Objective To observe the influence of the indomethacin on the proliferative and invasive activity of OCM-1 human choroidal melanoma cells. Methods OCM-1 cells were cultured with different concentrations of indomethacin (25, 50, 100, 200, 400 mu;mol/L ), and their proliferation were assessed by methyl thiazolyl tetrazolium（MTT）, invasive behaviors were examined by cell invasion assays, expression of survivin and VEGF were evaluated by reverse transcriptase polymerase chain reaction（RT-PCR）, immunofluorescence staining, ELISA and western blot analysis. Result All concentrations of indomethacin in this study can inhibit the proliferation and invasion of OCM-1 cells in a time and dosage-dependant manner（MTT/24 h：F=19.642，P＜0.01；MTT/48 h：F=136.597，P＜0.01；MTT/72 h：F=582.543，P＜0.01；invasion assays：F=54.225，P＜0.01）. Immunofluorescence staining indicated that survivin and VEGF mainly expressed in the cytoplasm of OCM-1 cells. Survivin mRNA in OCM-1 cells was inhibited by 100, 200, 400 mu;mol/L indomethacin（F=16.679，P＜0.01）. The concentrations of survivin were (787.3plusmn;47.37), (257.0plusmn;26.21), (123.3plusmn;8.02) pg/ml in control group and 100, 400 mu;mol/L indomethacin groups, respectively. Survivin expression was also significantly down-regulated in indomethacin-treated cells by Western blot analysis.Indomethacin had no effects on VEGF expression in OCM-1 cells.Conclusions Indomethacin can inhibit proliferation and invasion of OCM-1 cells in vitro,down-regulated expression of survivin may be the mechanism.
ObjectiveTo observe the effects of NDRG1 on proliferation, migration and lumen formation of retinal vascular endothelial cells (RF/6A cells) in monkeys under high glucose condition. MethodsRF/6A cells were divided into normal group, mannitol group, high glucose group, small interfering RNA (siRNA) negative control group without target gene (siRNA group), 30 nmol/L siRNA down-regulated NDRG1 genome (siNDRG1 group) and 50 nmol/L siNDRG1 group. Normal group cells were cultured conventionally. The mannitol group was added with 25 mmol/L mannitol, and the high-glucose group was added with 25 mmol/L glucose. In the siRNA group, 25 mmol/L glucose was added, and then blank siRNA was added for induction. The 30 and 50 nmol/L siNDRG1 groups were added with 25 mmol/L glucose and induced with 30 and 50 nmol/L siRNDRG1, respectively. All cells were incubated for 24 h for follow-up experiments. Cell proliferation was observed by 4', 6-diaminidine 2-phenylindole staining. Cell counting kit-8 staining was used to detect cell activity. The expression level of NDRG1 mRNA and protein was detected by Western blot and real-time quantitative polymerase chain reaction. Cell migration was observed by cell scratch assay. Cell lumen formation assay was used to detect lumen formation. The two-tailed Student t test was used to compare the two groups. One-way analysis of variance was used to compare groups. ResultsThere were significant differences in cell proliferation rate (t=36.659, 57.645) mobility rate (t=24.745, 33.638) and lumen formation number (t=41.276, 22.867) between high glucose group and normal group and mannitol group (P＜0.01). Compared with normal group and mannitol group, the relative expression levels of NDRG1gene mRNA and protein in high glucose group were significantly decreased, with statistical significance (t=46.145, 21.541, 36.738, 32.976; P＜0.001). Compared with the siRNA negative group, the relative expression levels of NDRG1gene mRNA and protein in 30 nmol/L siNDRG1 group and 50 nmol/L siNDRG1 group were significantly decreased, and the differences were statistically significant (t=44.275, 40.7577, 57.167, 25.877; P＜0.01). Compared with normal group and siRNA group, cell mobility in 30 nmol/LsiNDRG1 group was increased, and the difference was statistically significant (t=57.562, 49.522; P＜0.01). Compared with normal group and siRNA group, the number of cell lumen formation in 30 nmol/LsiNDRG1 group was significantly increased in the same field of vision, and the difference was statistically significant (t=63.446, 42.742; P＜0.01). ConclusionDown-regulation of NDRG1 gene can improve the activity, migration and lumen formation of RF/6A cells under hyperglycemia.
