,Gleiter CH,Z€o chling R,Boissl KW,Leblhuber F, Riederer P(1999)Persistence of haloperidol in human brain tissue.Am J Psychiatry156: 885–890
Korpi ER,Kleinman JE,Costakos DT,Linnoila M,Wyatt RJ(1984)Reduced haloperidol in the post-mortem brains of haloperidol-treated patients.Psychiatry Res11:259–269 Lieberman JA(1999)Is schizophrenia a neurodegenerative disorder?A clinical and neurobio-logical perspective.Biol Psychiatry46:729–739
Marks N,Berg MJ,Guidotti A,Saito M(1998)Activation of caspase-3and apoptosis in cerebellar granule cells.J Neurosci Res52:334–341
Implication of Akt-mediated signal changes by haloperidol681 Matsumoto H,Simmons A,Williams S,Hadjulis M,Pipe R,Murray R,Frangou S(2001) Superior temporal gyrus abnormalities in early-onset schizophrenia:similarities and differ-ences with adult-onset schizophrenia.Am J Psychiatry158:1299–1304
Meltzer HY,Matsubara S,Lee JC(1989)Classi?cation of typical and atypical antipsychotic drugs on the basis of dopamine D-1,D-2and setotonin2pK i values.J Pharmacol Exp Ther 251:238–246
Minon CC,Andreasen NC,Arndt S,Swayze VZ2,Cohen GA(1991)MRI abnormalities in tardive dyskinesia.Psychiatry Res40:157–166
Ninan I,Kulkarni SK(1999)Quinpirole,8-OH-DPATand ketanserin modulate catalepsy induced by high doses of atypical antipsychotics.Meth Find Exp Clin Parmacol21:603–608 Noh JS,Kang HJ,Kim EY,Sohn S,Chung YK,Kim SU,Gwag BJ(2000)Haloperidol-induced neuronal apoptosis:role of p38and c-Jun-NH2-terminal protein kinase.J Neurochem75: 2327–2334
Ogawa N,Tanaka K,Asanuma M,Kawai M,Masumizu T,Kohno M,Mori A(1994) Bromocriptine protects mice against6-hydroxydopamine and scavenges hydroxyl free radicals in vitro.Brain Res657:207–213
Peuskens J(1995)Risperidone in the treatment of patients with chronic schizophrenia:a multi-national,multi-centre,double-blind,parallel-group study versus haloperidol.Br J Psychiatry 166:712–726
Raff MC,Barres BA,Burne J,Coles HS,Ishizaki Y,Jacobson MD(1993)Programmed cell death and the control of cell survival:lessons from the nervous system.Science262:695–700 Segal RA,Greenberg ME(1996)Intracellular signaling pathways activated by neurotrophic factors.Annu Rev Neurosci19:463–489
Skaper SD,Floreani M,Negro A,Facci L,Giusti P(1998)Neurotrophins rescue cerebellar granule neurons from oxidative stress-mediated apoptotic death:selective involvement of phosphatidylinositol3-kinase and then mitogen-activated protein kinase pathway.J Neu-rochem70:1859–1868
Sokoloff P,Martres MP,Schwartz JC(1980)Three classes of dopamine receptor(D-2,D-3,D-4) identi?ed by binding studies with3H-apomorphine and3h-domperidone.Drug-induced movement disorders.Naunyn Schmiedebergs Arch Pharmacol315:89–102
Song F(1997)Risperidone in the treatment of schizophrenia:a meta-analysis of randomized controlled trials.J Psychopharmacol11:65–71
