ObjectiveTo investigate the expression of miR-195 and the underlying molecular mechanisms of miR-195 regulating HMGB1 in diabetic retinopathy (DR). MethodsExtract 5 ml venous blood from DR patients, diabetes mellitus (DM) patients and normal subjects, then extract and perificate plasma total RNA. MicroRNA array and real time polymerase chain reaction (RT-PCR) was used to screen out miRNAs which were expressed with significant differences in the serum of patients with DR. Bioinformatics was employed to predict the miR-195 related to high mobility group box 1 (HMGB1) regulation. Next, miR-195 was down-regulated or up-regulated in umbilical vein endothelial cells through transfection of miR-195 inhibitor and miR-29b mimics respectively.Then we analyzed expression of HMGB1 mRNA and protein by RT-PCR and Western blot. ResultsMicroRNA array results showed the expression of miR-195 in DR group is decreased by 8.34 times and 11.47 times compared with DM group and the normal group. RT-PCR verification results conforms to the microRNA array results. Compared with the DM group (F=0.034, t=8.057) and the normal group (F=0.370, t=9.522), the expression of miR-195 in DR group were significantly reduced, the differences were statistically significant (P < 0.05). RT-PCR showed that the expression of HMGB1 mRNA was significantly decreased in up-regulation group, compared with blank (F=0.023, t=11.287) and negative control group (F=0.365, t=7.471), the difference was statistically significant (P < 0.05). The expression of HMGB1 mRNA was significantly increased in down-regulation group, compared with blank (F=0.053, t=10.871) and negative control group (F=0.492, t=6.883), the difference was statistically significant (P < 0.05). Western blot showed that the expression of HMGB1 protein was significantly decreased in up-regulation group, compared with blank (F=0.021, t=8.820) and negative control group (F=0.039, t=7.401), the difference was statistically significant (P < 0.05); and significantly increased in down-regulation group, compared with blank (F=0.186, t=10.092) and negative control group (F=0.017, t=12.923), the difference was statistically significant (P < 0.05). ConclusionMiR-195 can inhibit the expression of HMGB1, reduce the inflammation and angiogenesis, thereby delaying or inhibiting the occurrence and development of DR.
Objective To investigate the expression of induced heat shock protein (HSP) 70 in ratprime;s retinal neurons (RNs) and Muuml;ller cells, and evaluate the protective effect of HSP 70 on RNs injured with glucose deprivation and glutamate. Methods Ratprime;s RNs and Muuml;ller cells cultured in vitro were treated with heat shock (42℃ for 1 hour), and duration of the expression of HSP70 was detected by immunocytochemical techniques. Viability of the cells was measured by methyl thiazolyl tetrazolium (MTT) chromatometry after incitant toxic injury with glucose deprivation (0.56 mmol/L glucose for 6 hours) and glutamate (100 mu;mol/L for 6 hours). Simultaneously, the expression was interdicted by HSP70. Results Hypereffective expression of HSP70 was found in cultured RNs and Muuml;ller cells after heat shock. The viability of RNs pretreated by heat shock after injured with glucose deprivation and glutamate significantly increased which could be interdicted by HSP70 antibody. Conclusion Hypereffective expression of HSP 70 may be induced by heat shock, which enhances the ability of tolerance of RNs to the incitant toxic injury by glucose deprivation and exitotoxicity. (Chin J Ocul Fundus Dis, 2005,21:110-113)
ObjectiveTo study the hydrophilicity and the cell biocompatibility of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) coated with a fusion protein polyhydroxyalkanoates granule binding protein (PhaP) fused with Arg-Gly-Asp (RGD) peptide (PhaP-RGD). MethodsPHBV and PHBHHx films were fabricated by solvent evaporation.Scanning electronic microscope (SEM) was used to study the morphology of the films.PhaP-RGD fusion proteins were expressed and purified by the technology of protein engineering; PHBV and PHBHHx films were immersed in the PhaP-RGD with an amount of 3.5 mg/mL protein/per sample respectively.The hydrophilicity of the surface were detected by the contact angle measurements.Septal cartilage cells obtained from human septal cartilage were cultured in vitro.The 2nd passage chondrocytes were incubated on PHBV unmodified with PhaP-RGD in group A1,PHBV modified with PhaP-RGD in group A2,PHBHHx unmodified with PhaP-RGD in group B1,PHBHHx modified with PhaP-RGD in group