【Abstract】ObjectiveTo investigate the growth effect of methylprednisolone （MP） on human hepatocellular carcinoma cell line HepG2.Methods Periodic distribution of cells and cellular apoptosis were detected by using cell culture,immunofluorescence staning of Annex Ⅴ and flow cytometric analysis in hepatocellular carcinoma cell.Results Compared with control group, methylprednisolone increased Ｇ0/Ｇ1 phase cell, decreased S phase cell on human hepatocellular carcinoma cell line HepG2 ,which had positive correlation with the time.The apoptosis rate and the necrosis rate of cells had the relation of dose-dependent with the concentration of MP, the cell membrane of early cellular apoptosis was stained green fluorescence. Conclusion Methylprednisolone can induce Ｇ0/Ｇ1 arrest , may play a proliferation-inhibition effect on the hepatocellular carcinoma cell line HepG2.
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.
ObjectiveTo study the effects of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) on the proliferation and differentiation of human bone marrow mesenchymal stem cells (hBMSCs).
MethodshBMSCs at passage 4 were divided into 4 groups according to different culture conditions:cells were treated with complete medium (α-MEM containing 10%FBS, group A), with complete medium containing 10 ng/mL LIF (group B), with complete medium containing 10 ng/mL bFGF (group C), and with complete medium containing 10 ng/mL LIF and 10 ng/mL bFGF (group D). The growth curves of hBMSCs at passage 4 in different groups were assayed by cell counting kit 8; cellular morphologic changes were observed under inverted phase contrast microscope; the surface markers of hBMSCs at passage 8 including CD44, CD90, CD19, and CD34 were detected by flow cytometry.
ResultsThe cell growth curves of each group were similar to the S-shape; the cell proliferation rates in 4 groups were in sequence of group D > group C > group B > group A. Obvious senescence and differentiation were observed very early in group A, cells in group B maintained good cellular morphology at the early stage, with slow proliferation and late senescence; a few cells in group C differentiated into nerve-like cells, with quick proliferation; and the cells in group D grew quickly and maintained cellular morphology of hBMSCs. The expressions of CD44 and CD90 in groups A and C at passage 8 cells were lower than those of groups B and D; the expressions of CD19 and CD34 were negative in 4 groups, exhibiting no obvious difference between groups.
ConclusionLIF combined with bFGF can not only maintain multiple differentiation potential of hBMSCs, but also promote proliferation of hBMSCs.
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 observe the effects of SARS-CoV-2 infection on the morphology, proliferation, apoptosis, cell cycle, and immune response function of mouse retinal photoreceptor cells (661w cells). MethodsA cell experiment. Logarithmic growth phase 661w cells were cultured in vitro and transfected with angiotensin-converting enzyme 2 (ACE2) overexpressing lentivirus to construct ACE2 overexpressing 661w cells that could be infected with SARS-CoV-2 pseudovirus (hereafter referred to as ‘pseudovirus’). The 661w cells were divided into three groups: the normal group (untreated), the siACE2 group (overexpressing ACE2 and not infected with the pseudovirus) and the infected group (overexpressing ACE2 and infected with the pseudovirus), in which the infected group was 5 TU/ml pseudovirus group, 15 TU/ml pseudovirus group, 30 TU/ml pseudovirus group and 50 TU/ml pseudovirus group, and the cells were infected with the pseudovirus for 12, 24, 48 and 72 h, respectively. The infected group was infected with 5 TU/ml pseudovirus group, 15 TU/ml pseudovirus group, 30 TU/ml pseudovirus group and 50 TU/ml pseudovirus group, respectively, for 12, 24, 48 and 72 h. Fluorescence microscopy was used to observe the transfection efficiency of ACE2; protein immunoblotting (Western blot) was used to detect the relative expression level of ACE2 in the cells; light microscope was used to observe the morphology of the cells in the normal