Objective To observe the expression of p53, bcl-2 genes, vascular endothelial cell growth factor（VEGF）, basic fibroblast growth factor（bFGF）, insulin-like growth factor-I （IGF-I）, and the receptors of these factors of retinal vascular endothelial cells (VECs) of 1- to 20-week diabetic rats, and the relationship between the expressions and cell cycle arrest.Methods Retinal sections of diabetic rats induced by alloxan were immunohistochemically stained and observed by light microscopy (LM) and electron microscopy (EM). Dot blotting and Western blotting were used to determine the expression of mRNA, proteins of p53 and bcl-2. Results Under LM, immunohistochemical positive expression of p53 and bcl-2 were found on the vessels of ganglion cell layer and inner nuclear layer of retinae of 8- to 20-week diabetic rats; under EM, these substances were observed depositing in VECs. The retinal VECs also expressed VEGF, bFGF, IGF-I and their receptors. There was no positive expression of other cell types in these retinae, all cell types of retinae in control group, or all cells of retinae of diabetic rats with the course of disease of 1 to 6 weeks. The result of dot blotting revealed that retinal tissue of 20-week diabetic rat expressed p53 and bcl-2 mRNA, and the result of Western blotting revealed that they also expressed p53 and bcl-2 proteins. But retinal tissues of control group did not. Positive expression of bax was not found in the retinae in control group or 1- to 20-week diabetic rats. Conclusion p53, bcl-2 may introduce cell cycle arrest of VECs of retinae in 8- to 20-week diabetic rats. High glucose might stimulate the expression of VEGF, bFGF, IGF-I and their receptors, and the growth factors may keep VECs surviving by self-secretion. (Chin J Ocul Fundus Dis，2003，19：29-33)
ObjectiveTo observe the effect of pyrimidine bundle-binding protein-associated splicing factors (PSF) on the function of hypoxia-induced human retinal microvascular endothelial cells (hRMECs).MethodsA three-plasmid system was used to construct lentivirus (LV)-PSF. After LV-PSF infected hRMECs in vitro, the infection efficiency was measured by flow cytometry. Real-time quantitative PCR (RT-PCR) was used to detect the expression of PSF mRNA in hRMECs infected with LV-PSF. The experiment was divided into two parts, in vivo and in vitro. In vivo experiments: 20 healthy C57B/L6 mice at the age of postnatal 7 were randomly divided into normal group, oxygen-induced retinopathy (OIR) group, OIR+LV-Vec group, and OIR+LV-PSF group, each group has five mice. Mice in 3 groups were constructed with OIR models except the normal group and the mice in OIR group were not treated. The mice in the OIR + LV-Vec group and the OIR+LV-PSF group were injected with an empty vector (LV-Vec) or LV-PSF in the vitreous cavity, respectively. The effect of LV-PSF on the formation of retinal neovascularization (RNV) was observed then. In vitro experiments: hRMECs were divided into normal group, hypoxia group, vector group, and PSF high expression group. HRMECs in the normal group were cultured in vitro; hRMECs in the hypoxic group were restored to normal culture conditions for 3 h after 3 h of hypoxia stimulation; hRMECs in the vector group and PSF high expression group were infected with LV-Vec and LV-PSF for 48 h, and hRMECs were returned to normal culture conditions for 24 h with hypoxia stimulation for 3 h. The effect of PSF on cell proliferation was observed by MTT colorimetry. Cell scratch test and Transwell migration experiment were used to observe the effect of PSF on cell migration ability under hypoxia stimulation. RT-PCR was used to observe the mRNA expression of HIF-1α, VEGF and PSF in each group of cells.ResultsThe LV-PSF of stably expressing PSF was successfully constructed. The infection efficiency was 97% determined by flow cytometry. The level of PSF mRNA in hRMECs infected with LV-PSF was significantly increased and detected by RT-PCR. In vivo experiments: The RNV area of the mice in the OIR group and the OIR + LV-Vec group was significantly