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Objective@#To construct a hit-deficient mutant strain of S. mutans ATCC25175 and verify its cell cycle regulatory function.@*Method @# Genomic DNA was extracted from S. mutans ATCC25175 strains, and then the upstream and downstream DNA fragments of the hit gene were cloned into the pFW5 vector (spectinomycin resistant) to construct recombinant plasmids using PCR amplification. Third, employed by natural genetic transformation in S. mutans ATCC25175 strains, the linearized recombinant plasmids were transformed into their genetic competence, induced by the synthesized competence-stimulating peptide (CSP), and then, homologous recombination was utilized to produce crossover and noncrossover products. Fourth, the hit-deficient mutant strains of S. mutans ATCC25175 were screened through the spectinomycin-resistance marker and identified by the electrophoresis of PCR products and PCR Sanger sequencing. Finally, its growth rate in vegetative BHI medium was also investigated.@* Results @# The upstream (856 bp) and downstream (519 bp) DNA fragments of the hit gene from the genomic DNA materials of S. mutans ATCC25175 were cloned into two multiple cloning sites (MCS-I and MCS-II) of the pFW5 vector, respectively, and the recombinant plasmid pFW5_hit_Up_Down was constructed and identified by double-emzyme digestion and PCR Sanger sequencing. The linearized recombinant plasmids were transformed into their genetic competence, induced by the synthetic CSP, and then, homologous recombination was utilized to produce various products. The hit-deficient mutant strains of S. mutans ATCC25175 were screened through the spectinomycin resistance marker and identified by the electrophoresis of PCR products and Sanger sequencing. The growth rate of the hit-deficient mutant strains versus their parental S. mutans ATCC25175 strains was increased greatly (P<0.001).@* Conclusion@# The hit-deficient mutant strains of S. mutans ATCC25175, having heritable traits, were successfully constructed, and the encoding Hit protein is growth-phase regulated in the cell cycle.
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Lung cancers are the leading cause of cancer deaths worldwide and pose a grave threat to human life and health. Non-small cell lung cancer (NSCLC) is the most frequent malignancy occupying 80% of all lung cancer subtypes. Except for other mutations (
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Objective The use of targeting therapy for the treatment of gastric glandular cancer has been a hot topic in recent years. This study aims to clarify that through what ways the histone deacetylase inhibitor MS-275 completes its selectively killing effect on gastric glandular cancer cell line SGC-7901. Methods SGC-7901 cells and GES-1 cells were respectively cultured for 24h, with(10-100) μmol/L concentrations of MS-275. (1) The survival rate of SGC-7901 cells, GES-1 cells and the normal cells were analyzed by WST-1; (2) The change of the mitochondrial membrane potential in SGC-7901 was estimated by flow cytometry;(3) The expression levels of p21, p27, p57, cyclinB1, cyclinD1 were determined by Western blot and PCR methods. Results (1) MS-275 could decrease the survival rate of SGC-7901 cells, the effect was significantly enhanced with the increasing of the concentration (P<0.05), but MS-275 showed no obvious effect on normal gastric mucosa epithelial cells GES-1; (2) MS-275 treatment could decreased the mitochondrial membrane potential of SGC-7901 cells (P<0.05); (3) MS-275 treatment could increase the relative contents of p21, p27, p57 genes and their protein and, at the same time, decrease the relative contents of CyclinB1 and CyclinD1 (P<0.05). Conclusion MS-275 treatment can selectively kill gastric glandular cancer cells SGC-7901 through several possible ways, such as inducing mitochondrial apoptosis and regulating the expression levels of cell cycle-related genes and proteins.
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AIM:ToinvestigatetheeffectofIkarosisoformsontheproliferationofhumanovariancancerSK-OV3 cells.METHODS:Three isoforms of Ikaros, IK1, IK2 and IK6, were transfected into ovarian cancer SKOV3 cells. CCK-8 assay and cell counting were used to detect the effects of Ikaros isoforms on the proliferation of SKOV 3 cells.The cell cycle was analyzed by flow cytometry .The cell cycle-related proteins were detected by Western blot .RESULTS:IK1 and IK2 expression inhibited SKOV 3 cells proliferation .Flow cytometry analysis indicated that IK 1 and IK2 induced SK-OV3 cell cycle arrest at the G 1 phase.IK6 isoform exerted no obvious effect on the proliferation or cell cycle of SKOV 3 cells.Compared with control EV group , IK1 group and IK2 group showed a dramatic elevation in the expression of the cell cycle inhibitor p21, along with a substantial decrease in the expression of the cell cycle inducers cyclin D 1 and cyclin D2, which did not change in IK 6 group.CONCLUSION:IK1 and IK2 significantly inhibit the proliferation of ovarian cancer SKOV3 cells and induce cell cycle arrest at G 1 phase by regulation of cell cycle-related proteins cyclin D1, cyclin D2 and p21, while IK6 isoform exerts no obvious effect on the proliferation and cell cycle of SKOV 3 cells.