Stefanis L,Burke RE,Greene LA(1997)Apoptosis in neurodegenerative disorders.Curr Opin Neurol10:299–305
Sunderland T,Cohen BM(1987)Blood to brain distribution of neuroleptics.Psychiatry Res20: 299–305
Weinberger DR(1987)Implications of normal brain development for the pathogenesis of schizophrenia.Arch Gen Psychiatry44:660–669
Xu H,Qing H,Lu Wenfu,Keegan D,Richardson JS,Chlan-Fourney J,Li XM(2002)Quetiapine attenuates the immobilization stress-induced decrease of brain-derived neurotrophic factor expression in rat hippocampus.Neurosci Lett321:65–68
Yamada M,Ohnishi H,Sano S,Nakatani A,Ikeuchi T,Hatanaka H(1997)Insulin receptor substrate(IRS)-1and IRS-2are tyrosine-phosphorylated and associated with phosphatidy-linositol3-kinase in response to brain-derived neurotrophic factor in cultured cerebral cortical neurons.J Biol Chem272:30334–30339
Yan Z,Feng J,Fienberg AA,Greengard P(1999)D(2)dopamine receptors induce mitogen-activated protein kinase and cAMP response element-binding protein phosphorylation in neurons.Proc Natl Acad Sci USA96:11607–11612
Authors’address:https://www.doczj.com/doc/7c2946209.html,ai,Department of Neuropsychiatry,School of Medicine,Sapporo Medical University,S-1,W-16,Chuo-ku,Sapporo,060-8543Japan,e-mail:ukai@sapmed.ac.jp
细胞凋亡试验常用的方法（MTT法、荧光法、DNA琼脂糖凝胶电泳法与流式细胞仪检测法） （一）药物对肿瘤细胞的抑制效应的MTT法： 用培养基将肿瘤细胞调整至2 X108个/L，在96孔板中每孔加入100ul细胞悬液于37℃、5% CO2下培养过夜。 次日每孔加入不同浓度的药物100mg/L作为试验组，设加完全培养基不加药物的阴性对照，并用功能明确的药物为阳性对照和0.5%的乙醇溶剂对照，每组均设4-6个复孔（平行孔）、37℃、5% CO2继续培养。 培养至12h、24h、48h、实验终止前4-6h加入10ulMTT（5g/L），培养4-6h后，阴性对照孔中已形成明显的蓝紫色颗粒结晶时加100ul/孔SDS-HCl终止反应，于37℃存放过夜。 用酶标仪在A570波长下测吸光度值，按下式计算抑制率 抑制率（%）=（1-试验组平均吸光度值/阴性对照组平均吸光度值）x 100%。 （二）荧光法： 选用上述最佳浓度作用于肿瘤细胞，培养细胞48h后，收货细胞用PBS洗2-3次后用0.4%多聚甲醛室温下固定30min。 弃去固定液，并用PBS洗2次后，用1%Triton X-100作用4min加入适量的0.5mg/L DAPI 荧光染色60min，用PBS冲洗3次，取10ul滴片，干燥后于荧光显微镜下检测断裂的颗粒和片状荧光。 （三）DNA琼脂糖凝胶电泳法： 1、DNA提取： 用大方瓶培养肿瘤细胞，每瓶10ml，细胞浓度为3 x 108个/ml，每隔药物浓度、作用时间均设2瓶，共分3个时间段，4个药物浓度。共培养26瓶细胞。 分别于细胞中加入不同浓度的药物，于37℃、5% CO2中分别培养12h、24h、48h，收货细胞，用PBS洗2-3次。 于-20℃将细胞冷却处理10min后将细胞收集至离心管中，加1ml细胞裂解液，再加蛋白酶K，轻轻振摇使悬液混匀，成黏糊状，50℃过夜。 冷却后加入等体积的饱和酚溶液，混合后10000r/min离心10min，吸出上层水相，移至另一离心管中，再加入等体积饱和酚溶液重复抽提一次，直到无蛋白为止。 吸上清加入氯仿/异戊醇（24：1）按上述方法再抽提一次。 吸取水相层加入1/10体积的3mol/L的醋酸钠溶液，混匀。 再加入2.5倍体积冷无水乙醇，混合置-20℃处理30min后，10000r/min离心10min，沉淀部分为提供的DNA，弃去无水乙醇后用70%乙醇漂洗2次，将离心管倒扣在吸水纸上，吸干乙醇。 加入200ulTE缓冲液融解DNA，再加入25ul的RNA酶，置37℃作用30min，置4℃冰箱保存。 2、琼脂糖凝胶电泳： TBE缓冲液配制1.8%琼脂糖凝胶。在微波炉内煮沸至琼脂糖融解，待冷却至60℃时，加入溴化乙锭，使其终浓度为0.5mg/ml，混匀后灌胶。 待凝胶固定后放入含TBE电泳液的电泳槽内，使TBE电泳液盖过凝胶。 取10-15ul提取的各组DNA样品液与上样缓冲液按4：1比例混匀后点样。 60V电泳1h，用紫外透射仪观察梯形条带。
题目：秀丽线虫生殖细胞凋亡检测 实验目的: 1. 掌握检测凋亡细胞的方法 2. 学习使用荧光染料活体染色的方法和步骤 ．实验原理 1. 秀丽隐杆线虫( Caenorhabditis elegans )：是一种无毒无害、可以独立生存的 线虫。其个体小，成体仅 1.5mm 长，为雌雄同体 ( hermaphrodites )，雄性个体仅占群体的 0.2%，可自体受精或双性生殖；在20℃下平均生活史为 3.5 天，平均繁殖力为 300-350 个；但若与雄虫交配，可产生多达 1400 个以上的后代。 1976 年， Sulston 和 Horvitz 利用秀丽隐杆线虫 ( Caenorhabditis elegans ) 研究发现，其约 13%的体细胞在胚胎发育中注定死亡，使得人们认识到细胞凋亡的遗传基础。 2. 荧光染料活体染色：本实验使用吖啶橙( Acridine orange )作为染色剂，该染 料对细胞具有慢性毒性，致癌性强，由于凋亡细胞因 DNA片段化可结合更多染料，荧光显微镜下呈亮绿色，可在荧光显微镜下快速方便的检测出，适用于多数品系。