and the infected groups; cell proliferation was detected by Cell Counting Kit-8 (CCK8) assay; flow cytometry was used to detect the cell cycle; Western blot and real-time quantitative polymerase chain reaction (qPCR) were used to detect the relative expression of interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), B lymphocytoma-2 (Bcl-2), Bcl-2-associated X-protein (Bax) proteins and mRNA in the cells of siACE2 group, infected group (30 TU/ml pseudovirus group); qPCR was used to detect the relative expression of nuclear factor (NF)- κB1 and NF-κB2, as well as NF- kB enhancer (P65) and precursor protein (P100) in cells of the siACE2 group and the infected group (30 TU/ml pseudovirus group). One-way ANOVA was used for comparison between multiple groups; t-test was used for comparison between two groups. Results Compared with the siACE2 group, the cells in the infected group showed different degrees of crumpling, and with the increase of the concentration and time of pseudovirus induction, the crumpling of the cells worsened, and the number of cells decreased. Compared with the normal group, the cells in the infected group showed a gradual decrease in cell viability with the prolongation of pseudovirus induction time, and the difference was no statistically significant (F=0.840, 0.412, 1.498, 1.138; P＞0.05), and the apoptotic index of the cells induced in the 30 and 50 TU/ml pseudovirus group was significantly elevated, and the difference was statistically significant (F=2.523, 6.716, 3.477, 3.421; P＜0.05). At 72 h of pseudovirus induction, compared with the siACE2 group, the G1 phase cells in the 30 TU/ml pseudovirus group were significantly increased, and the difference was statistically significant (t=3.812, P＜0.05); the relative expression of IL-6, TNF-α, Bax protein and mRNA in the cells was up-regulated (t=7.601, 6.039, 3.088, 5.193, 6.427, 7.667; P＜0.05), the relative expression of Bcl-2 protein and mRNA was down-regulated (t=3.614, 6.777; P＜0.05), and the relative expression of NF-κB1, NF-κB2, P65, and P100 mRNA was significantly up-regulated with statistically significant differences (t=3.550, 3.074, 3.307, 4.218; P＜0.05). ConclusionSARS-CoV-2 infection may inhibit photoreceptor cell proliferation, promote apoptosis and cycle blockade by activating the NF-κB signalling pathway.
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.
Objective To study the effects of L-arginine （L-Arg） on cell proliferation, inducible nitric oxide synthase (iNOS) expression and cell cycle in human colon carcinoma cell line LS174 through nitric oxide (NO) pathway. Methods LS174 cells were cultured in medium with L-Arg at different concentrations for different times. MTT method was employed to evaluate the level of the cell proliferation. The production of NO in culture supernatants of LS174 cell was detected with enzyme reduction of nitrate. The distribution of the cell cycle was detected with the flow cytometry (FCM). The expression level of iNOS in the cells was determined by Western blot and SP immunocytochemical staining method. Results The growth of LS174 was promoted by the L-Arg at low concentration (0.125 mmol/L) and inhibited at high concentrations (0.5， 2， 8 and 32 mmol/L). The level of NO was increased with the increasing concentration of L-Arg in culture medium. To compare with the control group, the ratio of cells at S phase was increased after 48 hours’ treatments with high concentrations (0.5, 2, 8 and 32 mmol/L) of L-Arg (P＜0.05, P＜0.01); while there was no obvious difference after treatments with low concentration (0.125 mmol/L) of L-Arg (Pgt;0.05). With the increase of the concentration of L-Arg, the expression of iNOS was increased as compared with control group. The higher the concentration of L-Arg was, the better the effect. Conclusion L-Arg can induce the expression of iNOS resulting in increase the production of nitric oxide (NO). Low concentration of L-Arg can promote the growth of LS174 cells, while high concentration ones can inhibit growth and proliferation. The high concentration of L-Arg could induce S phase arrestion in the cell cycle.