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BACKGROUND:Amniotic cells are mainly composed of amniotic epithelial cells and amniotic mesenchymal cells, which have multi-differentiation potential and can be transformed into neurons as wel as synthesize and release biological y active substances and neurotrophic factors. In preliminary studies, amniotic cells that are transplanted into the brain can significantly promote the regeneration of brain neurons. OBJECTIVE:To explore the role of amniotic cells in mouse brain cells after ischemia-reperfusion injury. METHODS:The model of cerebral ischemia-reperfusion injury was established in Babl/c mice using occlusion of bilateral common carotid arteries, and then brain cells were separated from mice. Amniotic cells were isolated from mouse placenta. Brain cells from Balb/C mice co-cultured with amniotic cells served as experimental group, and brain cells cultured with PBS as control group. RESULTS AND CONCLUSION:The viability of brain cells in the experimental group was significantly higher than that in the control group (P0.05);after 48 hours co-culture, however, the necrotic rate of brain cells was significantly lower in the experimental group than the control group (P<0.05). In cellcycle, the experiment group showed increased S phase cells;while, the control group exhibited increased G 1 phase cells and decreased S phase cells. G 2 phase cells had no changes in number in both two groups. Through the above results, amnion cells can be proved to protect and promote the regeneration of brain cells of Balb/C mice with ischemia-reperfusion injury, and inhibit cellnecrosis and apoptosis.
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OBJECTIVE Toinvestigatethedifferenceofcytotoxiceffectsofhydroxycamptothecin(HCPT)onhuman lungcancercelsA549andhumanembryolungfibroblastcelsMRC-5.METHODS A549celsandMRC-5celswere treated with HCPT 20-200 μmol·L-1 for 24,48 and 72 h,or pulse treated with HCPT 50-400 μmol·L-1 for 24 h along with 5 d release.cellsurvival was detected by MTT assay.Morphological changes for both types of cells were observed under an inverted phase-contrast microscope.cellcycle and apoptosis in both cells treated with HCPT 50 μmol·L-1 for 48 h weredeterminedbyflowcytometry.RESULTS HCPT20-200μmol·L-1inhibitedthesurvivalofbothcelsinaconcen-tration-dependent manner and more cytotoxicity was observed in A549 cells for 48 h.The concentration-effect correlation coefficient(r)of HCPT in A549 and MRC-5 cells for 48 h was 0.898 (P=0.015)and 0.996 (P=2.56E-5)respectively. The inhibition rates were significantly different between A549 and MRC-5 cells with treatment of HCPT 20,50,80,1 00, 1 60 and 200 μmol·L-1 for 48 h (P<0.05).The IC50 of HCPT on A549 and MRC-5 cells was (24.00 ±0.69)μmol·L-1 and (1 23.63 ±3.89)μmol·L-1 respectively,indicating that A549 cells were 5-fold more sensitive to HCPT than MRC-5 cells at 48 h.After exposure to HCPT 50 μmol·L-1 for 48 h,some A549 cells were rounded up and shrank dramatical y, and some cells underwent membrane blebbing or lysing while MRC-5 cells had no obvious changes.cellcycle and apop-tosis analysis showed that A549 cells were arrested at both S and G2/M phases and apoptosis occurred but MRC-5 cells were just arrested at S phase.In the recovery growth curve,the growth of A549 cells was inhibited to a larger extent than MRC-5 cells and the growth retardation stil existed for 24 h in both cells.The survival of MRC-5 cells was faster than that ofA549cels,althoughtherewasnocompleterecoveryineithercel.CONCLUSION A549celsaremoresensitiveto HCPT than MRC-5 cells due to the fact that HCPT induces cellcycle arrest at both S and G2/M phases and apoptosis in A549 cells,but only triggers S phase arrest in the MRC-5 cells.
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AsoneoftheCdc25phosphatasefamilymembers,Cdc25Cplaysanimportantroleinregu-lating mitosis of eukaryotic cells.In eukaryotic cells,CDK1-cell cycle protein B (CyclinB)compound mainly control the process of G2-M.The activity of Cdc25 C is the key in cell cycle into M phase.It activates CDK1-cyclinB complexes to promote cells from G2 to M phase .Improving Cdc25 C activity can promote the G2-M phase transition,and remove the G2-M phase retardation induced by ionizing radiation,preventing the damaged DNA from repaired into the phase of cell division,resulting in cell death caused by excessive cell proliferation, thus enhance radiosensitivity.