increased compared to the normal group (t=18.31, 43.71), and the RNV area of the mice in the OIR + LV-PSF group was smaller than that in the OIR group (t=11.30) and OIR + The LV-Vec group (t=15.47), and the differences were statistically significant (P＜0.05). In vitro experiments: MTT colorimetry results showed that the proliferative capacity of hRMECs in the hypoxic group was significantly enhanced compared with the normal group (t=2.57), and the proliferative capacity of hRMECs in the PSF high expression group was significantly lower than that of the normal, hypoxic, and vector groups (t=5.26, 5.46, 3.73), the differences were statistically significant (P＜0.05). The results of cell scratch test showed that the hRMECs could be stimulated by the hypoxia stimulation for 3 hours to restore the normal condition for 24 hours or 48 hours (t=8.35, 13.84; P＜0.05). Compared with the vector group, cell migration rate in the PSF-high expression group was not significant (t=10.99, 18.27, 9.75, 8.93, 26.94, 7.01; P＜0.05). Transwell experiments showed that the number of cells stained on the microporous membrane was higher in the normal group and the vector groups, while the number of cells stained in the PSF high expression group was significantly reduced (t=9.33, 6.15; P＜0.05). The results of RT-PCR showed that the mRNA expression of HIF-1α and VEGF in hRMECs in the hypoxic and vector groups increased significantly compared with the normal group (t=15.23, 21.09; P＜0.05), but no change in the mRNA expression of PSF (t=0.12, 2.15; P>0.05); compared with the hypoxia group and the vector group, the HIF-1α and VEGF mRNA expression in hRMECs in the PSF high expression group were significantly decreased (t=10.18, 13.10; P＜0.05), but the PSF mRNA expression increased (t=65.00, 85.79; P<0.05).ConclusionPSF can reduce the RNV area in OIR model mice. PSF may inhibit hypoxia-induced proliferation and migration of hRMECs through the HIF-1α/VEGF signaling pathway.
OBJECTIVE :To investigate ocular vessel flow velocity in normal eyes by color Doppler imaging(CDI). METHODS: Ninety people (180 normal eyes)had flow velocity measured by CDI in three vessels,ophthalmic artery (OA),central retinal artery(CRA) and posterior ciliary artery (PCA),and the relations between velocity and affecting factors were analysed. RESULT: The diastolic and systolic velocity of OA,CRA and PCA were (31.7plusmn;10.9)cm/s, (7.2plusmn;2.6)cm/,s, (10.2plusmn;3.4)cm/s, (2.8plusmn;1.2)cm/s and (11.3plusmn;3.6)cm/s, (3.2plusmn;1.4)cm/s respectively. The velocity of OA had possitive correl-ativity with RBC,HCT and negative correlativity with age,while it revealed no relationship with sex, laterality of right or left eye,and normal IOP. CDI of ocular vessels in normal eyes is a triangle with three peaks and double sunkens in its frenquency spectum, revealed highly resistant,and both the pulsatility and resistive indexes were relatively high. The width of each frequency band basically was the same,and there was no obvious frequency window. CONCLUSION: The ocular application of CDI might lay the foundation of a comparatively comprehensive knowledge of the ocular hemodynamics. （Chin J Ocul Fundus Dis,1997,13: 99-101）
ObjectiveTo observe the effect of epinephrine in intraocular irrigation solution on retinal vascular caliber and macular thickness. MethodsA prospective control study. 32 eyes of 32 patients with macular hole who underwent vitrectomy were enrolled in this study. The patients including 14 males (14 eyes) and 27 females (18 eyes), with the average age of (64.0±4.5)years. Uncorrected visual acuity, corrected visual acuity, slit lamp biomicroscopy, indirect ophthalmoscopy, fundus color photography and optical coherence tomography were performed in all patients. Retinal vascular caliber located in 0.5-1.0 disc diameter from optic disk was measured from digital fundus photographs and summarized as central retinal artery (CRAE) and vein (CRVE) equivalents in all eyes at baseline and at the 1 month, 3 months follow-up visit. The macular thickness is the distance