Objective To observe the effect of epidermal growth factor (EGF) on integrin alpha;5 expression and its influence on human retinal pigment epithelium (RPE) cells.Methods Human RPE cells were treated in vitro with 0.1,1.0,10.0,20.0 and 100.0 ng/ml of EGF, the mRNA and protein of integrin alpha;5 was measured by reverse transcription polymerase chain reaction（RT-PCR）and flow cytometry. Human RPE cells were cultured under 4 conditions including DMEM/F12,DMEM/F12+10 ng/ml EGF, DMEM/F12+10 ng/ml EGF+rabbit antihuman integrin alpha;5 antibody (1∶100)，DMEM/F12+10 ng/ml EGF+rabbit antihuman vimentin antibody (1∶100), and their proliferation and migration were measured by methylthiazole tetrazolium（MTT）and Boyden chamber.Results The integrin alpha;5 mRNA level of human RPE cells was not changed after 12 hours of EGF stimulation (F=0.618, P=0.687), however it was induced in a dosedependent manner after 24 and 48 hours of EGF stimulation (F=465.303, 212.340; P=0.000,0.000).The protein level of integrinalpha;5 was higher in 10 ng/ml EGF stimulation compared with the control group and 0.1 ng/ml group（P＜0.01）.MTT and Boyden chamber showed that the integrin alpha;5 expression increased the proliferation and migration of human RPE cells. Conclusion EGF can induce integrin alpha;5 expression,thus increase the proliferation and migration of human RPE cells.
Objective To observe the effect of Twist gene interference on the migration and pAkt protein expression of Rhesus retinal vascular endothelial cell line. Methods The Rhesus retinal vascular endothelial cells (RF/6A) were divided into Twist interference plasmid group, negative control group, and phosphate buffered solution (PBS) group; plasmid vectors were transfected via liposome gene transfection method. Migrated endothelial cells was detected and counted by Transwell chamber assay. Matrigel was used in endothelialcell tube formation; the inhibitory effect of Twist gene interference on endothelial cell tube formation was observed.The effect of Twist gene interference on the expression of pAkt protein in RF/6Acells was measured by Western blot. Results The number of migrated endothelial cells in Twist interference plasmid group was lower than that in the negative control and PBS group (F=23.786，P=0.000).The number of endothelial cell tubes in Twist interference plasmid group was apparently less than that in the negative control and PBS gorup (F=7.159，P=0.014). The expression of pAkt protein in Twist interference plasmid group decreased markedly.Conclusion Twist gene interference can suppress the migration of retinal endothelial cells via inhibiting the expression of pAkt protein.
ObjectiveTo address the effect and mechanism of interleukin 17 (IL-17) on the proliferation, migration and apoptosis of human retinal vascular endothelial cells (HREC). MethodsIL-17 receptor (IL-17R) mRNA and protein expression in human retinal vascular endothelial cells (HREC) were quantified by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation of HREC was examined using CCK-8 assay in the presence of different concentrations of IL-17. Cell migration of HREC was detected using wound scratch assay. Flow cytometry was used to test the effect of IL-17 on the apoptosis of HREC. The effects of IL-17 on HREC expression of basic fibroblast growth factor (bFGF), Caspase-3 and thrombospondin-1 (TSP-1) were quantified by reverse transcriptase polymerase chain reaction (RT-PCR). The effect of IL-17 on HREC expression of Caspase-3 was examined using Western blot. ResultsIL-17 receptor (IL-17R) expressed in HREC as quantified by RT-PCR and Western blot. The proliferation of HREC in the presence of IL-17 was promoted in a dosage-dependent manner (t=-3.235, -6.276;P=0.032, 0.000). Wound scratch assay showed a significant increase in the migrated distance of HREC with IL-17 stimulation under the concentration of 100μg/L(t=-3.551, -2.849; P=0.006, 0.019), 200μg/L(t=-10.347, -4.519; P=0.000, 0.001) and 500μg/L (t=-3.541, -2.607; P=0.008, 0.036). The intervention of 200μg/L IL-17 can effectively inhibit the apoptosis of HREC, compared with the control group using flow cytometry (t=5.682, P=0.047). RT-PCR results showed that IL-17 can promote the expression of bFGF and inhibit the expression of Caspase-3 and TSP-1. Western blot result also showed that IL-17 can suppress the protein expression of Caspase-3. ConclusionThe mechanism of IL-17 promoting proliferation, migration but suppress apoptosis of HREC may via regulating the expression of bFGF and Caspase-3.
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.
ObjectiveTo observe the effect of bone morphogenetic protein 4 (BMP4) on the proliferation and migration of human retinal microvascular endothelial cells (hRMEC) under oxidative stress. MethodsThe hRMEC cultured in vitro were divided into control group, 4-hydroxynonenal (HNE) treatment group (4-HNE group), 4-HNE+BMP4 group (BMP4 group). Cell culture medium of 4-HNE treatment group was added with 10 μmmol/L 4-HNE; cell culture of BMP4 group was cultured with 10 μmmol/L 4-HNE, and after stimulation for 6 h, 100 ng/ml recombinant human BMP4 was added. The effects of 4-HNE and BMP4 on hRMEC viability was detected by thiazole blue colorimetric method. The effects of 4-HNE and BMP4 on cell migration was determined by cell scratch test. The relative expression of BMP4 mRNA in the cells of the control group and 4-HNE treatment group and the mRNA expression of the control group, the fibronectin (FN) of BMP4 group, laminin (Laminin), α-smooth muscle contractile protein (α-SMA), and collagen type Ⅰ (Collagen Ⅰ), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) were detected by real-time quantitative polymerase chain reaction (qRT-PCR). Western blot was used to detect the relative expression of BMP4 protein in the control group and 4-HNE group. The control group and 4-HNE group were compared by t test. ResultsCompared with the control group, cell viability (t=12.73, 16.26, P=0.000 2, <0.000 1), cell migration rate (t=28.17, 37.48, P<0.000 1, <0.000 1) in 4-HNE group and BMP4 group were significantly increased, and the difference was statistically significant; the relative expression of BMP4 mRNA and protein in the 4-HNE group was significantly increased, and the difference was statistically significant (t=16.36, 69.35, P=0.000 1, <0.000 1). The qRT-PCR test results showed that compared with the control group, the relative expression of VEGF, FN, Laminin, α-SMA, Collagen Ⅰ, and CTGF mRNA in the cells of the BMP4 group was significantly increased, and the difference was statistically significant (t=10.61, 17.00, 14.85, 7.78, 12.02, 10.61, P=0.0004, <0.000 1, 0.000 1, 0.001 5, 0.000 1, 0.000 4). ConclusionBMP4 can induce the proliferation and migration of hRMEC; it can also regulate the expression of angiogenesis factors and fibrosis-related factors in hRMEC.