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BACKGROUND:The researches about the effect of retinoic acid on the proliferation of adipose-derived stem cells are rare, and the researches on the testosterone are mainly on the inhibition of cellaging. OBJECTIVE: To study the effects of retinoic acid and testosterone or combination on the cellcycle of adipose derived stem cells. METHODS:Adipose derived stem cells were isolated from adult female Sprague Dawley rats with 2 months age and cultured in vitro til passage 3 adipose derived stem cells, and then the 3rd passage adipose-derived stem cells were performed with adipogenic induction, osteogenic induction and surface marker identification. The cells were divided into six groups:(1) Control group;(2) 10-5 mol/L retinoic acid group;(3) Retinoic acid group;(4) 10-5 mol/L retinoic acid+testosterone group;(5) 10-6 mol/L retinoic acid+testosterone group;(6) Testosterone group. The adipose-derived stem cells in the control group were cultured with Dulbecco’s modified Eagle’s medium+10%fetal bovine serum culture medium, and the adipose-derived stem cells in the other five groups were induced with corresponding dose of retinoic acid and testosterone on the basis of control group. After cultured for 36 hours, the flow cytometry was used to detect the changes of cellcycle. RESULTS AND CONCLUSION:Compared with the control group, cellproportions in phase G 1 of 10-5 mol/L retinoic acid group and 10-6 mol/L retinoic acid group were increased significantly, and the cellproportions in phase S were decreased. Compared with control group, the cellproportion in phase G 1 of testosterone group was significantly reduced, and the cellproportion in phase S was increased. Compared with 10-5 mol/L retinoic acid group and 10-6 mol/L retinoic acid group, cellproportions in phase G 1 of 10-5 mol/L retinoic acid+testosterone group and 10-6 mol/L retinoic acid+testosterone group were reduced significantly and the cellproportions in phase S were increased. Retinoic acid can inhibit the cellcycle of adipose-derived stem cells in phase G 1 , and delay the process of the cellcycle from phase G1 to phase S;while testosterone can promote the cellcycle of adipose-derived stem cells from phase G1 to phase S;the combination induction of retinoic acid and testosterone can accelerate the process of the cellcycle of adipose-derived stem cells from phase G 1 to phase S.
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New progress in the area of nanoparticle research are the studies on interactions between nanoparticles (NPs) and biological molecules in body fluid,cellular microenvironment,intracellular components or secreted cellular proteins such as cytokines,growth factors and enzymes and the use of engineered NPs to target various signal transduction pathways in cancer therapy.NPs-induced toxicological mechanism has become one of the most studied topics.Oxidative stress result is not sufficient to explain all the biological effects.This article reviews the latest research progress in nanotoxicology from the perspective of cellular mechanisms,including the interactions between NPs and the cell membrane receptor,cell oxidative stress result,cell apoptosis induction and the effects of NPs on cell cycle.
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Renal epithelial cells damaged by ischemia/reperfusion (I/R) can be restored by timely and appropriate treatment. Recent studies have reported that intra renal adult kidney stem cells contribute to the restoration of tubules damaged by I/R. Here, we determined the role of adult tubular cells in the restoration of damaged tubules. We labeled slow cell-cycle cells (SCCs) with 5-bromo-2'-deoxyuridine (BrdU) and investigated their location in the kidneys as well as their contribution to the restoration of tubular cells damaged by I/R injury in mice. Thirty minutes of bilateral ischemia resulted in severe disruption of tubular epithelial cells along with a decline in renal function. The post-ischemic disruption of tubular epithelial cells was most severe in the S3 segment of the outer stripe of the outer medulla. Damaged tubules demonstrated gradual recovery of renal function over time. BrdU-labeled SCCs were mainly observed in tubules located at the junction of cortex and outer medulla, as well as in the inner medulla. The tubular SCCs expressed functional tubule cell markers such as Na/K-ATPase, Na-K-Cl cotransporter-2, and aquaporin 1 and 2. BrdU-labeled SCCs survived I/R injury and proliferated. These results demonstrate that SCCs present in tubules contribute to the restoration of tubular epithelial cells injured by I/R.
