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Objective To evaluate the effect of try psin on the proliferation,migration and adhesion of a skin squamous cell carcinoma cell line A431.Methods Cultured A431 cells were divided into several experimental groups treated with trypsin at concentrations of 0.1,1,10 and 100 nmol/L for 24,48 and 72 hours respectively,and a control group treated with DMEM complete medium only.Cell counting kit-8 (CCK8) assay was conducted to evaluate cellular proliferative activity to select the optimal concentration of trypsin.Then,some A431 cells treated with trypsin at the selected concentration for 24,48 and 72 hours respectively (or 48 hours only) served as the experimental groups (or group),and other A431 cells treated with DMEM complete medium served as the control group.Flow cytometry was performed to assess cell cycle distribution and proliferation index,fibronectin-based adhesion assay to estimate cell adhesive capacity,and wound healing assay and Transwell assay were conducted to evaluate the migratory capacity of cells in two-and three-dimensional space.Statistical analysis was carried out by using analysis of variance,paired samples t test and chi-square test.Results The proliferative activity of A431 cells increased along with the increase of trypsin concentrations,with the strongest increasing effect observed at 100 nmol/L.After treatment with 100 nmol/L trypsin,the experimental group showed a decrease in the percentage of G1-phase cells,but an increase in the percentage of S-phase cells,proliferation index,migratory and adhesive capacity compared with the control group (all P ＜ 0.05).Conclusion Trypsin can promote the proliferation,migration and adhesion of A431 cells.
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Objective To analyze the expression characteristics of annexin A2 in dermal papilla cells (DPCs) with aggregative behavior.Methods Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were performed to measure the mRNA and protein expressions of annexin A2 respectively in DPCs with or without aggregative behavior.Results The mRNA expression level of annexin A2 was significantly higher in DPCs with aggregative behavior than in those without aggregative behavior (0.50 ± 0.15 vs.0.35 ± 0.19, t =8.26, P ＜ 0.05).Western blot showed that annexin A2 had two isoforms, including one isoform with a relative molecular mass of 40 000 and the other one with a relative molecular mass of 36 000.The annexin A2 isoform with a relative molecular mass of 40 000 was highly expressed in both DPCs with aggregative behavior and those without aggregative behavior, while the other isoform was only expressed in DPCs with aggregative behavior.Conclusion Annexin A2 may be closely related to the aggregative growth of DPCs.
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Objective To investigate the effect of hyperoxia on growth of type Ⅱ alveolar epithelial cells (AECⅡ).MethodsLungs of fetal rats at 19 days of pregnancy were collected,and AEC Ⅱ was isolated and cultured by differential adherence method.Cells were randomly divided into air group and hyperoxia group.In air group,cells were cultured in 5％ CO2 incubator.And cells in hyperoxia group were cultured in 5％ CO2+95％ O2 incubator.The growth,activity,cell cycle,cell apoptosis of AEC Ⅱ were observed at 2,4,6 and 8 days of culture.The interaction between different time and groups were analyzed by ANOVA of factorial design.Comparison of means was done by two-sample independent t test and one-way analysis of variance.Bonferroni correction was used during the comparisons.Results(1) Cell growth situation:in hyperoxia group,cell number was decreased from2 hto 8 h [(7.29±0.43)×105/ml,(2.68±0.37)×105/ml,(0.23±0.10)×105/ml and (0.00±0.00) × 105/ml],and lower than those in air group [(10.41 ± 0.24) × 105/ml,(27.90±1.91) × 105/ml,(27.12±0.85) ×105/ml and (26.29±1.59) × 105/ml](t=10.992,38.912,94.166and 49.696,P=0.000 respectively). (2) Cell activity:the living cells ratio in hyperoxia group at 2 d[(79.00±0.71) ％],4 d [(52.80±1.14)％] and 6 d [(31.60±1.52)％] was lower than those [(97.00±0.71)％,(97.20±0.84)％ and (95.00±0.71)％] ir air group (t=31.213,70.519 and 84.722,P=0.000 respectively).(3) Cell cycle:the cell ratios of G1 phase and S phase in hyperoxia group at day 4 [(66.82±1.20) ％ and (27.31±1.16) ％] and day 6 [(70.22±1.27) ％ and (30.31±1.40) ％] were significantly higher than that at day 2 and that in air group (P＜0.05 respectively).(4) Cell apoptosis:in hyperoxia group,the cell ratio of Annexin-V+/PI- subgroup at 4 h was the highest [(23.89 ± 0.52)％],followed by those at day 2 and 6 [(21.32 ± 0.43)％ and (1.47 ±0.61)％].While the cell ratio of Annexin-V+/PI+ was the highest at 6 h [(53.92± 1.64)％],followed by those at 4 h and 2 h [(45.03±1.01)％ and (12.17±0.60)％],which were all different with those in air group(P＜0.05 respectively).ConclusionsHyperoxia might inhibit cell activity and cell cycle of AEC Ⅱ and promote apoptosis.