B2,and on the cell culture plates in group C.After cultured for 3 days,the proliferation of cells was detected by the DAPI staining; the proliferation viability of cells was detected by the MTT assay after cultured for 3 and 7 days; after cultured for 7 days,the adhesion and morphology of the cells on the surface of the biomaterial films were observed by SEM and the matrix of the cells was detected through the toluidine blue staining. ResultsSEM observation showed that PHBV and PHBHHx films had porous structures.The contact angle of the surface of the PHBV and PHBHHx films modified with PhaP-RGD fusion proteins were significantly reduced when compared with the films unmodified with PhaP-RGD fusion proteins (P<0.05).Chondrocytes of human nasal septal cartilage incubated on the films could grow in all groups.After 3 days of cultivation in vitro,the cell proliferation and viability of group B2 were the strongest among all groups (P<0.05); the cell proliferation after cultured for 7 days was significantly stronger than that after cultured for 3 days in groups A1,A2,B1,and B2 (P<0.05); and the cell proliferation was significantly stronger in groups B1 and B2 than groups A1,A2 and C,in group B2 than group B1,and in group A1 than group A2 (P<0.05).The results of toluidine blue staining showed that blue metachromasia matrixes were observed in groups A1,A2,B1,and B2; group A1 and group A2 had similar staining degree,and the staining of group B2 was deeper than that of group B1.The adhesion of cells in all groups was good through SEM observation; and the connection of cells formed and stretched into the pores of the materials. ConclusionThe biomaterial films of PHBHHx modified with PhaP-RGD fusion protein can promote its biocompatibility with chondrocytes.
ObjectiveTo observe the expression of heat shock protein 47 (HSP47) and transforming growth factor-β2(TGF-β2) in vitreous specimens and epiretinal membranes of patients with proliferative vitreoretinopathy diseases. MethodsVitreous specimens and epiretinal membranes were obtained from 48 patients (48 eyes) with proliferative vitreoretinopathy (PVR) and 50 patients (50 eyes) with proliferative diabetic retinopathy (PDR). Vitreous specimens and internal limiting membranes were collected from 20 patients (20 eyes) with idiopathic macular hole (IMH) as control group. The expression of HSP47 and TGF-β2 in the vitreous specimens was evaluated using enzyme linked immunosorbent assay. The expression of HSP47, TGF-β2, typesⅠandⅢcollagen in epiretinal membrane and internal limiting membrane specimens were observed for immunohistochemical staining method. The correlation between the positive expression of HSP47 and TGF-β2, typesⅠandⅢcollagen in epiretinal membrane specimens of patients with PVR and PDR were analyzed. ResultsThe expression of HSP47 in vitreous specimens of patients with PVR, PDR and IMH were (212.35±23.32), (231.30±26.79), (171.06±28.91) pg/ml, respectively. The expression of TGF-β2 in vitreous specimens of patients with PVR, PDR and IMH were (1919.96±318.55), (1939.39±177.57), (1194.61±234.20) pg/ml, respectively. The expression of HSP47, TGF-β2 in the vitreous specimens of patients with PVR and PDR were significantly increased compared with patients with IMH and the difference was statistically significant (F=12.952, 34.532;P < 0.01). The epiretinal membrane of patients with PVR and PDR showed markedly increased expression of HSP47, TGF-β2, typesⅠandⅢcollagen in the cytoplasm and extracellular matrix. The expression of HSP47 and typeⅢcollagen was negative and the expression of TGF-β2 was weakly positive and the expression of typesⅠcollagen was positive in internal limiting membrane of patients with IMH. The expression of HSP47, TGF-β2, typesⅠandⅢcollagen in the epiretinal membrane of patients with PVR and PDR were significantly increased compared with patients with IMH and the difference was statistically significant (F=13.469, 18.752, 12.875, 20.358; P < 0.01). The expression of HSP47 was positively correlated with the positive expression of TGF-β2, typesⅠandⅢcollagen in epiretinal membrane specimens of patients with PVR (r=0.475, 0.556, 0.468; P < 0.05) and PDR (r=0.484, 0.589, 0.512; P < 0.05). ConclusionsThis study showed increased consistent expression of HSP47 and TGF-β2 in vitreous and epiretinal membrane specimens of patients with PVR and PDR. Both HSP47 and TGF-β2 were expressed in the cytoplasm and extracellular matrix. HSP47 and TGF-β2 may be involved in the pathological process of PDR and PVR by promoting collagen synthesis.
