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@#Objective To analyze the effect of different treatment conditions on cells synchronization in G0/G1 phase to get the best con-dition, and to explore its effect on neural differentiation of bone marrow mesenchymal stem cells (BMSCs) induced by basic fibroblast growth factor (bFGF). Methods BMSCs were isolated and cultured in 5%, 1%, 0.5%, 0.1%, 0 fetal bovine serum (FBS) respectively, for 24 hours and 48 hours. After PI staining, cell cycle proportions of each phase were detected by flow cytometry, and were compared with the normal group (10%FBS). After the optimal treatment condition was got, 20 ng/ml bFGF was added into synchronization group and unsyn-chronization group 3 days and 7 days, respectively. The expression of Nestin and Tuj-1 were detected with immunofluorescence. Results Adult rat BMSCs were isolated from bone marrow and cultured, after passage, the cells were with long spindle shape. Compared with the normal group, the cell proportion of G1/G0 phase increased under different treatments, peaked with (94.274 ± 0.468)%under 1%FBS, 48 hours (F=39.91, P<0.001). After bFGF induction for 3 days, the Nestin+cell number was higher in the synchronization group than in the un-synchronization group [(80.3 ± 2.4)%vs. (12.1 ± 1.5)%] (F=28.25, P<0.001). After bFGF induction for 7 days, the Tuj-1+cell number was higher in the synchronization group than in the unsynchronization group [(74.8±3.2%)%vs. (19.3±2.5)%] (F=17.95, P<0.001). Conclusion 1%FBS, 48 hours is the optimal condition to BMSCs synchronization in G0/G1 phase, which can promote the neural differentiation of BM-SCs.
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Objective: To observe the protection of Testudinis Carapax et Plastrum extracts (TCPE) on serum starvation-induced PC12 cell apoptosis and explore its mechanism. Methods: The PC12 apoptosis model was established by serum starvation for 3 d. The cells were randomly divided into four groups: control group, model group, low-dose and high-dose (3 and 30 μg/mL) TCPE groups. In the three days of the treatment, cell absorbance was determined by MTT, ratio of cell apoptosis was examined by Annexin V/PI double stain flow cytometry (FCM), Caspase-3, BMP4, BMPR-IA, and p-Smad1/5/8 signaling molecular expression were detected by Western blotting, and the anti-apoptotic effect of TCPE was observed after blocking BMPs signal pathway. Semi-quantitative analysis of bands was carried out by Bio-Rad Quantity One gel analysis system. Results: MTT and FCM analyses demonstrated that TCPE could increase PC12 cell viability and decrease their apoptotic ratios in a dose dependent manner. Western blotting results showed that TCPE could decrease Caspase-3 expression, promote the expression of BMP4, BMPR-IA, and p-Smad1/5/8. There was statistically significant difference between TCPE (3 and 30 μg/mL) groups and model group (P<0.05, P<0.01) in all above results. While TCPE had no effect on the expression of BMP2, BMP7, and BMPR-II. BMPR-IB hadn't been detected. The anti-apoptotic activity was partially mitigated by neutralizing BMP4 antibody. Conclusion: TCPE has the capacity to inhibit the apoptosis of PC12 induced by serum starvation in a dose dependent manner and its mechanism may be associated with partially activating and up-regulating the expression of BMP4 signaling pathway.
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Objective:To investigate the expression of vascular endothelial growth factor (VEGF) in oral squamous cell carcinoma by serum-starvation and hypoxia. Methods:The expression of VEGF mRNA was evaluated in two oral squamous cell carcinomas cells TSCCa and GNM treated with serum-starvation and hypoxia.using semi-quantitative RT-PCR. The activities of VEGF in two cell lines were detected by enzyme linked immunosorbent assay (ELISA). Results:Contrast to TSCCa cells, GNM cells expressed significantly higher levels of mRNA of VEGF by RT-PCR detection. Serum-starvation and hypoxia could increased expression of VEGF mRNA in both two cell lines in vitro, the levels of mRNA of VEGF increased more in TSCCa cell lines than that in GNM cell lines when treated with serum-starvation and hypoxic. After 4 hours hypoxic treatment, the activities of VEGF increased significantly and approached the peak after 8 hours; the activities of VEGF in GNM cell lines was dramatically twice higher than that of the control group, while the activities of VEGF increased by 6 times in TSCCa cell lines. The activities of VEGF in TSCCa cell lines were higher than that of in GNM cell lines when treated with serum-starvation. Conclusion: Serum-starvation and hypoxia can up-regulate VEGF mRNA expression and activities in oral squamous cell lines in vitro,which may play an important role in the angiogenesis of oral squamous cell carcinoma.