实验材料及设备 1. 实验材料： a) 各品系秀丽隐杆线虫：N2(实验组) , ced-1::gfp (方法对照组)，ced- 3(阴性对照) b) OP50 c) M9培养基 d) NGM培养基 2. 实验设备： a) 普通光学显微镜 b) 载玻片若干，盖玻片若干，铂金丝 c) 暗箱 d) 吸水纸、滴管等 e) 荧光显微镜 四．实验方法及步骤 1. 线虫接种、同步化 2. 取样：在 12 孔板培养板上，每孔吸取 900μL 预先接入少量 OP50 的 M9 培养基，每孔用铂金丝挑取培养 20~30 条成体线虫 3. 染色：向 N2与 ced-3 品系中每孔加入 250μg/mL 吖啶橙 100μL, 混匀后 置于培养箱(避光)染色 45~60min。 4. 方法对照组观察：向 ced-1::GFP 品系中加入 1 滴盐酸左旋咪唑，麻痹线
细胞凋亡实验步骤及注意事项 一、实验目的 1、掌屋凋亡细胞的形态特征 2、学会用荧光探针对细胞进行双标记来检测正常活细胞、凋亡细胞与坏死 细胞的方法 二、实验原理 细胞死亡根据其性质、起源及生物学意义区分为凋亡与坏死两种不同类型。凋亡普遍存在于生命界,在生物个体与生存中起着非常重要的作用。它就是细胞在一 定生理条件下一系列顺序发生事件的组合,就是细胞遵循一定规律自己结束生命 的自主控制过程。细胞凋亡具有可鉴别的形态学与生物化学特征。 在形态上可见凋亡细胞与周围细胞脱离接触,细胞变园,细胞膜向内皱缩、胞浆浓缩、内质网扩张、细胞核固缩破裂呈团块状或新月状分布、内质网与细胞膜进一步融合将细胞分成多个完整包裹的凋亡小体,凋亡小体最后被吞噬细胞吞噬消化。在凋亡过程中细胞内容物并不释放到细胞外,不会影响其它细胞,因而不引起炎症反应。 在生物化学上,多数细胞凋亡的过程中,内源性核酸内切酶活化,活性增加。核DNA 随机地在核小体的连接部位被酶切断,降解为180-200bp或它的整倍数的各种片断。如果对核DNA进行琼脂糖电泳,可显示以180-200bp为基数的DNA ladder(梯状带纹)的特征。 相比之下,坏死就是细胞处于剧烈损伤条件下发生的细胞死亡。细胞在坏死早期 即丧失质膜完整性,各种细胞器膨胀,进而质膜崩解释放出其中的内容物,引起炎症反应,坏死过程中细胞核DNA虽也降解,但由于存在各种长度不等的DNA片断,不能形成梯状带纹,而呈弥散状。 一些温与的损伤刺激及一些抗肿瘤药物可诱导细胞凋亡,通常这些因素在诱导凋亡的同时,也可产生细胞坏死,这取决于损伤的剧烈程度与细胞本身对刺激的敏感 程度。 三尖杉酯碱(HT)就是我国自行研制的一种对急性粒细胞白血病,急性单核白血病等有良好疗效的抗肿瘤药物。研究表明HT在0、02~5μg/ml范围内作用2小时,即可诱导HL-60细胞凋亡,并表现出典型的凋亡特征。本实验用1μg/ml HT在体外诱导培养的HL-60细胞发生凋亡,同时也有少数细胞发生坏死。用 Hoechst33342与碘化丙啶(propidium iodide,PI)对细胞进行双重染色,可以区别凋亡、坏死及正常细胞。 细胞膜就是一选择性的生物膜,一般的生物染料如PI等不能穿过质膜。当细胞坏死时,质膜不完整,PI就进入细胞内部,它可嵌入到DNA或RNA中,使坏死细胞着
常用细胞凋亡检测方法（图） 转载请注明来自丁香园 发布日期：2012-02-16 13:41 文章来源：丁香通 关键词：丁香园生物专题义翘神州细胞培养点击次数：951 一、细胞凋亡的形态学检测 1、光学显微镜和倒置显微镜 ①未染色细胞：凋亡细胞的体积变小、变形，细胞膜完整但出现发泡现象，细胞凋亡晚期可见凋亡小体。贴壁细胞出现皱缩、变圆、脱落。 ②染色细胞：常用姬姆萨染色、瑞氏染色等。凋亡细胞的染色质浓缩、边缘化，核膜裂解、染色质分割成块状和凋亡小体等典型的凋亡形态。 2、荧光显微镜和共聚焦激光扫描显微镜 一般以细胞核染色质的形态学改变为指标来评判细胞凋亡的进展情况。常用的DNA 特异性染料有：HO 33342 (Hoechst 33342)，HO 33258 (Hoechst 33258), DAPI。三种种染料与DNA的结合是非嵌入式的，主要结合在DNA的A-T碱基区。紫外光激发时发射明亮的蓝色荧光。Hoechst是与DNA特异结合的活性染料，储存液用蒸馏水配成1mg/ml的浓度，使用时用PBS稀释，终浓度为10 ug/ml。DAPI为半通透性，用于常规固定细胞的染色。储存液用蒸馏水配成1mg/ml的浓度，使用终浓度一般为10 ug/ml。结果评判：细胞凋亡过程中细胞核染色质的形态学改变分为三期：Ⅰ期的细胞核呈波纹状（rippled）或呈折缝样（creased），部分染色质出现浓缩状态；Ⅱa期细胞核的染色质高度凝聚、边缘化；Ⅱb期的细胞核裂解为碎块，产生凋亡小体(图1)。 3、透射电子显微镜观察 结果评判：凋亡细胞体积变小，细胞质浓缩。凋亡Ⅰ期（pro-apoptosis nuclei）的细胞核内染色质高度盘绕，出现许多称为气穴现象（cavitations）的空泡结构(图2)；Ⅱa期细胞核的染色质高度凝聚、边缘化；细胞凋亡的晚期，细胞核裂解为碎块，产生凋亡小体。 二、磷脂酰丝氨酸外翻分析（Annexin V法） 磷脂酰丝氨酸（Phosphatidylserine, PS）正常位于细胞膜的内侧，但在细胞凋亡的早期，PS可从细胞膜的内侧翻转到细胞膜的表面，暴露在细胞外环境中(图3)。Annexin-V是一种分子量为35~36KD的Ca2+依赖性磷脂结合蛋白，能与PS高亲和力特异性结合。将Annexin-V进行荧光素（FITC、PE）或biotin标记，以标记了的Annexin-V作为荧光探针，利用流式细胞仪或荧光显微镜可检测细胞凋亡的发生。 碘化丙啶(propidine iodide, PI)是一种核酸染料，它不能透过完整的细胞膜，但在凋亡中晚期的细胞和死细胞，PI能够透过细胞膜而使细核红染。因此将Annexin-V 与PI匹配使用，就可以将凋亡早晚期的细胞以及死细胞区分开来。 方法
