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Objective: To investigate the role of aldehyde dehydrogenase-2 (ALDH-2) for regulating human endothelial progenitor cells (EPCs) oxidative stress reaction and its mechanism. Methods: Human EPCs were isolated from peripheral blood of healthy adults and the cells were cultured in 4 groups:①Blank control group,②Alda-1 group, the cells were treated by 1μmol/L Alda-1, a speciifc activator of ALDH-2,③tBHP (10μg/ml) group and④Alda-1 pretreatment+tBHP group. EPCs reactive oxygen species (ROS) levels were evaluated by DCFH-DA staining, mitochondrial membrane potentials were detected by JC-1 method, migration capacity was measured by transwell chamber method and the activation of p38 signal pathway was examined by Western blot analysis. Results: Compared with Blank control group, ROS levels in tBHP group and Alda-1 pretreatment+tBHP group were (441.7 ± 24.8) % and (237.4 ± 12.0) %, allP<0.05. In Blank control group, tBHP group and Alda-1 pretreatment+tBHP group, the proportion of EPCs lost their mitochondrial membrane potentials were (5.7 ± 2.1) %, (81.7 ± 3.7) % and (37.4 ± 3.2) % respectively, allP<0.05; the number of EPCs migration were (108 ± 9)/HP, (22 ± 4)/HP and (67 ± 7)/HP respectively, allP<0.05. Compared with Blank control group, the activation of p38 signal pathway increased to (259.1 ± 7.7) % in tBHP group, while it was reduced to (186.4 ± 8.0) % in Alda-1 pretreatment+tBHP group. Conclusion: ALDH-2 could reduce ROS level in human EPCs, it may decrease mitochondrial membrane damage, protect migration which might be related to p38 signal pathway.
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Objective To investigate the effect of different maturation methods on mitochondrial functions of oocytes and the possible mechanism. To explore novel ideas for developing assisted reproductive technology (ART). Methods Female mice were used as models and randomly allocated into three groups, COH, IVM and NC control. Oocytes maturated with different methods which were all simulated with those treatments in human IVF cycle. Immunofluorescence were used to measure the mitochondrial membrane potentials and analyze the cy-toskeleton. Results The mitochondrial membrane potential in the COH group was significantly lower than that in NC group and IVM group (P < 0.05). The proportion of normal cytoskeleton including spindle structure and chromosome configuration in the COH group and IVM group were significantly lower than that in the NC group (PCOH < 0.01, PIVM < 0.05). Conclusions Both COH and IVM can affect mitochondrial functions.
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Aim To investigate the mechanism of demethylation on adenosine (ADO )and homocysteine (HCY)-induced apoptosis in human hepatoma HepG2 cells .Methods HepG 2 cells were treated with differ-ent concentrations of ADO (1.0、2.0、4.0 mol · L-1 ) alone or in combination with HCY for 6h,12h and 24h,5-aza-2-deoxycytidine (5-Aza-CdR)as a positive control.Cell viabilities were assessed by CCK8 assay. Cell apoptosis was observed by AnnexinV-FITC/PI staining.The mitochondrial membrane potentials(ΔΨ) were measured by flow cytometry.The mRNA and pro-tein expressions of caspase-3,caspase-8,caspase-9, MDM-2,p53,Cytochrome C,DNMT1,DNMT3a,DN-MT3 b were detected by RT-qPCR and Western blot re-spectively.Results ADO alone or in combination with HCY significantly reduced the viability of HepG2 cells in a dose and time-dependent manner.The apoptotic rates of HepG2 cells after combination treatment with ADO and HCY at 1 .0,2.0,4.0 mol · L-1 for 24 h were (1 8.63 ± 1.25 )%,(29.42 ±2.37 )% and (42.47 ±3.09 )%,compared with the control group (1.30 ±0.82 )%,P <0.01;and the mitochondrial membrane potentials were decreased from 674.15 ± 82.8%(black control group)to (428.38 ±54.5)%, (297.78 ±30.5)%,(74.45 ±5.73)%,P<0.01, respectively.The expressions of caspase-3,caspase-8, caspase-9,MDM-2,p53,Cytochrome C were up-regula-ted and MDM-2 were down-regulated after combination treatment of ADO and HCY.The mRNA expressions of DNMT1 ,DNMT3 a and DNMT3 b were down-regulated after combination treatment with ADO and HCY or 5-Aza-CdR alone.Conclusion Combination treatment of ADO and HCY can cause cellular methylation chan-ges.The effects of demethylation of ADO and HCY may activate p53 gene and mitochondrial pathway, which at last leads to HepG2 cell apoptosis.