from retinal interface of inner plexiform layer to retinal pigment epithelium layer. The macula was divided into inner ring ( < 3 mm) and outer ring (3-6 mm) according to the distance from the fovea. The patients were divided into experiment group (include epinephrine in intraocular irrigation solution, 1:1000) and control group (without epinephrine in intraocular irrigation solution), 16 eyes in each group. The difference of CRAE and CRVE between two groups was not significant (P > 0.05). The difference of macular thickness between inner ring and outer ring was not significant (P > 0.05). The average follow-up was 3.5 months. CRAE, CRVE and macular thickness in inner ring and outer ring before and 1 month, 3 months after surgery were comparatively analyzed. ResultsThe differences of CRAE and CRVE before and 1, 3 months after surgery both in experiment group (tCRAE=0.322, 0.148; tCRVE=0.317, 0.005) and control group (tCRAE=0.226, 0.137; tCRVE=0.284, 0.151) were not significant (P > 0.05). The differences of CRAE (t=0.624, 0.424) and CRVE (t=0.015, 0.041) between experiment group and control group also were not significant (P > 0.05). The differences of macular thickness in inner ring and outer ring before and 1, 3 months after surgery both in experiment group (tinner=0.322, 0.148;touter=0.317, 0.005) and control group (tinner=0.226, 0.137;touter=0.284, 0.151) were not significant (P > 0.05). The differences of macular thickness in inner ring (t=1.568, 0.373) and outer ring (t=-1.697, 0.536) between experiment group and control group also were not significant (P > 0.05). ConclusionEpinephrine (1:1000) in intraocular irrigation solution has no effect on retinal vascular caliber and macular thickness in patients with macular hole.
The activities and distributions of succinate dehydrogenase(SDH),malic dehydrogenase(MDH),lactic dehydrogenase(LDH),acid phosphatase(ACP) and alkaline phosphatase(AKP) in retinal vessels were studied and observed with enzymatic histochemical techniques. The retinal vessels showed a b LDH activity, moderate SDH and MDH activity. The dehydrogenase activity described above was evenly and equally distributed in the microvasculature between arterioles and venules, and was the best in arteries. AKP showed predominant activity in the endothelial cells of capillaries and arterioles which were stained bly. No activity for ACP observed in the retinal vessels. The observations above indicate that the retinal vessels are metabolically active and have a great capacity for glycolysis. （Chin J Ocul Fundus Dis,1992,8:6-9）
0bjective To explore the effect of endothelin(ET)、nitrioxide (NO) in plasma on retinopathv in the pregnancy-induced hypertension(PIH)． Methods The 1evel of ET and NO in plasma of 75 cases of in-patient women with PIH and 20 cases of women with the full terms and normal pregnancy before and after delivery was determined by radioimmunoassay．The retinopathy of the patients with FIH before and after delivery was detected by appointed doctor．The levels of ET and N0 in both groups were compared and the relationship between ET and N0 in plasma and the retinopathy before and after the delivery was detected.Results The levels of ET[(145．oo±54.41)ng／L] in serious PIH patients were much higher than that in the control[(81．50±43．80)ng／L]，the minor[(85.30±33.33)ng／L]and middling PIH group[(90．20±39．25)ng／L]．The levels of ET in plasma before and after pregnancy were not changed in PIH patients [(118．70±33．44)ng／L]，but were higher than that in the control group. The levels of plasma NO in serious[(87．56±35．58)ng／L]and middling[(78.11±28.96)ng／L] PIH group were both higher than that in the control group[(46．70±32.64)ng/L]，and the levels in minor(52．56±28．35)ng／L]and middling PIH group were lower than that in the serious PIH group.The level of N0 in plasma of PIH patients after the delivery was much lower than that before the delivery，while higher than that in the control．The positive correlation between levels of ET and NO and retinopathy was found in PIH patients.Conclusions The 1evels of plasma ET and N0 in PIH patients are related to the extent of the disease，and the level of ET in plasma is highly related to the retinopathy in PIH patients, ET and NO might be played an important role in pathogenesis of retinopathy and ET might be a good index in reflecting the rank of retinopathy in PIH.(Chin J Ocul Fundus Dis，2004，20：12-15)