Objective To explore the effects of bile from patients with cholecystolithiasis on the growth of human gallbladder carcinoma cells GBC-SD and the potential correlation between cholecystolithiasis and gallbladder carcinoma. Methods Cholecystolithiasis bile (CB) and normal bile (NB) specimens were used for this study. The proliferative effects of bile were measured by methabenzthiazuron (MTT) assay and cell cycle and apoptosis were analyzed by flow cytometry. Results CB can significantly promote the proliferation of GBC-SD cells, GBC-SD proliferative index increased significantly after treated with 1% CB for 48 h （P＜0.05）.The Sphase fraction of CB 〔（49.26±8.07）%〕 increased remarkably (P＜0.05) compared with that of NB 〔（25.54±6.57）%〕, and the CB percentage of G0/G1 phase 〔（40.59±9.12）%〕 decreased remarkably (P＜0.05) compared with NB 〔(60.64±13.42）〕%. Conclusion CB can promote the proliferation of human gallbladder carcinoma GBC-SD cells.
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 explore the effect of bone morphogenetic protein 4 (BMP4) on the glycolysis level of human retinal microvascular endothelial cells (hRMECs). MethodsA experimental study. hRMECs cultured in vitro were divided into normal group, 4-hydroxynonenal (HNE) group (4-HNE group) and 4-HNE+BMP4 treatment group (BMP4 group). 4-HNE group cell culture medium was added with 10 μmmol/L 4-HNE; BMP4 group cell culture medium was added with recombinant human BMP4 100 ng/ml after 6 h stimulation with 10 μmol/L 4-HNE. The levels of intracellular reactive oxygen species (ROS) were detected by flow cytometry. The effect of 4-HNE on the viability of cells was detected by thiazole blue colorimetry. Cell scratch test and Transwell cell method were used to determine the effect of 4-HNE on cell migration. The relative expression of BMP4 and SMAD9 mRNA and protein in normal group and 4-HNE group were detected by real-time quantitative polymerase chain reaction and Western blot. Seahorse XFe96 cell energy metabolism analyzer was used to determine the level of intracellular glycolysis metabolism in normal group, 4-HNE group and BMP4 group. One-way analysis of variance was used for comparison between groups. ResultsThe ROS levels in hRMECs of normal group, 4-HNE group and BMP4 group were 21±1, 815±5, 810±7, respectively. Compared with the normal group, the levels of ROS in the 4-HNE group and the BMP4 group were significantly increased, and the difference was statistically significant (F=53.40, 50.30; P＜0.001). The cell viability in the normal group and 4-HNE group was 1.05±0.05 and 1.28±0.05, respectively; the migration rates were (0.148±0.005)%, (0.376±0.015)%; the number of cells passing through the pores were 109.0±9.6, 318.0±6.4, respectively. Compared with the normal group, the 4-HNE group had significantly higher cell viability, cell migration rate, and the number of cells passing through the pores, and the differences were statistically significant (F=54.35, 52.84, 84.35; P＜0.05). The relative expression levels of BMP4 and SMAD9 mRNA in the cells of the 4-HEN group were 1.680±0.039 and 1.760±0.011, respectively; compared with the normal group, the difference was statistically significant (F=53.66, 83.54; P＜0.05). The relative expression levels of BMP4 and SMAD9 proteins in the cells of the normal group and 4-HEN group were 0.620±0.045, 0.860±0.190, 0.166±0.049, 0.309±0.038, respectively; compared with the normal group, the differences were statistically significant (F=24.87, 53.84; P＜0.05). The levels of intracellular glycolysis, glycolytic capacity and glycolytic reserve in normal group, 4-HNE group and BMP4 group were 1.21±0.12, 2.84±0.24, 1.78±0.36, 2.59±0.11, 5.34±0.32, 2.78±0.45 and 2.64±0.13, 5.20±0.28, 2.66±0.33. Compared with the normal group, the differences were statistically significant (4-HNE group: F=86.34, 69.75, 58.45; P＜0.001; BMP4 group: F=56.87, 59.35, 58.35; P＜0.05). There was no significant difference in intracellular glycolysis, glycolysis capacity and glycolysis reserve level between 4-HNE group and BMP4 group (F=48.32, 56.33, 55.01; P＞0.05). ConclusionBMP4 induces the proliferation and migration of hRMECs through glycolysis.