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Objective To investigate the role of mitochondrial permeability transition pore (mPTP) of hippocampal neurons in process of hydrogen-rich saline attenuating global cerebral ischemia-reperfusion (I/R) injury in rats.Methods Seventy-two male Sprague Dawley rats,weighing 250-300 g,were randomly divided into six groups ( n =12 each):sham operation group (group S),cerebral ischemia-reperfusion group (group IR),normal saline group (group NS),hydrogen-rich saline group (group H),atractyloside group (group A) and hydrogen-rich saline + atractyloside group (group HA).Global cerebral I/R injury was produced by four-vessel occlusion method.Bilateral vertebral arteries were cauterized.Then bilateral common carotid arteries were occluded for 15min and followed by reperfusion.In groups H and HA,hydrogen-rich saline 5 ml/kg was injected intraperitoneally immediately after reperfusion,while equal volume of normal saline was injected in the other four groups.The rats in groups A and HA received intracerebroventricular injection of atractyloside 15 μl 10 min before reperfusion,while groups NS and H received intracerebroventricular injection of equal volume of normal saline.After the neurological behavior was evaluated at 24 h of reperfusion,8 rats in each group were sacrificed and the hippocampi were immediately isolated and homogenized followed by density gradient centrifugation.The opening degree of mPTP was assayed with spectrophotometry and the mitochondrial membrane potential (MMP) was detected with Rhodamine 123 method.Four rats in each group were killed at 72 h of reperfusion and the brains were removed for microscopic examination of the area CA1 of the hippocampus and determination of the number of normal pyramidal neurons.Results Compared with group S,the neurological behavior was compromised,MMP was decreased and mPTP opening degree was enhanced in the other five groups ( P ＜ 0.05).The neurological behavior was better,MMP was increased and mPTP opening degree was decreased in groups H and HA as compared with group IR ( P ＜ 0.05).Compared with group H,the neurological behavior was compromised,MMP was decreased and mPTP opening degree was enhanced in group HA ( P ＜ 0.05).Compared with group IR,the number of normal pyramidal neurons at 72 h of reperfusion in the CA1 region of the hippocampus was higher in group HA ( P ＜0.05).The injury of the CA1 region of the hippocampus at 72 h of reperfusion was attenuated in group H as compared with groups IR,NS,A and HA.Conclusion Hydrogen-rich saline can attenuate global cerebral I/R injury throngh inhibiting the mPTP opening and reducing the dissipation of MMP,thus maintaining the mitochondrial function.
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Objective To investigate the effects of ischemic postconditioning on mitochondrial permeability transition and mitochondrial transmembrane potential(△Ψm)following hepatic ischemia-reperfusion(I/R)in rats.Methods Forty male SD rats weighing 220-260 g were randomly divided into 5 groups with 8 animals in each group:sham operation group(group S);atractyloside+sham operation group(group A+S);I/R group;ischemic postconditioning group(group IPO)and atractyloside+ischemic postconditioning group(group A+IPO).The animals were anesthetized with intramuscular injection of atropine 0.05 mg/kg.Hepatic I/R was produced by occlusion of hepatic blood flow for 60 min followed by 6 h reperfusion.In group A+S,atractyloside 5 mg/kg was injected intravenously before abdomen Was closed.In group IPO,the animals were subjected to 3 cycles of 1 min reperfusion interspersed with 1 min hepatic isehemia at the end of 60 min hepatic ischemia.In group A+IPO,atractyloside 5 mg/kg was injected intravenously before reperfusion. Venous blood samples were collected for determination of serum ALT and AST activities immediately before ischemia and at 6 h of reperfusion. The animals were then sacrificed.Their livers were removed for microscopic examination, detection of apoptosis and determination of cytochrome c (Cyt c) expression, △Ψm and mitochonerial permeability transition pore (MPTP)activity. Apoptosis index (AI) was calculated. Results There was no significant difference in serum ALT and AST activities, AI, Cyt c expression, △Ψm and MPTP activity between S and A + S groups (P＞0.05). Compared with group S, serum ALT and AST activities and AI were significantly increased, Cyt c expression was up-regulated, △Ψm was decreased and MPTP activity was increased in groups I/R, IPO and A+IPO(P＜0.05).Compared with group I/R, serum ALT and AST activities and AI were significantly decreased,Cyt c expression was down-regulated, △Ψm was increased and MPTP activity was decreased in group IPO(P＜0.05), while no significant change was found in group A+IPO(P＞0.05).Compared with group IPO,serum ALT and AST activities and AI were significantly increased, Cyt c expression was up-regulated, △Ψm was decreased and MPTP activity was increased in group A + IPO(P＜ 0.05).Microscopic examination showed that hepatic injury was reduced in group IPO compared with group I/R, while aggravated in group A+ IPO compared with group IPO. Conclusion Ischemic postconditioning can protect liver from I/R injury by attenuating the I/R-induced increase in MPTP opening and decrease in △Ψm in rats.