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Adulto , Animais , Humanos , Camundongos , Injúria Renal Aguda , Células-Tronco Adultas , Aquaporina 1 , Bromodesoxiuridina , Células Epiteliais , Isquemia , Rim , Túbulos Renais , Regeneração , Células-TroncoRESUMO
Objective To investigate and compare the different effects of soluble adult wornl antigen(SWA)and soluble egg antigen(SEA)of Schistosoma japonicum on the apoptosis and cell-cycle of routine CD4~+T cells.Methods Purified CD4~+T ceUs from normal C57BL/6 mice were cultured with CFSE labeled antigen presenting clls in the presence of different stimuli for 36 h.Flow cytometry(FCM)was used to detect the apoptosis of CD4~+T cells by fluorescence conjugated caspase-3 antibodie staining.The flow cytometry was used to analyze the cell-cycle of CD4~+T cells cultured as described above for 96 h by propidium iodide staining.Results Compared with the apoptosis percentage of CD4~+T cells[(1.24±0.29)%]in the SEA stimulated group,that in the SWA stimulated group[(1.52±0.38)%]did not show statistically significant difference(P>0.05).Compared with the cell percentages in G1 phase[(78.91±2.98)%],S phase[(7.39±0.85)%]and G2/M phase[(10.69±1.05)%] in the SWA stimulated group,that of the G1 phase[(59.42±1.32)%]was significantly lower,but those in the S phase[(21.07±O.88)%] and G2/M phase[(18.88±1.21)%]were significantly increased in the SEA stimulated group(P<0.01).Conclusions There is no statistically significant difference between the apoptosis levels of CD4~+T ceHs stimulated by SWA and SEA.However,SEA significantly promotes the progression of the cell-cycle of CD4~+T cells compared with SWA.
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Objective To investigate the effects of granulocyte colony-stimulating factor on proliferation of adipose-derived adult stromal cells(ADASc) in rats. Methods Sixteen SD rats were randomly divided into G-CSF group( n = 8) and control group( n = 8). The rats were subjected to subcutaneous injections of G-CSF at a dose of The ADASc were separated and cultured. Then: ( 1 ) The surface antigens of the ADASc were analyzed by flow cy- tometry. (2)The growth information of the ADASc were observed in vitro. (3)The generation cycle of the ADASc were investigated with the flow cytometry. Results ( 1 ) Flow cytometic detection of ADASe surface marks showed CD44+CD105-CD31-CD45-(2)The doubling generation time, the maximum proliferation multiple and the cell cycle distri- bution of ADASc in G-CSF group had significant difference from that of the control group. Conclusion The discrep- ancy adherence was a practical method to culture the ADASc;G-CSF treatment could promote ADASc re-entering into cell cycle.
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[Objective] To investigate into the cellular mechanism of growth promotion due to shear stress by studying G1-phase events responsible for the suppression of cell transition from the G1 to S phase of the cell cycle,and to establish the most suitable physiological stress to stimulate bone formation.[Methods]The osteoblasts derived from Kunming murine's calvaria were exposed to Fliud shear stress(FSS:12 dyn/cm2)for 0,0.25,0.5,1,2,4 h,respectively.In the flow chamber,its impact on cell proliferation,differentiation and the effection of cell cycle's G1/S checkpoint were recorded.The cell proliferation was studied by MTT assay.The cell differentiation was assessed through alkaline phosphatase(ALP)activity assay.Flow-cytometry,immunofluorescence and RT-PCR techniques were used to evaluate the proportion of S phase in cell cycle,the activity of CDK2,CDK4 and the expression of E2F-1,p27mRNA,which demonstrate how FSS underlying multiple mechanisms to enhance the cell cycle progression from G1 to S phase.[Results]FSS increased proliferation and advanced the time in cell growth curve,but after 1,2,4 h,the proliferation was inhibited.The FSS also increased the ALP activity,which were significantly stimulated at 0.25 and 0.5 h after shear stress(128% and 158 % of control);but the FSS decreased ALP activity at 1,2,and 4 hs.The proportion of S phase in cell cycle raised within the early period.The S phase rate significantly increased at 0.5 h(P
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BACKGROUND: The effect of genistein on different types of cells has been investigated. However, its effect on the nervous system is still unclear. The aim of the present work is to explore the effect of genistein on rat neuroblastoma B35 cells. METHODS: The effect of genistein on the proliferation of B35 cells, its cytotoxicity, the cell-cycle distribution, the ultra-structural changes and the induction of apoptosis were determined using MTT assay, LDH assay, Flow-cytometric analysis, transmission electron microscopy and Hoechst staining, respectively. Furthermore, Real-time quantitative RT-PCR and Western blotting were used to examine the transcriptional and post-translational alterations of the G2/M cell-cycle arrest marker cyclin-dependent kinase inhibitor p21(waf1/cip1) and the apoptosis-related genes after genistein treatment. RESULTS: Genistein significantly inhibits cell survival, slightly elevates the release of lactate dehydrogenase and induced apoptosis in B35 cells. Genistein increased the number of cells at S-phase and induced cells to accumulate at the G2/M phase. These G2/M arrested cells are associated with a marked up-regulation of p21(waf1/cip1) at both the mRNA and protein levels. We observed that genistein up-regulates pro-apoptotic Bax with concurrent down-regulation of the anti-apoptotic Bcl-2 protein. CONCLUSION: These observations suggest that the anticancer effect of genistein on B35 neuroblastoma cells is mediated through multiple cellular pathways including G2/M cell-cycle arrest and the induction of apoptosis.