ObjectiveTo investigate the influence of high activity of CYP2C9 (Cytochrome P450 proteins 2C9)and VKORC (Vitamin K epoxide reductase C)on warfarin anticoagulation of patients after heart valve replacement (HVR). MethodsFrom February 2010 to May 2013, 40 patients with high activity of CYP2C9 and VKORC underwent HVR in the Department of Cardiac Surgery, the First Affiliated Hospital of Zhengzhou University. There were 18 male and 22 female patients with their age of 40-51 (45.18±2.93)years. There were18 patients receiving mitral valve replacement (MVR), 14 patients receiving MVR and tricuspid valvuloplasty (TVP), and 8 patients receiving double valve replacement (DVR). Depen-ding on whether they received preoperative genetic polymorphism detection of CYP2C9 and VKORC1, all the patients were divided into 2 groups with 20 patients in each group. Patients in group A didn't receive preoperative genetic polymorphism detection of CYP2C9 and VKORC1, while patients in group B received preoperative genetic polymorphism detection of CYP2C9 and VKORC1. Postoperatively, periodic examination of international normalized ratio (INR)was performed to adjust warfarin dosage. Time to reach expected INR value and morbidity were collected. All the patients were followed up for 3-12 months after discharge. Monthly telephone follow-up was performed to record INR values, morbidity and general recovery. ResultsPostoperatively, in group A, 2 patients had cerebral infarction, 2 patients had popliteal artery throm-bosis, 1 patient had pulmonary embolism, and 1 patient had thrombosis in the annulus. Expected INR was achieved 15-20 days after warfarin treatment among the other 14 patients without thromboembolism. Three months after surgery, CYP2C9 and VKORC1 gene polymorphism was examined to find 17 patients with positive CYP2C9*1/*1 (*2CC/*3AA)and positive VKORC1-1639 GA, and 3 patients with positive CYP2C9*1/*1 (*2CC/*3AA)and positive VKORC1-1639 GG. In Group B, patients received aspirin (100 mg/d)and low molecular heparin (0.4 ml/d)in addition to warfarin since the second posto-perative day. Expected INR was achieved 5-9 days after warfarin treatment, and then aspirin and low molecular heparin were discontinued. During the 6 months follow-up period, no obvious thromboembolism was found, and only 1 patient had epistaxis who was cured with nasal tamponade. ConclusionPreoperative detection of genetic polymorphisms of CYP2C9 and VKORC1 can provide important guidance for warfarin anticoagulation after HVR.
ObjectiveTo observe the short-term effects of intravitreal injection of aflibercept (IVA) for initial treatment and dressing change on exudative age-related macular degeneration (eAMD). MethodsA retrospective clinical study. From June 2018 to February 2021, forty-nine eAMD eyes of 38 patients who underwent IVA treatment in Department of Ophthalmology of Central Theater Command Hospital of People’s Liberation Army were included in the study. Among them, there were 24 males with 29 eyes and 14 females with 20 eyes; the average age was 66.82±8.71 years. All affected eyes were treated with IVA. The initial loading dose was 2.0 mg, which was injected once a month for 3 consecutive months, followed by monthly review and treatment as needed. Of the 49 eyes, 26 eyes were initially treated (initial treatment group), they were diagnosed within 3 months of the first onset and followed by IVA treatment, and no intraocular drugs and surgery were performed from the onset to the first diagnosis. Twenty-three eyes were treated with drug exchange therapy (dressing change group), they received intravitreal injection of ranibizumab and/or conbercept more than 4 times 6 months before the replacement therapy, during which there was persistent interlaminar cystoid edema and/or subretinal fluid (SRF) in the macular area and no improvement in pigment epithelial detachment (PED). Before IVA treatment, there were no statistically significant differences in the best corrected visual acuity (BCVA), foveal thickness (CMT), PED height (PEDH), and PED volume (PEDV) of the two groups of eyes before IVA treatment (P＞0.05). The same equipment and methods as before treatment were used for related examinations, and the changes of BCVA, CMT, PEDH, PEDV and complications of the two groups of eyes were recorded in 1, 3, and 6 months after treatment. The comparison of BCVA, CMT, PEDH, and PEDV between the two groups were used repeated measures analysis of variance. ResultsSix months after treatment, the number of IVA injections in the eyes of the initial treatment group and dressing change group were 4.15±0.73 and 4.39±0.72 times, respectively, and the difference was not statistically significant (t=?1.141, P=0.260). The BCVA, CMT, PEDH, and PEDV of the the initial treatment group (F=5.345, 22.995, 6.764, 5.425) and the dressing change group (F=12.519, 15.576, 8.843, 9.406) were significantly improved compared before treatment with 1, 3, and 6 months. All were statistically significant (P＜0.05). There was no significant difference in BCVA, CMT, PEDH, and PEDV between the initial treatment group and the dressing group at each time point after treatment (F=1.741, 0.069, 0.876, 3.455; P＞0.05). During the follow-up period, none of the affected eyes had complications such as persistent intraocular pressure increase, endophthalmitis, and retinal pigment epithelial tear. ConclusionsIVA can improve eyesight of patients with eAMD and reduce CMT, PEDH, and PEDV. The initial treatment and dressing change have the same effect.