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Objective To investigate the effects of pretreatment with Shen-fu injection on mitochondrial permeability transition and mitochondrial transmembrane potential (△ψm) following myocardial ischemiareperfusion (IR) injury in rats. Methods Thirty SD rats of both sexes weighing 250-300 g were randomly divided into3 groups with 10 animals in each group:Ⅰ sham operation group (group S); Ⅱ IR group and Ⅲ Shen-fu injection group (group SFI). The animals were anesthetized with intraperitoneal 20% urethane 5 ml/kg. The chest was opened and the heart exposed. Myocardial IR was induced by temporary ligation of the anterior descending branch of left coronary artery maintained for 30 min, followed by 120 min reperfusion. Myocardial ischemia was confirmed by decoloration of apex and elevation of S-T segment (＞ 0.1 mV) or erection of T wave. In group SFI,SFI 10 ml/kg was infused at 15 min before ischemia, while in group S and IR equal volume of normal saline was infused instead of SFI. At 120 min of reperfusion, blood samples were collected from right internal carotid artery for determination of serum concentration of cTnI. The animals were then sacrificed and the hearts were immediately removed for measurement of myocardial mitochonerial permeability transition pore (MPTP) activity (by spectrophotometry at 540 nm) and myocardial △ψm (by fluorospectrophotometer using rhodamine 123 as fluorescent probe). Results Compared with group S, serum cTnI concentration and MPTP activity were significantly increased and △ψm was decreased in group IR. SFI pretreatment significantly attenuated the IRinduced increase in serum cTnI concentration and the MPTP activity and decrease in △ψm. Conclusion SFI pretreatment can protect myocardium from IR injury by attenuating the IR induced increase in MPTP opening and decrease in △ψm.
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BACKGROUND: Mitochondrial dysfunction plays an important role in Abeta-induced neuronal toxicity in Alzheimer's disease (AD). We measured the membrane potentials of mitochondria (delta psim) and assessed the genetic expressions of A beta(25-35)-induced neurotoxicity in the human neuroblastoma cell line, SK-N-SH cell. METHODS: SK-N-SH cells were incubated with a single dose of 25 micrometer A beta(25-35) for 0-24 hours, and kinetic study was done. delta psim was measured by flow cytometry. Messenger RNA expressions of cytochrome c oxidase (COX), cytochrome c, succinate dehydrogenase (SDH), amyloid-beta alcohol dehydrogenase (ABAD), caspase 9, and Bcl-2 were measured by quantitative real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). Cell death rate was measured by MTT reduction assay. RESULTS: delta psim was reduced at 24 hours. mRNA expression for COX gradually decreased by about 29% (p<0.05) while-expressions for cytochrome c, SDH, ABAD, and caspase 9 increased (p<0.05) progressively during the 24-hour time period. Bcl-2 expression decreased (p<0.05) gradually; and apoptotic cell death rate was about 24% (p<0.01) by 24 hours. CONCLUSION: Extracellular administration of A beta(25-35) contributes directly to mitochondrial dysfunction in SK-N-SH cells with the enzymatic impairment of the tricarboxylic acid cycle and electron transport chain, and eventually leading to apoptotic cell death.