细胞凋亡检测方法 一、细胞凋亡的形态学检测 1 光学显微镜和倒置显微镜 （1）未染色细胞：凋亡细胞的体积变小、变形，全面皱缩，细胞膜完整但出现发泡现象，细胞凋亡晚期可见凋亡小体，凋亡小体为数个圆形小体围绕在细胞周围。贴壁细胞出现皱缩、变圆、脱落。 （2）染色细胞： 姬姆萨（Giemsa）染色、瑞氏染色等：正常细胞核色泽均一；凋亡细胞染色质浓缩、边缘化，核膜裂解、染色质分割成块状和凋亡小体等典型的凋亡形态；坏死细胞染色浅或没染上颜色。 苏木素-伊红（HE）染色：细胞核固缩碎裂、呈蓝黑色、胞浆呈淡红色（凋亡细胞），正常细胞核呈均匀淡蓝色或蓝色，坏死细胞核呈很淡的蓝色或蓝色消失。 2 荧光显微镜和共聚焦激光扫描显微镜 一般以细胞核染色质的形态学改变为指标来评判细胞凋亡的进展情况。 常用的DNA特异性染料有：Hoechst 33342，Hoechst 33258，DAPI。三种染料与DNA 的结合是非嵌入式的，主要结合在DNA的A-T碱基区。紫外光激发时发射明亮的蓝色荧光。 Hoechst是与DNA特异结合的活性染料，能进入正常细胞膜而对细胞没有太大细胞毒作用。Hoechst 33342在凋亡细胞中的荧光强度要比正常细胞中要高。 DAPI为半通透性，用于常规固定细胞的染色。 PI和Hoechst33342双标：PI、Hoechst33342均可与细胞核DNA（或RNA）结合。但PI不能通过正常细胞膜，Hoechst则为膜通透性荧光染料，故细胞在处于坏死或晚期调
亡时细胞膜被破坏，这时可为PI着红色。正常细胞和中早期调亡细胞均可被Hoechst着色，但是正常细胞核的Hoechst着色的形态呈圆形，淡兰色，内有较深的兰色颗粒；而调亡细胞的核由于浓集而呈亮兰色，或核呈分叶，碎片状，边集。故PI着色为坏死细胞；亮兰色，或核呈分叶状，边集的Hoechst着色的为调亡细胞。 凋亡细胞体积变小，细胞质浓缩。细胞凋亡过程中细胞核染色质的形态学改变分为三期：Ⅰ期的细胞核呈波纹状（rippled）或呈折缝样（creased），部分染色质出现浓缩状态；Ⅱa期细胞核的染色质高度凝聚、边缘化；Ⅱb期的细胞核裂解为碎块，产生凋亡小体(图1)。 3 透射电子显微镜观察 凋亡细胞体积变小，细胞质浓缩。凋亡Ⅰ期（pro-apoptosis nuclei）的细胞核内染色质高度盘绕，出现许多称为气穴现象（cavitations）的空泡结构(图2)；Ⅱa期细胞核的染色质高度凝聚、边缘化；细胞凋亡的晚期，细胞核裂解为碎块，产生凋亡小体。 二、磷脂酰丝氨酸外翻分析（Annexin V法） 磷脂酰丝氨酸（Phosphatidylserine, PS）正常位于细胞膜内侧，但在细胞凋亡早期，PS可从细胞膜内侧翻转到细胞膜表面，暴露在细胞外环境中。磷脂酰丝氨酸的转位发生在凋亡早期阶段，先于细胞核的改变、DNA断裂、细胞膜起泡。体内的吞噬细胞可通过识别
细胞凋亡主要发生机制及相关作用研究 摘要 细胞凋亡是一种有序的或程序性的细胞死亡方式，是细胞接受某些特定信号刺激后在基因调控下所发生的一系列细胞主动死亡过程，通常来说是一种正常生理应答反应。目前认为细胞凋亡信号传导通路主要包括三种：内源性途径、外源性途径以及内质网途径。细胞凋亡的研究已成为当前生命科学研究热点之一。研究细胞凋亡的信号传导通路及其调控对进一步认识和治疗凋亡相关疾病有重要意义。 关键词：细胞凋亡信号传导通路疾病治疗
ABSTRACT Apoptosis is an orderly or programmed cell death way, is a series of cells active death process under gene regulation that after cell accepted certain specific signal stimulation, it is a normal physiological response. At presently, the cell apoptosis signaling pathways mainly includes three types: intrinsic pathway, extrinsic pathway, and the way of endoplasmic reticulum. The research of apoptosis has become the life science research hotspot. Researching cell apoptosis signaling pathways and regulation can get further understanding and also have the important meaning to treatment of apoptosis related diseases. Key words: A poptosis Signal conduct pathway Treatment of diseases
实验14 细胞凋亡的诱导和检测 20世纪60年代人们注意到细胞存在着两种不同形式的死亡方式：凋亡（apoptosis）和坏死（necrosis）。细胞坏死指病理情况下细胞的意外死亡，坏死过程细胞膜通透性增高，细胞肿胀，核碎裂，继而溶酶体、细胞膜破坏，细胞容物溢出，细胞坏死常引起炎症反应。 细胞凋亡apoptosis一词来源于古希腊语，意思是花瓣或树叶凋落，意味着生命走到了尽头，细胞到了一定时期会像树叶那样自然死亡。凋亡是细胞在一定生理或病理条件下遵守自身程序的主动死亡过程。凋亡时细胞皱缩，表面微绒毛消失，染色质凝集并呈新月形或块状靠近核膜边缘，继而核裂解，由细胞膜包裹着核碎片或其他细胞器形成小球状凋亡小体凸出于细胞表面，最后凋亡小体脱落被吞噬细胞或邻周细胞吞噬。凋亡过程中溶酶体及细胞膜保持完整，不引起炎症反应。细胞凋亡时的生化变化特征是核酸切酶被激活，染色体DNA被降解，断裂为50～300 kb长的DNA片段，再进一步断裂成180～200bp整倍数的寡核苷酸片断，在琼脂糖凝胶电泳上呈现“梯状”电泳图谱(DNA Ladder)。细胞凋亡在个体正常发育、紫稳态维持、免疫耐受形成、肿瘤监控和抵御各种外界因素干扰等方面都起着关键性的作用。 1．细胞凋亡的检测方法 凋亡细胞具有一些列不同于坏死细胞的形态特征和生化特征，据此可以鉴别细胞的死亡形式。细胞凋亡的机制十分复杂，一般采用多种方法综合加以判断，同时不同类型细胞的凋亡分析方法有所不同，方法选择依赖于具体的研究体系和研究目的（表？）。