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Objective To confirm whether or not let-7b and miR-199a were significantly associated with malignant melanoma growth and proliferation. Methods An over -expression plasmid and an inhibitor, which targeted on let-7b and miR-199a, was constructed. B16F10 cells were divided into seven groups: control group, let-7b plasmid group, miR-199a plasmid group, empty plasmid group, let-7b inhibitor group, miR-199a inhibitor group, inhibitor control group. Foreign gene was transfected into B16F10 cells, let-7b and miR-199a expression were validated from RNA level, protein level and cell level. Results The relative let-7b or miR-199a gene expression of the let-7b plasmid group (3.8776±0.1372)and miR-199a plasmid group (2.8660±0.2821)were significantly higher than control group (P＜0.05), the relative let-7b or miR-199a gene expression of the let-7b inhibitor group (0.2057±0.0263) and miR-199a inhibitor group(0.2656±0.0253) were significantly lower than control group(P＜0.05). The cyclinD1 expression of the let-7b plasmid group(2.023±0.315) and let-7b inhibitor group (1.857±0.377) were significantly higher than control group (0.997±0.041) (P＜0.05), whereas, the Met expression of themiR-199a plasmid group (5.19±0.309) and miR-199a inhibitor group (4.87±0.044) were significantly higher than control group (2.2±0.198) (P＜0.05). The let-7b plasmid group and miR-199a plasmid group B16F10 cell growth rate were slower than control group, especially on the third day after transfection, the growth rate gradually dropped to the lowest value (P＜0.05). In addition, the apoptosis rates of the let-7b plasmid group and miR-199a plasmid group reach to (11.8±1.19)% and (11.3±1.59)%,which were significantly higher than control group (P＜0.05). Conclusions let-7b and miR-199a may be a negative regulator on the B16F10 cell growth and proliferation.
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Objective To investigate the effects of caveolin-1 overexpressing on the growth of cervical squamous cell cancer Hela cell line. Methods Eukaryotic expression vector of human caveolin-1 gene was introduced into Hela cells by Lipofectamine. The clones stably overexpressed caveolin-1 were identiffed by RT-PCR, immunofluorescence cell staining techniques and Westernblotting. Cells proliferation viabihty was tested by MTT assay, and flow cytometry was used to assay the cell cycle and apoptosis, and the relative phosphorylation level of extracellular regulated protein kinases (Erk1/2) were detected by Westernblotting. Results The clones stably overexpressed caveolin-1 were obtained. Compared with the parental Hela cells, the tranfected cells exhibited a slower rate of growth. FAGS analysis results revealed that overexpression of caveolin-1 resulted in the cell cycle arrest in the G0/G1 [ ( 68. 04 ± 2. 57 ) % vs ( 53.41 ±1.01)%] phase and increased the apoptotic cell fraction[ (19. 18 ±2.20)% vs (5.63 ±0.55)%, P <0. 05 ]. Western blotting results showed that overexpression of caveolin-1 reduced the phosphorylation of Erk1/2(0.28 ±0.05 vs 0.81 ±0.07, P <0.05). Conclusions Overexpression of caveolin-1 suppressed the growth of Hela cells and induced apoptosis, down-regulation of Erk1/2 phosphorylation might be involved in its mechanism.
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Objective: To study the inhibitory effects of periplocin from cortex periplocae (CPP) on proliferation of human esophageal carcinoma TE-13 cells and the related mechanism for cell cycle arrest. Methods: The inhibitory effects of CPP on the growth of TE-13 cells were measured by MTT assay. The morphological changes of TE-13 cells were analyzed by Gimsa staining. Detection of cell apoptosis and cell cycles were performed by flow cytometry. Protein expressions of cyclin-dependent kinase CDK2 and CDK4 were analyzed by Western blotting assay. Results:CPP significantly inhibited the proliferation of TE-13 cells in a dose- and time-dependent manner (P 0.05). CDK4 expression was inhibites by CPP at different concentrations, but the expression of CDK2 had no marked changes (P > 0.05). Conclusion: CPP significantly inhibites the growth of TE-13 cells. The action mechanism may be related with induction of cell cycle blocking and apoptosis.