ObjectiveTo observe the expression of Toll-like receptor 4 (TLR4) and inflammatory cytokines, leucocytic density and permeability in retina of diabetic rat. MethodsA total of 106 Brown Norway rats were randomly divided into experimental group and control group with 53 rats in each group. Diabetic model was established in experimental group by intraperitoneal injection of streptozotocin, and control rats received intraperitoneal injection of an equal volume of citric acid-sodium citrate buffer. Four weeks later, the retinas were collected for further analysis. TLR4 RNA and protein expression were measured by quantitative polymerase chain reaction and Western blot. Inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemo-attractant protein-1 (MCP-1), were measured by enzyme-linked immunosorbent assay in rat retina homogenate. Leukocyte density in the retina was measured by acridine orange fundus angiography. The retinal permeability was evaluated by Evans blue (EB) staining. ResultsTLR4 expression was significantly increased in diabetic rats of experimental group compared with non-diabetic rats of control group (F=1.606, 0.789; P < 0.05). Inflammatory cytokines (TNF-α, IL-1β and MCP-1) were significantly increased in retina of diabetic rats of experimental group versus non-diabetic rat of control group (F=24.622, 5.758, 4.829; P < 0.05). The retinal leukocyte density was (6.2±0.5)×10-5, (2.2±0.3)×10-5 cells/pixel2 in experimental and control group respectively, the difference was statistically significant (F=2.025, P < 0.05). The amount of retinal EB leakage was (23.41±4.47), (13.22±3.59) ng/mg in experimental and control group respectively, the difference was statistically significant (F=21.08, P < 0.05). ConclusionTLR4 and inflammatory cytokines expression, leucocytic density and permeability increased significantly in retina of diabetic rat.
Objective To investigate the effects of knocking down Rac1 gene (ras-related C3 botulinum toxin substrate 1) by small hairpin RNA (shRNA) on retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Methods One hundred and eight 7-day-old C57BL/6J mice were divided into three groups randomly.The OIR was induced by Smith protocol in 2 groups. OIR mice received an intravitreal injection of Rac1-shRNA plasmid or the nonsense plasmid in the geneintervention group and control group respectively at the age of postnatal day 11 (P11). Non-OIR mice also received an intravitreal injection of Rac1-shRNA plasmid at P11 as the blankintervention group which lived in the normoxic environment.Retinal neovascularization was investigated on flat-mounts after fluorescence angiography at P15 and P17. Endothelial cell nuclei breaking through the internal limiting membrane were counted on pathological section at P17.The expression of Rac1 and NF-kappa;B p65 subunit was measured by immuohistochemistry, Western blot, real-time polymerase chain reaction (RT-PCR) and in situ hybridization. Results Compared with the blank-control group,the level of Rac1 mRNA in the gene-intervention group decreased obviously（t=4.500，P=0.001）;the retinal non-perfusion areas,fluorescence leakage, neovascularization and the number of endothelial cell nuclei breaking through the internal limiting membrane were reduced significantly（t=6.521,P＜0.001）; the level of NF-kappa;B p65 nuclear translocation decreased（t=16.008,P＜0.001）while the expression of NF-kappa;B p65 mRNA was reduced obviously（t=3.354,P=0.006）, which was positively correlated with the expression of Rac1-mRNA (P=0.012).Conclusion Intravitreal injection of Rac1-shRNA with liposome in mice can effectively inhibit the expression of Rac1,and inhibit the retinal neovascularization under relative hypoxia via blocking the ROS-NF-kappa;B pathway.