形态学观察方法：利用各种染色法可观察到凋亡细胞的各种形态学特征： （1）DAPI时常用的一种与DNA结合的荧光染料。借助于DAPI染色，可以观察细胞核的形态变化。 （2）Giemsa染色法可以观察到染色质固缩、趋边、凋亡小体形成等形态。 （3）吖啶橙(AO)染色，荧光显微镜观察，活细胞核呈黄绿色荧光，胞质呈红色荧光。凋亡细胞核染色质呈黄绿色浓聚在核膜侧，可见细胞膜呈泡状膨出及凋亡小体。 （4）吖啶橙(A())／溴化乙啶(EB)复染可以更可靠地确定凋亡细胞的变化，AO只进入活细胞，正常细胞及处于凋亡早期的细胞核呈现绿色；EB只进入死细胞，将死细胞及凋亡晚期的细胞的核染成橙红色。 （5）台盼蓝染色对反映细胞膜的完整性，区别坏死细胞有一定的帮助，如果细胞膜不完整、破裂，台盼蓝染料进入细胞，细胞变蓝，即为坏死。如果细胞膜完整，细胞不为台盼蓝染色，则为正常细胞或凋亡细胞。使用透射电镜观察，可见凋亡细胞表面微绒毛消失，核染色质固缩、边集，常呈新月形，核膜皱褶，胞质紧实，细胞器集中，胞膜起泡或出“芽”及凋亡小体和凋亡小体被临近巨噬细胞吞噬现象。 （6）木精-伊红（HE）染色是经典的显示细胞核、细胞质的染色方法，染色结果清晰。发生凋亡的细胞经HE染色后，其细胞大小的变化及特征性细胞核的变化：染色质凝集、呈新月形或块状靠近核膜边缘，晚期核裂解、细胞膜包裹着核碎片“出芽”凸出于细胞表面形成凋亡小体等均可明显显示出来。 DNA凝胶电泳：细胞发生凋亡或坏死，其细胞DNA均发生断裂，细胞小分子 质量DNA片段增加，高分子DNA减少，胞质出现DNA片段。但凋亡细胞DNA断裂点均有规律的发生在核小体之间，出现180～200 bp DNA片段，而坏死细胞的DNA断裂点为无特征的杂乱片段，利用此特征可以确定群体细胞的死亡，并可与坏死细胞区别。
细胞凋亡的检测 细胞凋亡与坏死是两种完全不同的细胞凋亡形式，根据死亡细胞在形态学、生物化学和分子生物学上的差别，可以将二者区别开来。细胞凋亡的检测方法有很多，下面介绍几种常用的测定方法。 一、细胞凋亡的形态学检测 根据凋亡细胞固有的形态特征，人们已经设计了许多不同的细胞凋亡形态学检测方法。 1 光学显微镜和倒置显微镜 （1）未染色细胞：凋亡细胞的体积变小、变形，细胞膜完整但出现发泡现象，细胞凋亡晚期可见凋亡小体。 贴壁细胞出现皱缩、变圆、脱落。 （2）染色细胞：常用姬姆萨染色、瑞氏染色等。凋亡细胞的染色质浓缩、边缘化，核膜裂解、染色质分割 成块状和凋亡小体等典型的凋亡形态。 2 荧光显微镜和共聚焦激光扫描显微镜 一般以细胞核染色质的形态学改变为指标来评判细胞凋亡的进展情况。 常用的DNA特异性染料有：HO 33342 (Hoechst 33342)，HO 33258 (Hoechst 33258), DAPI。三种染料与DNA的结合是非嵌入式的，主要结合在DNA的A-T碱基区。紫外光激发时发射明亮的蓝色荧光。 Hoechst是与DNA特异结合的活性染料，储存液用蒸馏水配成1mg/ml的浓度，使用时用PBS稀释成终浓度为2~5mg/ml。 DAPI为半通透性，用于常规固定细胞的染色。储存液用蒸馏水配成1mg/ml的浓度，使用终浓度一般为0.5 ~1mg/ml。 结果评判：细胞凋亡过程中细胞核染色质的形态学改变分为三期：Ⅰ期的细胞核呈波纹状（rippled）或呈折缝样（creased），部分染色质出现浓缩状态；Ⅱa期细胞核的染色质高度凝聚、边缘化；Ⅱb期的细胞核裂解为碎块，产生凋亡小体(图1)。
细胞凋亡机制的研究及其意义 摘要: 细胞凋亡是维持神经系统正常发育, 维持其免疫系统正常功能所必需过程。目前, 对细胞凋亡的研究已经成为生命科学领域研究的热点。本文就细胞凋亡的发生机制、基因调节机制等方面作一综述。 关键词: 细胞凋亡; 机制；意义 引言：细胞凋亡对机体的健康发育甚为重要，在生理条件下，它作为机体正常细胞群生长与死亡相协调的重要方式，有利于清除多余的细胞、无用细胞、发育不正常细胞、有害细胞、完成正常使命的衰老细胞；有利于维持机体细胞群的自身稳定，从而维持器官组织的正常发育。细胞凋亡过少时，机体易患肿瘤性疾病、自身免疫性疾病；细胞凋亡过多时，机体易患神经系统方面的疾病。人的艾滋病等疾病之所以发生，主要是由于机体细胞凋亡发生异常的结果。 正文： 1、细胞凋亡机制 1.1 信号传递机制 凋亡一般由细胞外的调节因素与其在细胞表面的受体结合而启动。经活化的受体又启动胞内第二信号系统，激活核酸内切酶，引起DNA裂解，进而引发细胞凋亡。细胞外的调节因素包括生理活性因子：如肿瘤坏死因子、转化生长因子及表皮生长因子等；非生理因素：如X射线、紫外线、一氧化氮、毒素及化疗药物等；感染因素：如EB病毒、腺病毒及HIV病毒等。有学者认为，细胞凋亡的信号传导能使用或部分利用细胞增殖和分化过程中的传统信号途径。传统信号途径包括G 结合蛋白信号途径和酶蛋白信号途径，前者可以调节第二信使cAMP和钙离子的生成，细胞内cAMP和钙离子浓度的变化可以对细胞凋亡产生影响；后者可通过酪氨酸蛋白激酶（PTK）、Ras-MAPK或JaK-STAT等途径参与凋亡信号的传导。但众多研究表明可直接启动细胞凋亡的信号途径或死亡信号途径是两种死亡因子，即肿瘤坏死因子和Fas配体与细胞膜表面的相应受体TNF受体和37? 结合以后所发生的凋亡反应。目前对TNF和FasL与相应受体结合所介导的细胞凋亡信号途径及其机制已取得了突破性进展 1.2 酶学机制 1.2.1 caspases蛋白酶 胱冬蛋白酶（caspases）是近几年研究的热点之一，属于ICE/CED3蛋白酶家族成员，目前发现至少有14种之多,分别命名为caspases1-caspases14。与细胞凋亡密切相关，它是通过级联反应，最终激活核酸内切酶来实现的。也有人认为凋亡并不总是引起caspases的释放，而caspases的释放也并不总是引起凋亡，很可能还与细胞的迁移和分化有关.。蛋白酶前体可在天冬氨酸位点上被切断成3部分，H2N端是抑制区域被移去，另一端COOH端断裂成一大一小亚单位
细胞凋亡的几种检测方法 1、形态学观察方法 （1）HE（苏木精—伊红染色法）染色、光镜观察：凋亡细胞呈圆形，胞核深染，胞质浓缩，染色质成团块状，细胞表面有“出芽”现象。 （2）丫啶橙（AO)染色，荧光显微镜观察：活细胞核呈黄绿色荧光，胞质呈红色荧光。凋亡细胞核染色质呈黄绿色浓聚在核膜内侧，可见细胞膜呈泡状膨出及凋亡小体。 （3）台盼蓝染色：如果细胞膜不完整、破裂，台盼蓝染料进入细胞，细胞变蓝，即为坏死。如果细胞膜完整，细胞不为台盼蓝染色，则为正常细胞或凋亡细胞。此方法对反映细胞膜的完整性，区别坏死细胞有一定的帮助。 （4）透射电镜观察：可见凋亡细胞表面微绒毛消失，核染色质固缩、边集，常呈新月形，核膜皱褶，胞质紧实，细胞器集中，胞膜起泡或出“芽”及凋亡小体和凋亡小体被临近巨噬细胞吞噬现象。 2、DNA凝胶电泳 细胞发生凋亡或坏死，其细胞DNA均发生断裂，细胞内小分子量DNA片断增加，高分子DNA减少，胞质内出现DNA片断。但凋亡细胞DNA断裂点均有规律的发