OBJECTIVE: To investigate the effects of Ginsenoside Rb1 on the proliferation of Schwann cell cultured. METHODS: The sciatic nerve from SD rats was cultured in vitro; 10 micrograms/ml, 20 micrograms/ml, 200 micrograms/ml and 1 mg/ml Ginsenoside Rb1 was applied on the fifth day of culture. The proliferation of Schwann cells of sciatic nerves was determined in different time by MTT assay and thymidine incorporation assay. RESULTS: 10 micrograms/ml of Ginsenoside Rb1 significantly induced Schwann cell proliferation better than DMEM cell culture medium, but higher concentrations of Ginsenoside Rb1 at 1 mg/ml significantly inhibited the proliferation of Schwann cells, whereas 200 micrograms/ml of Ginsenoside Rb1 had similar effects to DMEM culture medium. CONCLUSION: Ginsenoside Rb1 at the optimal concentration is effective on inducing the proliferation of Schwann cells, but at higher concentration is cytotoxic for Schwann cells.
ObjectiveTo investigate the effects of thrombospondin-1 active fragment (TSP-1) synthetical peptide VR-10 on proliferation and migration of rhesus choroidal-retinal endothelial (RF/6A) cell and the expressions of apoptosis relative genes in RF/6A cell. MethodsThe survival rate of RF/6A cell were detected by methyl thiazolyl tetrazolium, and migration ability was measured by transwell chamber after exposure to 1.0 μg/ml TSP-1 and synthetic peptide VR-10 (0.1, 1.0, 10.0 μg/ml) for different times (6, 12, 24, 48 hours). Caspase-3 and factor associated suicide (FAS) protein levels were measured by Western blot. The mRNA level of bcl-2 and FAS ligand (FASL) were measured by reverse transcription-polymerase chain reaction (RT-PCR). ResultsThe survival rate of RF/6A cells was determined by the treatment time and concentration of TSP-1(1.0 μg/ml) and the synthetic peptide VR-10 (0.1, 1.0, 10.0 μg/ml). The lowest survival ratio of RF/6A was 78% (P < 0.001) when cells were treated by 10 μg/ml synthetic peptide VR-10 after 48 hours. TSP-1 and synthetic peptide VR-10 could inhibit migration of RF/6A cells in transwell chamber (P < 0.001). 10.0 μg/ml synthetic peptide VR-10 had the strongest effect, 1.0 μg/ml TSP-1 was the next. Migration inhibition rate was increase with the increase of the concentration of VR-10 (P < 0.001). There was no significant differences between 0.1 μg/ml and 1.0 μg/ml VR-10 (P=0.114). Western bolt showed that RF/6A cell in control group mainly expressed the 32×103 procaspase-3 forms. To 10.0 μg/ml VR-10 treated group, it showed decreased expression of procaspase-3 (32×103) and concomitant increased expression of its shorter proapoptotic forms (20×103). Compared with control group, expression of FAS peptides were significantly increased in 10.0 μg/ml VR-10 treated group. Compared with control group, expression of FasL mRNA was significantly increased in 10.0 μg/ml VR-10 treated group(t=39.365, P=0.001), but the expression of bcl-2 mRNA was decreased(t=-67.419, P=0.000). ConclusionTSP-1 and synthetic peptide VR-10 had the ability to inhibit proliferation and migration of endothelial cell, and also induce apoptosis by increasing FAS/FASL expression and repressing bcl-2 expression.
Objective To investigate the influence of RNA interference targeting c-Jun gene on the proliferation of rat vascular smooth muscle cells (VSMCs). Methods The experiment was performed with c-Jun siRNA （c-Jun siRNA group）, control reverse sequence siRNA (control siRNA group) or no siRNA (control group). VSMCs were transfected with siRNA targeting c-Jun gene by liposome. Effects of c-Jun siRNA on mRNA and protein expressions of c-Jun were examined by RT-PCR analysis and Western blot respectively. MTT test and 3H-TdR incorporation were used to detect VSMCs proliferation. Cell cycle analysis of VSMCs in vitro was determined by flow cytometer. Results The expression levels of mRNA and protein of c-Jun in c-Jun siRNA group were significantly lower than those in control group （P＜0.05， P＜0.01). There was no significant difference between control group and control siRNA group （Pgt;0.05）. Proliferation activity of VSMCs decreased significantly in c-Jun siRNA group compared with that in control group （P＜0.05） and VSMCs was blocked in the G0/G1 phase of cell cycle significantly （P＜0.05）. There was no significant difference between control group and control siRNA group （Pgt;0.05）. Conclusion c-Jun gene silenced by RNA interference can inhibit VSMCs proliferation effectively in vitro.