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PURPOSE: In the present study, the effects of bFGF on the early responses of proliferation of UMR 106-01 osteoblast cells during cell cycle reentry from the latent(G0/G1) to the proliferative periods(S/M) were investigated. MATERIALS AND METHODS: The synchronized cell culture method using the serum starvation was utilized. After the addition of bFGF, the time courses of protein synthesis, DNA synthesis, thymidylate synthase(TS) activity, TS mRNA level and expression of c-fos were determined. RESULTS: 87% UMR 106-01 cells were synchronized to G0/G1 by serum starvation for seven days in the medium containing 0.1% serum. The protein level began to increase 3 hours after bFGF treatment and reached the maximum at 18 hours. TS activity began to increase 3 hours after the bFGF treatment and reached its peak at 6 hours while its mRNA level, determined by quantitative PCR, reached the maximum at 12 hours. The expression of c-fos protein, determined by western blot analysis and immunocytochemistry, increased 3 hours after bFGF treatment. On the contrary, these prominent changes and responses to bFGF were not observed in the case of using non-synchronized cells cultured in the medium containing 10% serum. CONCLUSION: Based on these data it can be concluded that bFGF-induced DNA synthesis in the early proliferative phase is due to increases in both TS activity and mRNA amount and that the increase in c-fos expression and TS activity occur before the increase in TS mRNA level.
Subject(s)
Blotting, Western , Cell Culture Techniques , Cell Cycle , DNA , Fibroblasts , Immunohistochemistry , Osteoblasts , Polymerase Chain Reaction , RNA, Messenger , Starvation , Thymidylate SynthaseABSTRACT
PURPOSE: In the present study, the effects of bFGF on the early responses of proliferation of UMR 106-01 osteoblast cells during cell cycle reentry from the latent(G0/G1) to the proliferative periods(S/M) were investigated. MATERIALS AND METHODS: The synchronized cell culture method using the serum starvation was utilized. After the addition of bFGF, the time courses of protein synthesis, DNA synthesis, thymidylate synthase(TS) activity, TS mRNA level and expression of c-fos were determined. RESULTS: 87% UMR 106-01 cells were synchronized to G0/G1 by serum starvation for seven days in the medium containing 0.1% serum. The protein level began to increase 3 hours after bFGF treatment and reached the maximum at 18 hours. TS activity began to increase 3 hours after the bFGF treatment and reached its peak at 6 hours while its mRNA level, determined by quantitative PCR, reached the maximum at 12 hours. The expression of c-fos protein, determined by western blot analysis and immunocytochemistry, increased 3 hours after bFGF treatment. On the contrary, these prominent changes and responses to bFGF were not observed in the case of using non-synchronized cells cultured in the medium containing 10% serum. CONCLUSION: Based on these data it can be concluded that bFGF-induced DNA synthesis in the early proliferative phase is due to increases in both TS activity and mRNA amount and that the increase in c-fos expression and TS activity occur before the increase in TS mRNA level.
Subject(s)
Blotting, Western , Cell Culture Techniques , Cell Cycle , DNA , Fibroblasts , Immunohistochemistry , Osteoblasts , Polymerase Chain Reaction , RNA, Messenger , Starvation , Thymidylate SynthaseABSTRACT
Objective:To construct a phage display antibody library of special antigen responding to serum starved U251 cell,from which the serum responding gene and protein would be gotten.Methods:U251 cells were cultured in serum-absent midium for 48 h.Its protein was extracted and used to immunize BALB/c mice.Total RNA of the spleenocytes of immunized mice was extracted.VH and VL were amplified by RT-PCR and were linked into ScFv(Single chain fragment of variation) with a linker.ScFv was recombined to pCANTAB5E vector and was transformed to TG1 strain.Results:The library capacity was up to 3?106 cfu/L.A positive clone was identified from 8 random clones of this library.Conclusion:The special ScFv phage library is constructed successfully.It is the basis for screening special antibodies which can recognize serum responding protein.
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Objective To evaluate influence of benzo(a)pyrene on cell cycle of the human embryonic lung fibroblast at different synchronous stage. Methods Cell cycle distribution were detected by flow cytometry(FCM). Cells were synchronized at cycle G0 stage through 48 hours serum starvation and reenter cell cycle together at a synchronous state after resupplied with whole growth medium contain good serum. Benzo(a)pyrene with a series concentrations were directly used or metabolized active by traditional and modified metabolize active methods through preincubation of S9 and were treated as regent to HELF cells mostly synchronized at G1, S or G2-M stage respectively. Then cell cycle distribution changes were detected 12 h after treatment. Results Serum starvation(48 hours) could synchronize HELF at G0-G1 stage effectively and 10-12 h, 16-18 h, 22-24 h were cycle phase change distinctly time after serum restimulated. Cells were synchronized at G1, S and G2-M stage mostly. Benzo(a)pyrene influence cycle distribution little without metabolize active, while modified methods could metabolize active benzo(a)pyrene well and avoided disturbing effect of S9 on cell cycle in traditional method. Except 2 ?mol/L benzo(a)pyrene treated at 22 h after restimulated caused the percentage of cells at S stage increase, most treatment induced the percentage of cells at S stage decrease which was associated obviously with the increasing dosages. The percentage of cell at S stage decrease at 16 h were more remarkable than other times and the percentage of cell at G2-M increase correspondingly. While the percentage of cell at G1 increase and the percentage of cell at G2-M decrease were obsered when benzo(a)pyrene treated at 10 h and 22 h after restimulated. Conclusion Serum starvation 48 hours and restimulate can synchronize HELF at different stage effectively. Modified metabolize active method is suitable for cell cycle research. Primary influence of benzo(a)pyrene on cell cycle is the decreased percentage of cells at S stage, G1 arrest, G2-M arrest and G1 arrest were occurred when benzo(a)pyrene were treated at G1, S and G2 stage respectively.