生在核小体之间，出现180－200bpDNA片断，而坏死细胞的DNA断裂点为无特征的杂乱片断，利用此特征可以确定群体细胞的死亡，并可与坏死细胞区别。正常活细胞DNA 电泳出现阶梯状（LADDER)条带；坏死细胞DNA电泳类似血抹片时的连续性条带 3、酶联免疫吸附法（ELISA)核小体测定 凋亡细胞的DNA断裂使细胞质内出现核小体。核小体由组蛋白及其伴随的DNA片断组成，可由ELISA法检测。 检测步骤 1、将凋亡细胞裂解后高速离心，其上清液中含有核小体； 2、在微定量板上吸附组蛋白体’ 3、加上清夜使抗组蛋白抗体与核小体上的组蛋白结合‘ 4、加辣过氧化物酶标记的抗DNA抗体使之与核小体上的DNA结合’ 4、加酶的底物，测光吸收制。 用途 该法敏感性高，可检测5*100/ml个凋亡细胞。可用于人、大鼠、小鼠的凋亡检测。该法不需要特殊仪器，
细胞在发生凋亡时，会激活一些DNA内切酶，这些内切酶会切断核小体间的基因组DNA。基因组DNA 断裂时，暴露的3'-0H 可以在末端脱氧核苷酸转移酶（Terminal Deox yn ucleotidyl Tran sferase,TdT）的催化下加上荧光素（FITC）标记的dUTP（fluorescein-dUTP），从而可以通过荧光显微镜或流式细胞仪进行检测，这就是TUNEL 法检测细胞凋亡的原理。 TUNEL法特异性检测细胞凋亡时产生的DNA断裂，但不会检测出射线等诱导的DNA断裂（和细胞凋亡时的断裂方式不同）。这样一方面可以把凋亡和坏死区分开，另一方面也不会把 射线等诱导发生DNA断裂的非凋亡细胞判断为凋亡细胞。 针对问题2（TUNEL法的实验原理是什么？）： 基本原理：对不同组织切片先增加细胞膜通透性，然后让rTDT和bio标记的dUTP进入细 胞内，在rTDT的辅助下dUTP与核断裂的DNA 3 -0H结合，再用HRP标记的链霉亲和素与dUTP 上的biot in 结合（每个链霉亲和素至少可以再结合3个biot in 分子），最后用DAB 过氧化氢与SP上的辣根过氧化物酶HRP发生氧化、环化反应，形成苯乙肼聚合物而呈现棕褐色，最终通过计数每张切片上不同视野中TUNEL阳性细胞的比例来判断细胞凋亡发生情 况。■ 1. TUNEL工作原理：简单说就是一一TUNEL细胞凋亡检测试剂盒是用来检测细胞在凋亡过程中细胞核DNA的断裂情况。 其原理是；生物素（biot in ）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdT En zyme）的 作用下，可以连接到凋亡细胞中断裂的DNA的3' - 0H末端，并可与连接了的辣根过氧化酶的 链霉亲和素（Streptavidin-HRP ）特异结合，在辣根过氧化酶底物二氨基联苯胺（DAB的存在下，产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在普通 显微镜下即可观察和计数凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA的断裂，因而没有3'-0H形成，很少能够被染色。 针对问题3 （TUNEL实验中几个关键步骤是什么？）： 1. 充分脱蜡和水化。脱蜡可以先60度20min，再用二甲苯两次5~10min ;而水化用梯度乙 醇从高浓度到低浓度浸洗，这些以便后面的结合反应充分、均匀； 2. 把握好细胞通透的时间。一般根据切片的厚薄，选择蛋白酶k的孵育时间，常用10~30min, 几um切片用短时间；几十um切片用长时间，通过摸索达到既不脱片，有能够使后面的酶和 抗体进入胞内。 3. 适当延长TUNEL反应液的时间。一般是37度1h,你也可以根据你的凋亡损伤程度，选择更长的时间，可长至2h,但要结合你最终的背景着色。 4. DAB显色条件的选择。一般DAB反应10分钟左右，结合镜下控制背景颜色，最长不超过 30min;我不喜欢用promega公司提供的DAB液（桃红色），不利于辨认棕褐色。 5. PBS的充分清洗。我个人认为，在TUNEL反应后和酶标反应后的清洗应十分严格，可增加 次数达5次，因为这些清洗直接决定最后切片的非特异性着色。 6. 此外，内源性POD的封闭也十分关键。对于肝脏、肾脏等血细胞含量多的组织，我的经 验是适当延长封闭时间和升高过氧化氢的浓度，可以达到很好的封闭效果，且不影响最终的 特异性染色。 针对问题5.细胞通透的原理、通透剂的浓度、孵育时间及其配制方法？ 1. 蛋白酶K是消化膜蛋白，从而起打孔作用，增加
(完整)常见细胞凋亡检测的方法与注意事项 编辑整理： 尊敬的读者朋友们： 这里是精品文档编辑中心，本文档内容是由我和我的同事精心编辑整理后发布的，发布之前我们对文中内容进行仔细校对，但是难免会有疏漏的地方，但是任然希望（(完整)常见细胞凋亡检测的方法与注意事项）的内容能够给您的工作和学习带来便利。同时也真诚的希望收到您的建议和反馈，这将是我们进步的源泉，前进的动力。 本文可编辑可修改，如果觉得对您有帮助请收藏以便随时查阅，最后祝您生活愉快业绩进步，以下为(完整)常见细胞凋亡检测的方法与注意事项的全部内容。
常见细胞凋亡检测的方法与注意事项 大家常把细胞凋亡和细胞坏死混淆,其实两者是不同的细胞死亡形式，大家可以在死亡细胞的形态、生化和分子指标上将二者区分开来，细胞凋亡检测的方法不少，这里就总结下几种常用的检测方法. 细胞凋亡检测更多详情，点击查看不可不知的细胞检测方法——MTT 一、细胞凋亡的形态学检测 根据凋亡细胞固有的形态特征，人们已经设计了许多不同的细胞凋亡形态学检测方法。 1 光学显微镜和倒置显微镜 （1) 未染色细胞：凋亡细胞的体积变小、变形，细胞膜完整但出现发泡现象，细胞凋亡晚期可见凋亡小体。 贴壁细胞出现皱缩、变圆、脱落. (2）染色细胞：常用姬姆萨染色、瑞氏染色等.凋亡细胞的染色质浓缩、边缘化，核膜裂解、染色质分割 成块状和凋亡小体等典型的凋亡形态。 2 荧光显微镜和共聚焦激光扫描显微镜 一般以细胞核染色质的形态学改变为指标来评判细胞凋亡的进展情况。 常用的DNA特异性染料有:HO 33342 (Hoechst 33342），HO 33258 (Hoechst 33258), DAPI。三种染料与DNA的结合是非嵌入式的，主要结合在DNA的A-T碱基区。紫外光激发时发射明亮的蓝色荧光。 Hoechst是与DNA特异结合的活性染料，储存液用蒸馏水配成1mg/ml的浓度，使用时用PBS稀释成终浓度为2~5mg/ml。 DAPI为半通透性，用于常规固定细胞的染色。储存液用蒸馏水配成1mg/ml的浓度，使用终浓度一般为0.5 ～1mg/ml。 结果评判：细胞凋亡过程中细胞核染色质的形态学改变分为三期：Ⅰ期的细胞核呈波纹状（rippled）或呈折缝样(creased)，部分染色质出现浓缩状态；Ⅱa期细胞核的染色质高度凝聚、边缘化;Ⅱb期的细胞核裂解为碎块,产生凋亡小体(图1）。 3 透射电子显微镜观察 结果评判：凋亡细胞体积变小,细胞质浓缩。凋亡Ⅰ期（pro—apoptosis nuclei)的细胞核内染色质高度盘绕，出现许多称为气穴现象（cavitations)的空泡结构(图2）；Ⅱa期细胞核的染色质高度凝聚、边缘化;细胞凋亡的晚期，细胞核裂解为碎块,产生凋亡小体。 图2
