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Objective:To explore combined detection of mad2 with anti-nuclear mitotic spindle apparatus antibody(MSA)and anti-centromere antibody(ACA)and their clinical value for the diagnosis of small cell lung cancer(SCLC).Methods:One hundred and twen-ty SCLC patients,110 non-small cell lung cancer(NSCLC)patients,and 115 pulmonary nodule(PN)patients were enrolled in this study. The expression of mad2 was analyzed by qt-PCR.MSA and ACA were detected by indirect immunofluorescence(IIF)staining.Results:mad2 was overexpressed in SCLC and NSCLC samples(P<0.05).There were significant differences between the results obtained for SCLC and NSCLC samples by qt-PCR(P<0.05).AUC in ROC curve for mad2 expression was 0.799 with an intermediate diagnostic value. In the correlative analysis,the odds ratio of MSA and ACA was 6.94 and 5.60,respectively.In the correlation analysis,Kappa value of mad2 with MSA was 0.49,and Kappa value of mad2 with ACA was 0.42.In the parallel analysis,the sensitivity and specificity was 83.31% and 79.34%,respectively,while the Youden Index was 0.62.Moreover,in the serial analysis,the sensitivity and specificity was 65.32% and 93.35%,respectively,and the Youden Index was 0.59.Conclusions:In comparison with the NSCLC and PN samples,mad2 was overexpressed in SCLC samples.Therefore,mad2 ought to play a critical role in the pathology of SCLC.The combined expression of mad2 with MSA and ACA may contribute to enhancing the sensitivity and specificity of detection;this expression may allow early diag-nosis and clinical diagnosis of SLCC and may be a promising treatment for SCLC.
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Objective To explore the molecular mechanism of BRAFV600E inducing chromosome instability in Sbcl2 and SK-MEL31 melanoma cells.Methods The endogenous Mps1 in stable Sbcl2-and SK-MEL31-B-RafV600E expression cells were depleted by siRNA approach.To test the effect of B-RafV600E on the centrosome amplification and the formation of multipolar spindles,cells at S-phase with HU-treatment were arrested and then the centrosomes and mitotic spindles structure were detected through immunofluoresence.Results The percentage of B-RafV600E expressing Sbcl2 and SK-MEL31 cells (Sbcl2-B-RafV600E and SKMEL31-B-RafV600E) with centrosome amplification and multipolar spindle was reduced from 36 ％ to 6 ％ when Mps1 was absent.Conclusion B-RafV600E leads to centrosome amplification and multipolar spindle through Mps1,thus results in chromosome instability in Sbcl2 and SK-MEL31 melanoma cells.
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Objective To explore the regulation of Bub1 mRNA expression in endometrial carcinoma cells by estrogen and paclitaxel. Methods The high differentiated endometrial adenocarcinoma cells ( Ishikawa cell line) were cultured in DMED/F12 supplemented with 10% fetal bovine serum(FBS) or phenol red-free DMED/F12 supplemented with 5% dextran-charcoal FBS (dcFBS). Firstly, the cells were stimulated by 10 nmol/L estradiol (17β-E2 ) or no hormonal stimulation as control group, and the cell proliferation was quantified at 24, 48 and 72 hours using cell counting kit-8 (CCK-8) method. Then the cells were stimulated with different concentrations of 17β-E2 (0.1, 10, 1000 nmol/L) at different periods (5,15,30 minites and 2,4,8,12,16,24,30 hours), the expression of Bub1 mRNA was detected by real-time quantitative PCR. Ishikawa cells were cultured with non-serum DMEM/F12 to be synchronized, and then were treated with different concentrations of paclitaxel( 10,100 nmol/L) for 8 and 24 hours. While, nonsynchronized Ishikawa cells were exposed to 100 nmoL/L paclitaxel for different periods(4, 8, 16, 24,48 hours), and real-time quantitative PCR was also used to detect the expression levels of Bub1 mRNA.Data were presented as folds change relative to control group, in which values ＜ 1 were down-regulated, and those ＞ 1 were up regulated. Results The proliferation rate of cells in the presence of 17β-E2 was significantly highter than that of the control group after treated 24 hours (A value: 0. 70 ±0. 08 vs. 0. 86 ±0.10, P = 0.049). Time-dependent experiments revealed that addition of 17β-E2 could increase cell numbers during 72 hours period, while the expression level of Bub1 mRNA was decreased in Ishikawa cell.Dose-dependent experiments revealed maximal estradiol stimulation effects at 10 nmol/L( P = 0. 