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Animais , Ratos , Apoptose , Western Blotting , Pontos de Checagem do Ciclo Celular , Ciclo Celular , Sobrevivência Celular , Regulação para Baixo , Genisteína , L-Lactato Desidrogenase , Microscopia Eletrônica de Transmissão , Sistema Nervoso , Neuroblastoma , Fosfotransferases , RNA Mensageiro , Regulação para CimaRESUMO
Objective To investigate the effects of wortmannin (WT) on leukemia cells cycle and BCL-2 protein expression. Methods 0 25?mol/L PI-3 kinase inhibitor wortmanin was used to treat K562 cells for 24 hours, and Arac was simultaneously used to induce the cell apoptosis. The specific fluoresent staining and FCM analysis were adopted to measure the cell cycle distribution and BCL-2 expression. Results Compared with the control cells, the proliferation index(PI) of the K562 cells treated by wortmannin was significantly lower, the cell number of G1 phase and the percentage of cell apoptosis increased, and the BCL-2 protein expression significantly decreased. Conclusion Wortmannin could inhibitedtheproliferationofK5 6 2cells ,andwascellcyclespecificagent (CCSA) .
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Objective To study the cell-cycle specificity of tamoxifen-induced apoptosis in the ER(+) breast cancer cells in vitro and in vivo. Methods ER(+) MCF-7 cell line (in vitro) and primary cultured ER(+) breast cancer cells (in vivo) were treated with tamoxifen, and the cell cycle specificity of cell apoptosis and the apoptotic rate were dertermined by flow cytometry. Results Both MCF-7 and primary cultured breast cancer cells were induced to apoptosis with G 0/G 1 phase specificity by tamoxifen. And the apoptotic rate in MCF-7 was higher than that in primary cultured breast cancer cells. Conclusion Tamoxifen could induced G 0/G 1 phase specific apoptosis in the ER(+) breast cnacer cells, and tamoxifen-induced apoptotic rate was higher in vitro than in vivo.
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Objective To study the effects of D-glucosamine hydrochloride on the apoptosis of gastric carcinoma cell line SGC-7901 and its mechanism.Methods SRB assay was used to examine the effects of D-glucosamine hydrochloride on the growth inhibition of SGC-7901.The cell-cycle and expression of cytochrome-C were observed by flow cytometry,the concentrations of intracellular Ca2+ was determined by CLSM and the expressions of cyclinB1,calcinerin were investigated by Western-Blot.Results D-glucosamine hydrochloride could block SGC-7901 cell-cycle at S phase of cell division,increase the concentration of intracellular Ca2+ and decrease the expression of cyclinB1,the expressions of calcinerin and cytochrome-c were increased respectively.Conclusion Block of cell-cycle may be one of the mechanism of inducing cell apoptosis by D-glucosamine hydrochloride.
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Aim To explore the antitumor active metabolites of marine-derived Streptomyces flavorectus Z4-007. Methods The separation procedure was guided by a flow cytometric bioassay using tsFT210 cells to examine cell cycle inhibitory activity, and various column chromatography using silica gel, Sephadex LH-20, and ODS were employed for the isolation and purification of bioactive compounds. Chemical structures were investigated by spectroscopic methods and biological activities were evaluated by flow cytometry using mouse cancer tsFT210 cells. Results Four bioactive compounds were isolated from the fermentation broth of Streptomyces flavorectus Z4-007, one of them had been identified as 1-(2,4-Dihydroxy-3,5-dimethyl-phenyl)-hexa- (E,E)-2,4-dien-1-one (1) and the other three compounds were considered to be peptide, Compound Ⅰ inhibited the cell-cycle of tsFT210 cells at the G_0/G_1 phase at higher concentrations, while at the lower concentrations compound Ⅰ inhibited the cell-cycle mainly at the G_2/M phase, accompanied with induction of apoptosis in the tsFT210 cells.Conclusion Compound Ⅰ was isolated from the metabolites of the genus Streptomyces for the first time and provided as a new cell-cycle inhibitor.