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Humans , Alcohol Dehydrogenase , Alzheimer Disease , Amyloid beta-Peptides , Apoptosis , Caspase 9 , Cell Death , Cell Line , Citric Acid Cycle , Cytochromes c , Electron Transport , Electron Transport Complex IV , Flow Cytometry , Gene Expression , Membrane Potentials , Mitochondria , Neuroblastoma , Neurons , Reverse Transcriptase Polymerase Chain Reaction , RNA, Messenger , Succinate DehydrogenaseABSTRACT
@#Objective To investigate the effects of morroniside on hydrogen peroxide (H2O2)-induced apoptosis in SH-SY5Y neuroblastoma cells. Methods SH-SY5Y cells were pre-incubated with morroniside (1, 10, and 100 μmol/L) for 24 h prior to exposure to H2O2 (300～500 μmol/L) for 18 h. The content of malondialdehyde (MDA)，mitochondrial membrane potentials (MMP) and apoptosis ratio were determined. Results Pretreatment the cells with morroniside (1, 10 and 100 μmol/L) lowered the H2O2-induced MDA concentration and MMP, and inhibited the apoptosis. Conclusion Morroniside may protect the neurocyte from H2O2-induced apoptosis by lowered MDA and inhibits MMP.
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Aim To explore the apoptotic effect of mimics of manganese superoxide dismutase(MnSODm)on human leukemia cell line K562 in vitro and the possible molecular mechanisms.Methods Human leukemia K562 cells were used as the target cells.The cell proliferating activity was examined by a MTT colorimetric assay,and the apoptosis of K562 cells was assessed with FITC-Annexin V and propidium iodide(PI)double staining and morphological changes.The expressions of bcl-2 and bax mRNA were detected by reverse transcription polymerase chain reaction(RT-PCR),and flow cytometry(FCM)was employed to measure the expressions of Bcl-2 and Bax protein,mitochondrial inner membrane potential(??m),Cytochrome C(Cyt C)release and Caspase-3 activity.Results The proliferation of K562 cells was obviously inhibited by 0.5~10 mg?L-1 MnSODm(P
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BACKGROUND AND OBJECTIVES: Auditory signal can be amplified by the organ of Corti and be detected in the auditory cortex. Outer hair cells (OHCs) play a pivotal role in this amplification process. The signal transduction mechanism of OHC relys on the precise control of intracellular calcium ([Ca2+]i). Membrane depolarization, induced by electrical stimulation or extracellular high concentration of KCl solution, increases [Ca2+]i through the opening of voltage-gated Ca2+ channel or the release from intracellular Ca2+ store which is stimulated by second messenger system. Efferent stimulation seems to inhibit the electromotility of OHC and modify the amplification process. Acetylcholine is the most promising neurotransmitter released from efferent synapse. In this study, we aimed to observe the effect of carbamylcholine (a non-hydrolyzable, non-selective cholinergic agonist) on the change of [Ca2+]i, and the modification of depolarization-induced [Ca2+]i increase in isolated OHCs of the guinea pig cochlea using the fluorescent indicator fluo-3. MATERIALS AND METHOD: Outer hair cells were isolated from the guinea pig cochlea and incubated in HBSS containing 5 M fluo-3 for 30 min. The [Ca2+]i was measured under inverted microscope equipped with epifluorescence system. The images were analysed and [Ca2+]i was calculated using Fmax, Fmin and Kd obtained from in vivo and in vitro calibration. RESULTS: [Ca2+]i increased by extracellular carbamylcholine application (1mM) in OHCs. KCl solution could induce [Ca2+]i increase when used more than 25mM and the responses were concentration-dependent. Preincubation of carbamylcholine, however, did not modify the depolarization-induced [Ca2+]i increase. CONCLUSION: Through these results, we speculate that the acetylcholine, released from the efferent synapse, can induce local increase of [Ca2+]i, but acetylcholine can not regulate the depolarization-induced [Ca2+]i in isolated OHCs of the guinea pig cochlea.