Objective To investigate the influence of enamel matrix proteins (EMPs) on the attachment, prol iferation and pre-mRNA of type I collagen synthesis of cultured human dermal fibroblast cells. Methods Human dermal fibroblast cells were obtained from human acrobystia and cultured in DMEM medium with 10% FBS. The 3rd to 6th passage cells were used. Ninety-six-well plates and 6-well plates were pre-coated with different concentrations of EMPs (50, 100, 150 and200 μg/ mL). ① The cell attachment experiment: 0.2 mL cells suspension at the concentration of 1 × 106/mL was added to the pre-coated 96-well plates as the experimental groups (groups A, B, C and D based on different concentrations of EMPs). At 1.5, 3.0, and 4.5 hours after inoculation, the attached cells were measured by MTT method. ② The cell prol iferation experiment: 0.2 mL cells suspension at the concentration of 5 × 104/mL was added to the pre-coated 96-well plates as the experimental groups (groups A1, B1, C1 and D1 based on the different concentrations of EMPs). At 2, 4, 6 and 8 days after inoculation, the cells were measured by MTT method. ③ The synthesis experiment of pre-mRNA: 2 mL cells at the concentration of 1 × 106/mL was added to the pre-coated 6-well plates as the experimental groups (groups A2, B2, C2 and D2 based on different concentrations of EMPs). At 5 days after inoculation, the synthesis of pre-mRNA was measured by RT-PCR method. Human dermal fibroblast cells were added to the un-coated plates as the control groups. Results ① The cell attachment experiment: There were significant differences in attachment cells between the control group, group A and the groups B, C and D (P lt; 0.05). There were no significant difference between group A and control group (P lt; 0.05). ② The cell prol iferation experiment: At 2 days, there were no significant differences in absorbance between the control group and the experimental groups (P gt; 0.05); at 4 days and 6 days, the absorbance of groups B1 (0.598 ± 0.020 and 0.639 ± 0.016 ), C1 (0.582 ± 0.017 and 0.641 ± 0.020) and D1 (0.574 ± 0.021and 0.635 ± 0.021) was significantly higher than that of the control group (0.548 ± 0.021 and 0.605 ± 0.019, P lt; 0.05); at 8 days, the absorbance of group B1 (0.629 ± 0.012) and group C1 (0.631 ± 0.014) was significantly higher than that of the control group (0.606 ± 0.031, P lt; 0.05). ③ The synthesis experiment of pre-mRNA: The synthesis of type I collage pre-mRNA of groups B2, C2 and D2 was significantly higher than that of the control group. Conclusion EMPs stimulate human dermal fibroblast cell attachment, prol iferation and synthesis of type I collage pre-mRNA, and its maximal effect can be achieved at the concentration of 100 μg /mL.
Objective To study the relationship between the expression of sonic hedgehog (Shh) and vascular endothelial growth factor (VEGF) in hypoxic human retinal pigment epithelial (hRPE) cells. Methods Cultured hRPE-19 cells (3rd - 6th generations) were used in this experiment. hRPE-19 cells were divided into three groups including the control group, the hypoxia experimental group (100 μmol/L CoCl2) and the inhibition group (pretreatment with 20 μmol/L cyclopamine 1 hour before hypoxia). After culturing for 4, 8, 12 and 24 hours, the mRNA level of Shh and VEGF genes in these cells were measured by fluorescence quantitative polymerase chain reaction, and the protein level of Shh and VEGF in the supernatants were measure by enzyme-linked immunosorbent assay. The relationship between the expression of Shh and VEGF was analyzed by Pearson?correlation?analysis. Results The control group expressed low levels of Shh and VEGF mRNA/protein. The expression of Shh and VEGF mRNA/protein in the hypoxia experimental group was significantly higher than that in the control group (F=178.364, 183.732, 77.456, 91.572; P＜0.01). The expression of Shh and VEGF mRNA in the inhibition group was significantly lower than that in the hypoxia experimental group (F=68.745, 121.834; P＜0.01). In the hypoxia experimental group, the expression of VEGF protein was positively correlated with the expression of Shh protein (r=0.942, P＜0.05); and the expression of VEGF and Shh mRNA was positively correlated (r=0.970, P＜0.01). However, there was no significant correlation in the expression of VEGF and Shh mRNA in the inhibition group (r=0.915, P＞0.05). Conclusion There is a positive correlation between the expression of Shh and VEGF in hypoxic hRPE cells.