020). After being treated with serum-free culture, Ishikawa cells were exposed to 10 nmol/L paclitaxel for 8 and 24 hours, and the expression of Bubl mRNA decreased (0. 403 ± 0. 008 vs. 0. 775 ± 0. 144, P = 0. 251 ).Compared to the control cells, the mRNA expression levels of Bub1 in cells treated by paclitaxel for 8 hours was significantly decreased ( P = 0. 009 ), while there was not significantly decreased at 24 hours ( P =0. 396). When exposed to 100 nmol/L paclitaxel for 8 and 24 hours, the expression of Bubl mRNA was also decreased(0. 697 ±0. 017 vs. 0. 850 ±0. 004, P =0. 061 ). Compared to the control cells, Bub1 mRNA expression was also significantly decreased (P = 0. 038 and P= 0. 019, respectively). While with serum freetreatment culture, when Ishikawa cells exposed to 100 nmoL/L paclitxel for 4, 8, 16, 24 or 48 hours, the expression of Bub1 mRNA significantly increased ( 1. 127 ± 0. 105 vs. 1. 614 ± 0. 154 vs. 2. 092 ± 0. 179vs. 1. 381 ± 0. 061 vs. 1. 519 ± 0. 182, P = 0. 002 ), of which was signicantaly increased at 16 hours treatment. Conclusion Bub1 exrpession could be regulated by estradiol and paclitaxel, in which deregulated Bubl expression may contribute to chemotherapeutic efficacy of paclitaxel.
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0bjectlve To examine the metaphase II spindle and chromosome configurations of human oocytes cultured for different times after thawing.MethodsUsing slow.cooling and raid—thawing protocol combined with 0.3 mol/L sucrose and 1.5 mol/L 1,2-propanedio 1(1,2-PROH)to cryoprotect human mature oocytes(n=102),the 64 survival oocytes without abnormal zona pellucida and cytoskeletal were randomly assigned to three groups after thawing:group A:culture 1 hour(n=20),group B:culture 3 hour(n=22),group C:culture 5 hours(n=22),the flesh oocytes served as control group(n=18).Immunocytochefifical staining and fluorescence microscopy were used to assess the morphology of the metaphase II spindle and chromosome.Results(1)The normal spindle rates of groups A,B and C were 10％(2/20),46％(10/22)and 41％(9/22)respectively,significantly decreased compared with control group(83％,15/18;P<0.05).The rates of absent spindle in group A(45％,9/20)was significantly higher than control group(6％,1/18;P<0.01).Also,the rates of absent spindle in group A was higher than groups B(14％,3/20)and C(14％,3/20;P<0.05).However,no significant differences were observed in groups B and C(P>0.05).(2)A significant increase in abnormal chromosome rate was observed in group A(30％,6/20)compared to groups B(68％,15/22),C(64％,14/22)and control group(78％,14/18;尸<0.05).No differences in chromosome morphology were observed in groups B,C and control group(P>0.05).Conclusions The cryoproteetant protocol leads to a deleterious effect on the organization of the meiotic spindle and chromosome at MI stage.The 3—5 hours post—thawing incubation could permit restoration of the meiotic spindles and chromosome.
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Objective To investigate the role of ECRG2,a novel tumor suppressor gene,in spindle assembly checkpoint. Methods Using siRNA approach to deplete the expression of ECRG2, using immunofluorescence to test the distribution of ECRG2,using Western blotting to examine the expression cell cycle proteins.Results ECRG2 localized to centrosomes during interphase and kinetochores during mitosis.Further analysis revealed that ECRG2 participates in the spindle assembly checkpoint.Depletion of ECRG2 abolished the spindle assembly checkpoint.Conclusion Our results indicated that ECRG2 is important for ensuring spindle assembly checkpoint,accurate chromosome segregation,and its depletion may contribute to chmmosome instability and aneuploidy in human cancers.
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Objective To evaluate the development of immature oocytes after freezing-thawing by conventional cryopreservation method for mature oocytes.Methods Immature oocytes were collected from stimulated ovaries of intracytoplasmic sperm injection(ICSI)cycles.Immature oocytes were in vitro matured directly or after slow freezing-fast thawing and immunostained for tubulin and chromatin and at last visualized by confocal microscopy.Results No statistical difference was found in maturity rate between freezing groups and the controls.There was a statistically significant increase in abnormalities of chromosome(23.7% vs. 50%)and spindle(28.9% vs.53.9%)in the GV freezing group compared with the GV control(P