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Animals , Acetylcholine , Auditory Cortex , Calcium , Calibration , Carbachol , Cochlea , Electric Stimulation , Guinea Pigs , Guinea , Hair , Membrane Potentials , Membranes , Neurotransmitter Agents , Organ of Corti , Second Messenger Systems , Signal Transduction , SynapsesABSTRACT
To explore the changes of mitochondrial membrane potential in rat cadiomyocyte apoptosis by fluorescent JC 1, H 2 O 2 was used to induce cadiomyocyte apoptosis, and JC 1 in combination with flow cyometry was used to detect the changes of mitochondrial membrane potential in the early stage of cadiomyocyte apoptosis. The results showed that living cardiomyocytes had a high mitochondrial membrane potential, JC 1 aggregates were formed in the inner membrane of mitochondria and emitted orange red fluorescence. H 2 O 2 caused the decrease of mitochondrial membrane potential, JC 1 aggregates were dissociated to monomer,which emitted green fluorenscence. So the red fluorescence decreased. It is suggested that JC 1 in combination with flow cyometry is an ideal method to detect the changes of mitochondrial membrane potential.
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AIM: To investigate the effects of salidroside on intracellular free calcium concentration (([Ca~(2+)]i)), apoptosis, mitochondrial membrane potential (MMP) and activity during injury induced by hypoxia/hypoglycemia in cultured SH-SY5Y cells. METHODS: Mitochondrial activity was measured by methylthiazolyl tetrazolium test. MMP, [Ca~(2+)]i and apoptosis were measured by flow cytometry. RESULTS: SH-SY5Y cells were cultured in a hypoxia/hypoglycemia condition for 2, 4, 6 and 12 h, [Ca~(2+)]i and apoptosis rate significantly increased compared with control group (P
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AIM: To investigate the role of nitric oxide (NO) in vascular hyporeactivity during prolonged hemorrhagic shock (HS). METHODS: Anesthetized Sprague-Dawley rats (180-220 g) were subjected to HS insult in which they were bled to a mean arterial pressure (MAP) of 40 mmHg (5.33 kPa) and arteriolar reactivity to norepinephrine in spinotrapezius was detected. The constant MAP of 40 mmHg was maintained until vascular hyporeactivity had occurred and then were resuscitated or sacrificed for further analysis. NO synthase (NOS) activity was measured ex vivo by the conversion of [3H]-arginine to [3H]-citrulline in homogenates from heart, lung, liver, spleen, duodunum, skeletal muscle. 24 h survival rates of resuscitated rats were observed with and without administration of aminoguanidine (AG), a selective inducible NOS (iNOS) inhibitor. Mesenteric arteriolar smooth muscle cells (ASMC) were isolated, and the effects of L-arginine (L-Arg) on membrane potential (MP) of ASMC were determined by fluorescent probe and confocal microscopy in the absence and presence of AG. RESULTS: When vascular hyporeactivity occurred, an increase of NOS activity was observed in liver and heart. Resuscitated rats with AG had a higher survival rate compared with that of control. The MP of ASMC was decreased (more negative) immediately following the addition of L-Arg, and the hyperpolarization effects of L-Arg were partially blocked in the presence of AG. CONCLUSION: These results suggest that excessive NO produced in HS is responsible for the occurrence of vascular hyporeactivity in prolonged hemorrhagic shock, and one of the mechanisms of which may be hyperpolarization of ASMC caused by NO.