Objective To investigate the effect of berbamine (BBM) on the proliferation and apoptosis of retinoblastoma (RB) HXO-RB44 cells and its possible mechanism in vitro.Methods RB cells in logarithmic growth phase were divided into BBM treated group and control group. RB cells in BBM treated group were cultured with different concentrations of BBM (2,4,8,16 and 32 mg/L) for 24,48 and 72 hours, respectively. The proliferation was assayed by methyl Thiazolyl tetrazolium (MTT). RB cells were cultured with different concentrations of BBM (4,8 and 16 mg/L) for 24 hours. The early apoptotic rates were detected by flow cytometry; the expression of bcl-2 and Bax were measured by enzyme-linked immunosorbent assay (ELISA) and the activity of Caspase-3 was detected by colorimetric assay.Results BBM could obviously inhibit the proliferation of RB cells in a time and dose dependent manner (24 hours: F=70.547,P＜0.01; 48 hours: F=603.438,P＜0.01; 72 hours: F=577.521,P＜0.01). The IC50 value at 24,48 and 72 hours were 25.26, 10.94 and 6.25 mg/L, respectively. Necrosis rates of control group and BBM treated group were (1.25plusmn;0.45)%, (4.10plusmn;2.95)%, (4.39plusmn;0.21)% and (10.54plusmn;4.38)% respectively; the difference between two groups was statistically significant (F=6.527,P＜0.05). Apoptotic and necrosis rates in advanced stage of control group and BBM treated group were (2.13plusmn;0.71)%, (5.45plusmn;2.31)%, (9.86plusmn;3.18)% and (11.10plusmn;1.70)%, respectively. The difference between two groups was statistically significant (F=10.845,P＜0.05). Early apoptotic rates of control group and BBM treated group were (0.51plusmn;0.26)%, (2.68plusmn;0.35)%, (5.97plusmn;0.50)% and (11.22plusmn;1.17)%, respectively. The difference between two groups was statistically significant (F=144.976,P＜0.01). In addition, BBM dose-dependently reduced bcl-2 level and increased Bax expression, causing the reduction of the bcl-2/Bax protein ratio as well as increased the Caspase-3 activity in RB cells remarkably (bcl-2: F=835.726,P＜0.01; bax: F=111.963, P＜0.01;Caspase-3:F=298.058,P＜0.01).Conclusions BBM can inhibit the proliferation and induce apoptosis or necrosis of RB cells in vitro, down regulating the expression of bcl-2, up regulating the expression of Bax. Along with increased Caspase-3 activity these may be the apoptotic mechanisms.
Objective To observe the effects of Galectin-3 on proliferation of vascular endothelial cells derived from peripheral blood endothelial progenitor cells. Methods The cultured peripheral blood endothelial progenitor cells in vitro were isolated and purified from human peripheral blood, and the cells were differentiated into vascular endothelial cells. Then the cells were cultivated with the galectin-3 of different concentrations, and to observe the proliferation of endothelial cells derived from peripheral blood endothelial progenitor cells. Results The abilities of proliferation of endothelial cells derived from peripheral blood endothelial progenitor cells of 0.1, 1.0, 2.5, 5.0, and 10.0 μg/ml groups were higher than that of 0 μg/ml group, there were not statistic significance of the differences between the 0.1，1.0, 2.5, and 0 μg/ml groups （P＞0.05）. But the abilities of proliferation of 5.0 and 10.0 μg/ml groups were obviously higher than that of 0, 0.1, 1.0, and 2.5 μg/ml groups （P＜0.05）, and the abilities of proliferation of 10.0 μg/ml group was also higher than that of 5.0 μg/ml group （P＜0.05）. Conclusion Galectin-3 can promote the proliferation of endothelial cells derived from peripheral blood endothelial progenitor cell.
ObjectiveTo observe the expression in vitro and the influence of adenovirus-mediated recombinant Tum5 gene to the proliferation, migration and tubing of Rhesus RF/6A cell under high glucose. MethodsTo construct the adenovirus vector of recombinant Tum5 gene (rAd-Tum5), and then infected RF/6A cell with it. The Flow Cytometry was used to detect the infection efficiency. RF/6A cells were divided into normal group, high glucose (HG)-control group (HG group), empty expression vector group (HG+rAd-GFP), and HG+rAd-Tum5 group. Western blot was used to detect the expression of Tum5. The CCK-8 test was applied to detect the proliferation of RF/6A cell, the Transwell test was applied to detect the migration and the Matrigel test was applied to detect the tubing of RF/6A cell under high glucose. The proliferation, migration and tubing of RF/6A were tested respectively by CCK-8 test, Transwell test and Matrigel test. ResultsThe adenovirus vector of recombinant Tum5 gene was successfully constructed. The infection efficiency of rAd-Tum5 in RF/6A cell was 50.31% and rAd-GFP was 55.13% by the Flow Cytometry. The results of Western blot indicated that Tum5 was successfully expressed in RF/6A cell. The result of CCK-8 test, Transwell test and Matrigel test indicated that there were statistical differences between all groups in proliferation, migration and tubing of the RF/6A cell (F=44.484, 772.666, 137.696;P < 0.05). The comparison of each group indicated that the HG group was higher than normal group (P < 0.05). There were no statistical differences between HG group and HG+rAd-GFP group (P > 0.05). However, the HG+rAd-Tum5 group was less than HG group (P < 0.05), and the same to HG+rAd-GFP (P < 0.05). ConclusionThe adenovirus vector of recombinant Tum5 gene can inhibit the proliferation, migration and tubing of RF/6A cell under high glucose.