题目：秀丽线虫生殖细胞凋亡检测 一．实验目的: 1.掌握检测凋亡细胞的方法 2.学习使用荧光染料活体染色的方法和步骤 二．实验原理 1.秀丽隐杆线虫（Caenorhabditis elegans）：是一种无毒无害、可 以独立生存的线虫。其个体小，成体仅 1.5mm长，为雌雄同体（hermaphrodites），雄性个体仅占群体的0.2%，可自体受精或双性生殖；在20℃下平均生活史为3.5天，平均繁殖力为300-350个；但若与雄虫交配，可产生多达1400个以上的后代。1976年，Sulston和Horvitz利用秀丽隐杆线虫（Caenorhabditis elegans）研究发现，其约13%的体细胞在胚胎发育中注定死亡，使得人们认识到细胞凋亡的遗传基础。 2.荧光染料活体染色：本实验使用吖啶橙（Acridine orange）作为 染色剂，该染料对细胞具有慢性毒性，致癌性强，由于凋亡细胞因DNA片段化可结合更多染料，荧光显微镜下呈亮绿色，可在荧光显微镜下快速方便的检测出，适用于多数品系。 三．实验材料及设备
1.实验材料： a)各品系秀丽隐杆线虫：N2（实验组）, ced-1::gfp（方法对照组），ced- 3（阴性对照） b)OP50 c)M9培养基 d)NGM培养基 2.实验设备： a)普通光学显微镜 b)载玻片若干，盖玻片若干，铂金丝 c)暗箱 d)吸水纸、滴管等 e)荧光显微镜 四．实验方法及步骤 1.线虫接种、同步化 2.取样：在12孔板培养板上，每孔吸取900μL预先接入少量OP50 的M9培养基，每孔用铂金丝挑取培养20~30条成体线虫 3.染色：向N2与ced-3品系中每孔加入250μg/mL吖啶橙100μL, 混匀后置于培养箱（避光）染色45~60min。 4.方法对照组观察：向ced-1::GFP品系中加入1滴盐酸左旋咪唑， 麻痹线虫后在荧光显微镜下观察。
细胞凋亡检测，细胞凋亡实验步骤，检测方法 一、定性和定量研究 只定性的研究方法：常规琼脂糖凝胶电泳、脉冲场倒转琼脂糖凝胶电泳、形态学观察（普通光学显微镜、透射电镜、荧光显微镜） 进行定量或半定量的研究方法：各种流式细胞仪方法、原位末端标记法、ELISA 定量琼脂糖凝胶电泳。 二、区分凋亡和坏死 可将二者区分开的方法：琼脂糖凝胶电泳，形态学观察（透射电镜是是区分凋亡和坏死最可靠的方法），Hoechst33342/PI双染色法流式细胞仪检测，AnnexinV/PI双染色法流式细胞仪检测等。 不能将二者区分开的方法：原位末端标记法、PI单染色法流式细胞仪检测等。 三、样品来源不同选择 组织：主要用形态学方法(HE染色，透射电镜、石蜡包埋组织切片进行原位末端标记，ELISA或将组织碾碎消化做琼脂糖凝胶电泳)。 四、细胞凋亡检测 1、早期检测: 1) PS(磷脂酰丝氨酸)在细胞外膜上的检测 2)细胞内氧化还原状态改变的检测 3)细胞色素C的定位检测 4) 线粒体膜电位变化的检测 2、晚期检测： 细胞凋亡晚期中，核酸内切酶在核小体之间剪切核DNA，产生大量长度在 180-200 bp 的DNA片段。 对于晚期检测通常有以下方法： 1) TUNEL(末端脱氧核苷酸转移酶介导的dUTP缺口末端标记) 2) LM-PCR Ladder (连接介导的PCR检测) 3) T elemerase Detection (端粒酶检测) 3、生化检测： 1）典型的生化特征：DNA 片段化 2）检测方法主要有：琼脂糖凝胶电泳、原位末端标记（TUNEL）等 3）TUNEL（末端脱氧核苷酸转移酶介导的dUTP缺口末端标记） 4）通过DNA末端转移酶将带标记的dNTP （多为dUTP）间接或直接接到DNA 片段的3’-OH端，再通过酶联显色或荧光检测定量分析结果。可做细胞悬液、福尔马林固定或石蜡处理的组织、细胞培养物等多种样本的检测。 4、LM-PCR Ladder (连接介导的PCR检测) 当凋亡细胞比例较小以及检测样品量很少（如活体组织切片）时，直接琼脂糖电泳可能观察不到核DNA的变化。通过LM-PCR,连上特异性接头，专一性地扩增梯度片段，从而灵敏地检测凋亡时产生梯度片段。此外，LM-PCR 检测是半定量的，因此相同凋亡程度的不同样品可进行比较。如果细胞量很少，还可在分离提纯DNA后，用32P-ATP和脱氧核糖核苷酸末端转移酶（TdT）使DNA标记，
细胞凋亡的几种检测方 法 Company number：【WTUT-WT88Y-W8BBGB-BWYTT-19998】
细胞凋亡的几种检测方法 1、形态学观察方法 （1）HE（苏木精—伊红染色法）染色、光镜观察：凋亡细胞呈圆形，胞核深染，胞质浓缩，染色质成团块状，细胞表面有“出芽”现象。 （2）丫啶橙（AO)染色，荧光显微镜观察：活细胞核呈黄绿色荧光，胞质呈红色荧光。凋亡细胞核染色质呈黄绿色浓聚在核膜内侧，可见细胞膜呈泡状膨出及凋亡小体。 （3）台盼蓝染色：如果细胞膜不完整、破裂，台盼蓝染料进入细胞，细胞变蓝，即为坏死。如果细胞膜完整，细胞不为台盼蓝染色，则为正常细胞或凋亡细胞。此方法对反映细胞膜的完整性，区别坏死细胞有一定的帮助。 （4）透射电镜观察：可见凋亡细胞表面微绒毛消失，核染色质固缩、边集，常呈新月形，核膜皱褶，胞质紧实，细胞器集中，胞膜起泡或出“芽”及凋亡小体和凋亡小体被临近巨噬细胞吞噬现象。 2、 DNA凝胶电泳 细胞发生凋亡或坏死，其细胞DNA均发生断裂，细胞内小分子量DNA片断增加，高分子DNA减少，胞质内出现DNA片断。但凋亡细胞DNA断裂点均有规
律的发生在核小体之间，出现180－200bpDNA片断，而坏死细胞的DNA断裂点为无特征的杂乱片断，利用此特征可以确定群体细胞的死亡，并可与坏死细胞区别。 正常活细胞DNA 电泳出现阶梯状（LADDER)条带；坏死细胞DNA电泳类似血抹片时的连续性条带 3、酶联免疫吸附法（ELISA)核小体测定 凋亡细胞的DNA断裂使细胞质内出现核小体。核小体由组蛋白及其伴随的DNA片断组成，可由ELISA法检测。 检测步骤 1、将凋亡细胞裂解后高速离心，其上清液中含有核小体； 2、在微定量板上吸附组蛋白体’ 3、加上清夜使抗组蛋白抗体与核小体上的组蛋白结合‘ 4、加辣过氧化物酶标记的抗DNA抗体使之与核小体上的DNA结合’ 4、加酶的底物，测光吸收制。 用途 该法敏感性高，可检测5*100/ml个凋亡细胞。可用于人、大鼠、小鼠的凋亡检测。该法不需要特殊仪器，