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AIM:We generated transgenic mice of NKCC1-/-(homozygous mutant),NKCC1+/-(heterozygous)and NKCC1+/+(wild-type)that have a targeted disruption in the NKCC1 gene to investigate the role of Na-K-2Cl(NKCC1)channel in auditory function of the inner ear.METHODS:Hearing threshold and endocochlear potential(EP)were measured in the NKCC1-/-,NKCC1+/-and NKCC1+/+ mice by auditory brainstem response(ABR)and EP recordings,respectively.The inner ears of the mice were removed and examined morphologically with the light microscope.RESULTS:The auditory function of NKCC1+/+ mice was normal,the mean value for ABR thresholds in response to click sound was [(23.13?3.78)dB,SPL],EP was(98?16)mV.The mean value for ABR thresholds to click sound was elevated in NKCC1+/-mice [(38.49?12.29)dB,SPL],relative to that significantly increased in NKCC1+/+ mice(P
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AIM: To explore the change of delayed rectifier potassium channel (K_V) activity in alveolar macrophages (AM) in chronic obstructive pulmonary disease (COPD) rats. METHODS: COPD model was established by exposure of the animals to cigarette smoke. With whole-cell voltage- or current-clamp techniques, K_V activity, membrane capacitance and resting membrane potential (Em) in AM from COPD model and control rats were compared. RESULTS: (1) Significant increases in total mononuclear cells and AM in bronchoal aveolar lavage fluid (BALF) were found in COPD group compared with in control group. (2) The AM K_V current altitude in COPD group [(520.5?38.7)pA, (+50) mV, n=30] was significantly lower than that in control group [(713.6?44.4)pA, (+50) mV, n=30, P0.05), but had more positive Em (P
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Objective To investigate the effects of lidocaine and ketamine on resting membrane potentials of primary cultured anoxic hippocampal neurons using patch-clamp technique. Methods Hippocampal neurons were isolated from newborn Wistar rats (
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Objective To study the effects on berberine hydrochloride on the mitochondria membrane potential and free intracellular calcium of HaCaT cells, and elucidate the mechanism of action of berberine on keratinocytes. Methods Rhodamine-123 fluorescence (very sensitive to mitochondria membrane potential) and Fluo-3/AM fluorescence (suitabe to detect free intracellular calcium in single HaCaT cell) were measured by laser scanning confocal technique. Results Fluo-3/AM fluorescence intensity of HaCaT cells was persistently increased after treating with berberine at concentrations of 5 ? 10-5M, 2.5 ? 10-5M and 1.25 ? 10-5M, and significant differences were observed as compared with the PBS control. The intensity of rhodamine-123 fluorescence in HaCaT cells was decreased immediately when exposed to berberine, with significant difference from that of the PBS control. Conclusions It is suggested that berberine could increase free intracellular calcium and decrease mitochondria membrane potential of HaCaT cells, induce overload of intracellular calcium, influence energy metabolism, and then inhibit the proliferation of keratinocytes.
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0.05).Zacopride at concentration of 1.0 ?mol/L showed the most potent activity on IK1 with approximately 30% increment both in inward current and outward current(P
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AIM: Through studying the differences between wild-type acidic fibroblast growth factor (waFGF) with recombinant aFGF (raFGF), the biological effect of raFGF concerned with mitogenic activity was evaluated. METHODS: NIH3T3 cell line was used. Cell proliferation with MTT method was used to study the mitogenic activity. Flow cytometry was also used for detection of apoptosis, cell membrane permeability and mitochondria potential. The role of heparin sulfate (HS) on aFGF biological effect was studied at the same time in this research. RESULTS: The enhancement of raFGF on cell proliferation was significantly lower than that of waFGF. The restriction of raFGF on apoptosis and the enhancement of it on cell membrane permeability were all lower than those of waFGF significantly. The enhancement of raFGF on mitochondria potential was lower than that of waFGF significantly. The HS improved the biological effect of aFGF. CONCLUSION: The mitogenic activity of raFGF is lower than that of waFGF and raFGF has little effect on apoptosis.
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AIM: To study the changes of mitochondrial membrane potential(△?m) and mitochondrial mass in apoptosis of Jurkat cells induced by camptothecin(CPT).METHODS: Jurkat cells were treated with CPT.Annexin V-FITC/propidium iodine(PI) double stainig was used to detected early stage of apoptosis and PI staining for analyzing the cell cycle.Jurkat cells were stained by annexin V-PE/DiOC_6(3) to detect changes of △?m.The mitochondrial mass was measured by cytometry with NAO staining.RESULTS: 6 h after treated with 10 ?mol/L CPT,the rate of early apoptotic cells(22.59?1.04)% had significantly difference compared with control group(3.93?0.73)%(P0.05).Apoptotic peak appeared obviously after treated with CPT,the percentage of late apoptotic cells(13.58?0.97)% had distinctly difference compared with control group(3.18?0.51)%(P