Objective To investigate the effect of carboxymethylated chitosan (CMCS) on the proliferation, cell cycle, and secretion of neurotrophic factors in cultured Schwann cells (SCs). Methods SCs were obtained from sciatic nerves of 20 Sprague Dawley rats (3-5 days old; male or female; weighing, 25-30 g) and cultured in vitro, SCs were identified and purified by immunofluorescence against S-100. The cell counting kit 8 (CCK-8) assay was used to determine the proliferation of SCs. The SCs were divided into 4 groups: 50 μg/mL CMCS (group B), 100 μg/mL CMCS (group C), 200 μg/mL CMCS (group D), and the same amount of PBS (group A) were added. The flow cytometry was used to analyze the cell cycle of SCs; the real-time quantitative PCR and Western blot analysis were used to detect the levels of never growth factor (NGF) and ciliary neurotrophic factor (CNTF) in cultured SCs induced by CMCS. Results The purity of cultured SCs was more than 90% by immunofluorescence against S-100; the CCK-8 results indicated that CMCS in concentrations of 10-1 000 μg/mL could promote the proliferation of SCs, especially in concentrations of 200 and 500 μg/mL (P lt; 0.01), but no significant difference was found between 200 and 500 μg/mL (P gt; 0.05). CMCS at a concentration of 200 μg/mL for 24 hours induced the highest proliferation, showing significant difference when compared with that at 0 hour (P lt; 0.01). The percentage of cells in phase S and the proliferation index were significantly higher in groups B, C, and D than in group A (P lt; 0.05), in groups C and D than in group B (P lt; 0.05); and there was no significant difference between group C and group D (P gt; 0.05). Real-time quantitative PCR and Western blot results showed that the levels of NGF and CNTF in groups B, C, and D were significantly higher than those in group A (P lt; 0.05), especially in group D. Conclusion CMCS can stimulate the proliferation, and induce the synthesis of neurotrophic factors in cultured SCs.
ObjectiveTo investigate the influences of lactic acid (LA), the final degradation product of polylactic acid (PLA) on the prol iferation and osteoblastic phenotype of osteoblast-l ike cells so as to provide theoretical basis for bone tissue engineering. MethodsRos17/2.8 osteoblast-l ike cells were harvested and divided into 3 groups. In groups A and B, the cells were cultured with the medium containing 4, 8, 16, 22, and 27 mmol/L L-LA and D, L-LA, respectively. In group C, the cells were cultured with normal medium (pH7.4). The cell prol iferation was determined with MTT method after 1, 3, and 5 days. The relative growth ratio (RGR) was calculated, and the cytotoxicity was evaluated according to national standard of China. In addition, the alkal ine phosphatase (ALP) activity of cells cultured with medium containing 4 mmol/L L-LA (group A), 4 mmol/ L D, L-LA (group B), and normal medium (group C) after 1 and 5 days were detected with ALP kits, and the relative ALP ratio (RAR) was calculated; after 21 days, the calcium nodules were tested with von Kossa staining method, and were quantitatively analyzed. ResultsWhen LA concentration was 4 mmol/L, the mean RGR of both groups A and B were all above 80%, and the cytotoxic grades were grade 0 or 1, which meant non-cytotoxicity. When LA concentration was 8 mmol/L and 16 mmol/ L, groups A and B showed cytotoxicity after 5 days and 3 days, respectively. When LA concentration was above 22 mmol/L, cell prol iferations of groups A and B were inhibited evidently after 1-day culture. At each LA concentration, RGR of group A was significantly higher than that of group B at the same culture time (P<0.05) except those at 4 mmol/L after 1-day and 3-day culture. After 1 day, the RAR of group A was significantly higher than that of group B on 1 day (144.1%±3.2% vs. 115.2%±9.8%, P<0.05) and on 5 days (129.6%±9.8% vs. 78.2%±6.9%, P<0.05). The results of von Kossa staining showed that the black gobbets in group A were obviously more than those of groups B and C. The staining area of group A (91.2%±8.2%) was significantly higher than that of groups B (50.3%±7.9%) and C (54.2%±8.6%) (P<0.05). ConclusionThe concentration and composition of LA have significant effects on the cell proliferation and osteoblastic phenotype of osteoblast-l ike cells.