生命科学学院专业生物技术 2016级生技班612组 姓名同实验者 2018年 4 月 23日 题目：秀丽线虫生殖细胞凋亡检测 一．实验目的: 1.掌握检测凋亡细胞的方法 2.学习使用荧光染料活体染色的方法和步骤 二．实验原理 1.秀丽隐杆线虫（Caenorhabditis elegans）：是一种无毒无害、可 以独立生存的线虫。其个体小，成体仅 1.5mm长，为雌雄同体（hermaphrodites），雄性个体仅占群体的0.2%，可自体受精或双性生殖；在20℃下平均生活史为3.5天，平均繁殖力为300-350个；但若与雄虫交配，可产生多达1400个以上的后代。1976年，Sulston和Horvitz利用秀丽隐杆线虫（Caenorhabditis elegans）研究发现，其约13%的体细胞在胚胎发育中注定死亡，使得人们认识到细胞凋亡的遗传基础。 2.荧光染料活体染色：本实验使用吖啶橙（Acridine orange）作为 染色剂，该染料对细胞具有慢性毒性，致癌性强，由于凋亡细胞因DNA片段化可结合更多染料，荧光显微镜下呈亮绿色，可在荧光显微镜下快速方便的检测出，适用于多数品系。
生命科学学院专业生物技术 2016级生技班612组 姓名同实验者 2018年 4 月 23日 三．实验材料及设备 1.实验材料： a)各品系秀丽隐杆线虫：N2（实验组）, ced-1::gfp（方法对照组），ced- 3（阴性对照） b)OP50 c)M9培养基 d)NGM培养基 2.实验设备： a)普通光学显微镜 b)载玻片若干，盖玻片若干，铂金丝 c)暗箱 d)吸水纸、滴管等 e)荧光显微镜 四．实验方法及步骤 1.线虫接种、同步化 2.取样：在12孔板培养板上，每孔吸取900μL预先接入少量OP50 的M9培养基，每孔用铂金丝挑取培养20~30条成体线虫 3.染色：向N2与ced-3品系中每孔加入250μg/mL吖啶橙100μL, 混匀后置于培养箱（避光）染色45~60min。
流式检测细胞凋亡 Annexin V 检测细胞凋亡 (2) 实验原理 (2) 实验用品 (2) 操作步骤 (3) Annexin V Blocking (5) 凋亡细胞的DNA 断裂片段分析 (7) 实验原理 (7) 实验用品 (8) 操作步骤 (9) BrdU Flow Kits 检测细胞增殖 (12) 实验原理 (12) BrdU Flow Kits 试剂盒 (12) 结果分析 (17) 流式仪器设置指南 (18) 线粒体膜电位变化检测细胞凋亡 (22) 实验原理 (22) 实验用品 (22) 样本制备 (23) 结果分析 (24) 注意事项 (24) Active Caspase-3 检测细胞凋亡 (26) 实验原理 (26) 实验步骤 (27) 结果分析 (28)
Annexin V 检测细胞凋亡 实验原理 Annexin V 是检测细胞凋亡的灵敏指标之一。它是一种磷脂结合蛋白，可以与早期凋亡细胞的胞膜结合，而细胞质膜的改变是细胞发生凋亡时最早的改变之一。在细胞发生凋亡时，膜磷脂酰丝氨酸(PS) 由质膜内侧翻向外侧。Annexin V 与磷脂酰丝氨酸有高度亲和力，因而与细胞外侧暴露的磷脂酰丝氨酸结合。由于在发生凋亡时，磷脂酰丝氨酸外翻的发生早于细胞核的改变，因此，与DNA 碎片检测比较，使用Annexin V 可以更早地检测到凋亡细胞。因为细胞坏死时也会发生磷脂酰丝氨酸外翻，所以Annexin V 常与鉴定细胞死活的核酸染料(如PI 或7-AAD)合并使用，来区分凋亡细胞(Annexin V+/核酸染料-) 与死亡细胞(Annexin V+/核酸染料+)。 实验用品 1. 一次性12×75mm Falcon试管。 2. PBS缓冲液：含0.1%NaN ，过滤后2-8°C保存。 3 3. 微量加样器和加样头。
细胞凋亡与坏死是两种完全不同的细胞凋亡形式，根据死亡细胞在形态学、生物化学和分子生物学上的差别，可以将二者区别开来。细胞凋亡的检测方法有很多，下面介绍几种常用的测定方法。 一、细胞凋亡的形态学检测 根据凋亡细胞固有的形态特征，人们已经设计了许多不同的细胞凋亡形态学检测方法。 1 光学显微镜和倒置显微镜 （1）未染色细胞：凋亡细胞的体积变小、变形，细胞膜完整但出现发泡现象，细胞凋亡晚期可见凋亡小体。 贴壁细胞出现皱缩、变圆、脱落。 （2）染色细胞：常用姬姆萨染色、瑞氏染色等。凋亡细胞的染色质浓缩、边缘化，核膜裂解、染色质分割 成块状和凋亡小体等典型的凋亡形态。 2 荧光显微镜和共聚焦激光扫描显微镜 一般以细胞核染色质的形态学改变为指标来评判细胞凋亡的进展情况。 常用的DNA特异性染料有：HO 33342 (Hoechst 33342)，HO 33258 (Hoechst 33258), DAPI。三种染料与DNA的结合是非嵌入式的，主要结合在DNA的A-T碱基区。紫外光激发时发射明亮的蓝色荧光。 Hoechst是与DNA特异结合的活性染料，储存液用蒸馏水配成1mg/ml的浓度，使用时用PBS稀释成终浓度为2~5mg/ml。 DAPI为半通透性，用于常规固定细胞的染色。储存液用蒸馏水配成1mg/ml的浓度，使用终浓度一般为0.5 ~1mg/ml。 结果评判：细胞凋亡过程中细胞核染色质的形态学改变分为三期：Ⅰ期的细胞核呈波纹状（rippled）或呈折缝样（creased），部分染色质出现浓缩状态；Ⅱa期细胞核的染色质高度凝聚、边缘化；Ⅱb期的细胞核裂解为碎块，产生凋亡小体(图1)。 3 透射电子显微镜观察 结果评判：凋亡细胞体积变小，细胞质浓缩。凋亡Ⅰ期（pro-apoptosis nuclei）的细胞核内染色质高度盘绕，出现许多称为气穴现象（cavitations）的空泡结构(图2)；Ⅱa期细胞核的染色质高度凝聚、边缘化；细胞凋亡的晚期，细胞核裂解为碎块，产生凋亡小体 二、磷脂酰丝氨酸外翻分析（Annexin V法） 磷脂酰丝氨酸（Phosphatidylserine, PS）正常位于细胞膜的内侧，但在细胞凋亡的早期，PS可从细胞膜的内侧翻转到细胞膜的表面，暴露在细胞外环境中(图3)。Annexin-V是一种分子量为35~36KD的Ca2+依赖性磷脂结合蛋白，能与PS高亲和力特异性结合。将Annexin-V 进行荧光素（FITC、PE）或biotin标记，以标记了的Annexin-V作为荧光探针，利用流式细胞仪或荧光显微镜可检测细胞凋亡的发生。 碘化丙啶(propidine iodide, PI)是一种核酸染料，它不能透过完整的细胞膜，但在凋亡中晚期的细胞和死细胞，PI能够透过细胞膜而使细胞核红染。因此将Annexin-V与PI匹配使用，就可以将凋亡早晚期的细胞以及死细胞区分开来。