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1.
Article in Chinese | WPRIM | ID: wpr-1038375

ABSTRACT

Objective@#To construct a human G protein-coupled receptor kinase 2 ( GRK2) eukaryotic expression system.@*Methods@#The primers were designed ,and the His-GRK2 target gene was amplified by PCR using the Pans-EGFP-GrK2 (full-length) gene as the template.The His-GRK2 target gene was connected to the pcDNA3.1EGFP eukaryotic expression vector. The pcDNA3. 1-EGFP-His-GRK2 plasmid was transfected into HEK 293T cells.48 h later,the expression of GRK2 protein was detected by Western blot,and the GRK2 protein was purified by nickel chelated magnetic bead method.The purification of GRK2 protein was detected by Coomassie bright blue staining and Western blot,and the activity of GRK2 protein was detected by His pull down. @*Results @#The results of double enzyme digestion and sequencing showed that pcDNA3. 1-EGFP-His-GRK2 eukaryotic expression plasmid was successfully constructed.Western blot analysis showed that the molecular weight of GRK2 protein was about 80 ku,indicating that GRK2 protein was successfully expressed in HEK 293T cells (t = 6. 433,P = 0. 003) .GRK2 protein was purified by nickel chelated magnetic beads.His pull down experiment results showed that GRK2 was bound to prostaglandin E2 receptor 4 (EP4) ,suggesting that GRK2 protein had biological activity (t = 13. 5,P = 0. 000 2) .@*Conclusion@#The pcDNA3.1-EGFP-His-GRK2 eukaryotic expression plasmid was correctly sequenced and the GRK2 recombinant plasmid was successfully constructed.The GRK2 recombinant plasmid was successfully expressed in eukaryotic cells HEK 293T and the protein expressed was biologically active.

2.
Article in Chinese | WPRIM | ID: wpr-908613

ABSTRACT

Objective:To investigate the inhibitory effects of rosmarinic acid (RA) on high glucose-induced angiogenesis of human retinal microvascular endothelial cells (HRMEC) and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome pathway-related proteins.Methods:The HRMEC were divided into control group, high glucose group, high glucose+ low concentration RA group, high glucose+ medium concentration RA group, and high glucose+ high concentration RA group, and were cultured in vitro with conventional medium, 30 mmol/L D-glucose medium, 30 mmol/L D-glucose+ 25 μmol/L RA medium, 30 mmol/L D-glucose+ 50 μmol/L RA medium and 30 mmol/L D-glucose+ 100 μmol/L RA medium accordingly.The 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to detect the cell proliferation.Transwell assay was performed to detect the cell migration.Matrigel assay was employed to determine the tube formation ability of cells.Western blot was utilized to detect the expression levels of NLRP3, apoptosis-associated speck like protein (ASC) and cysteinyl aspartate-specific protease-1 (Caspase-1). Enzyme-linked immunosorbent assay (ELISA) kit was used to detect the concentrations of interleukin (IL)-1β and IL-18 in supernatant of cell culture. Results:The cell proliferation rate, the number of migrated cells and the number of formed tubes were (100.00±0.92)%, 37.67±9.02 and 45.00±4.58 in the control group, (163.56±1.46)%, 117.33±7.23 and 95.00±9.54 in the high glucose group, (152.29±2.90)%, 78.67±4.04 and 84.67±1.53 in the high glucose+ low concentration RA group, (147.72±2.22)%, 65.33±4.16 and 71.00±3.61 in the high glucose+ medium concentration RA group, (132.47±0.74)%, 52.67±6.81 and 60.00±1.00 in the high glucose+ high concentration RA group, respectively.There were statistically significant differences in cell proliferation rate, the number of migrated cells and formed tubes among all groups ( F=537.07, 64.63, 45.58; all at P<0.001). Compared with the control group, the cell proliferation rate, the number of migrated cells and formed tubes were significantly increased in the high glucose group, high glucose+ low concentration RA group, high glucose+ medium concentration RA group and high glucose+ high concentration RA group, showing statistical significances (all at P<0.05). Compared with the high glucose group, the cell proliferation rate, the number of migrated cells and formed tubes were significantly decreased in the different concentrations RA groups (all at P<0.05). With the increase of RA concentration, the cell proliferation rate, the number of migrated cells and formed tubes were decreased, and there were statistical differences among high glucose+ low/medium/high concentrations RA groups (all at P<0.05). There were significantly differences in the relative expression levels of NLRP3, ASC and Caspase-1 proteins in cells and the concentrations of IL-1β and IL-18 in cell culture supernatant among all the five groups ( F=145.12, 422.82, 463.79, 2 019.96, 33 406.97; all at P<0.001). Compared with the control group, the relative expression levels of NLRP3, ASC and Caspase-1 proteins as well as the concentrations of IL-1β and IL-18 in cell culture supernatant were significantly increased in the high glucose group, high glucose+ low concentration RA group, high glucose+ medium concentration RA group and high glucose+ high concentration RA group (all at P<0.05). Compared with the high glucose group, the expression levels of NLRP3, ASC and Caspase-1 proteins as well as the concentrations of IL-1β and IL-18 were decreased in the different concentrations RA groups, and the differences were statistically significant (all at P<0.05). With the increase of RA concentration, the expression levels of NLRP3, ASC and Caspase-1 proteins as well as the concentrations of IL-1β and IL-18 were decreased, and there were statistically significant differences among high glucose+ low/medium/high concentrations RA groups (all at P<0.05). Conclusions:RA can inhibit proliferation, migration and tube formation of HRMEC induced by high glucose, and inhibit high glucose-induced activation of NLRP3 inflammasome signaling pathway.

3.
Article in Chinese | WPRIM | ID: wpr-609720

ABSTRACT

Objective To investigate the relationship between choroidal telangiectasia and subfoveal choroidal thickness in the patients with central serous chorioretinopathy.Methods Eighty-four patients (84 eyes) with central serous chorioretinopathy in our hospital from February 2015 to April 2016 were selected,based on the fluorescein angiography the patients were divided into 3 groups:Mild group (leakage at 9-10 minutes after injection,26 cases),moderate group (leakage at 5-8 minutes after injection,37 cases),severe group (leakage at 5 minutes after injection,21 cases).The choroidal capillary dilatation was assessed by OCTA,and subfoveal choroidal thickness in three groups and patients with different foveal choroidal expansion degree were measured by EDI-OCT,and correlation analysis was performed.Results The proportion of severe dilation of choriocapillae in mild group,moderate group,severe group were 7.69%,13.52% and 23.81%,respectively.The subfoveal choroidal thickness was sequentially increased in mild group,moderate group,severe group (P < 0.05).The subfoveal choroidal thickness in patients with mild,moderate and severe dilatation of choroidal capillaries were (306.59 ± 74.18) μm,(367.21 ± 85.04) μm and (416.27 ± 104.56) μm,respectively (P < 0.05).The degree of choroidal telangiectasia in patients with central serous chorioretinopathy had a positive correlation with the thickness of subfoveal choroid (r =0.812,P =0.037).Conclusion The degree of choroidal telangiectasia and subchondral choroidal thickness in patients with central serous chorioretinopathy are significantly increased with the severity of the disease,and there is a positive correlation between them.

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