The retinal vessel changes are the primary and major features of retinal vascular diseases. The retinal vessel is part of systemic vessels with its own characteristics to sustain normal retinal function. These basic characteristics are important to the correct understanding and proper treatment of retinal vascular diseases. Always keep in mind that the retinal vessels is one part of the systemic vascular system, thus retinal vascular diseases may have systemic etiology, and systemic drug administration may have a profound effects to the whole body. However retinal vascular system also has its own structural and functional characteristics, thus retinal vascular diseases are also different from the systemic diseases. Finally the main function of retinal vascular network is to maintain the neuro-retinal function, thus we should balance the vision protection and treatments against abnormal retinal blood vessels. Over-treatments may damage the retinal vision.
ObjectiveTo explore the effects of transthyretin (TTR) on biological behavior of retinal microvascular epithelial cell (RMVEC). MethodsRMVEC was cultured in medium with 0 μmol/L and 4 μmol/L TTR. The proliferation, migration and healing abilities (0, 24, 48 hours) of RMVEC with different concentrations of TTR were measured by methyl thiazol tetrazolium (MTT) assay, transwell assay and scarification test. ResultsMTT assay shows that RMVEC with the concentrations of 4 μmol/L TTR [absorbance (A) value=0.17±0.02] glows faster than with the concentrations of 0 μmol/L TTR (A value=0.40±0.03), the difference was statistically significant (t=15.47, P=0.000 1). The transwell assay shows RMVEC with the concentration of 4 μmol/L TTR [(140±7) cells] migrants faster than RMVEC with the concentration of 0 μmol/L TTR [(227±14) cells], the difference was statistically significant (t=5.44, P=0.000 6). The scarification test shows that the RMVEC with the concentration of 4 μmol/L TTR [(134.4±45.4) μm] heals faster than the RMVEC with the concentration of 0 μmol/L TTR [(330.0±23.1) μm], the difference was statistically significant (t=8.25, P<0.01). The cells in 48 hours and 4 μmol/L group were healed completely, but not healed in 0 μmol/L group. ConclusionTTR can promote the proliferation, migration and healing abilities of RMVEC.
ObjectiveTo observe the stoichiometry of vascular endothelial growth factor receptor 2 (VEGFR2) on the retinal vascular endothelial cell membrane by single-molecule fluorescence imaging.MethodsRhesus monkey retinal vascular endothelial cells (RF/6A) were divided into blank control group (normal culture) and plasmid transfection group [transfected with VEGFR2-green fluorescent protein (GFP) recombinant plasmid]. The expression of GFP in the plasmid transfected group was observed by confocal microscope, and the expression of VEGFR2 in the cells was detected by real-time fluorescent quantitative polymerase chain reaction (qPCR) and Western blot. The fluorescence intensity distribution and bleaching steps of single VEGFR2-GFP molecule on the cell membrane were recorded by single-molecule imaging. The distribution of fluorescence intensity and the number of fluorescence bleaching steps of GFP were recorded.ResultsGFP green fluorescence was observed in the transfected cells 12 hours after transfection. qPCR results showed that the expression of VEGFR2 and GFP mRNA in the plasmid transfected group was significantly higher than that in the blank control group (t=11.240, 12.330; P＜0.001, 0.001). Western blot results showed that the expression of VEGFR2 protein in the plasmid transfected group was significantly higher than that in the blank control group (t=8.346, P＜0.01). The results of single-molecule imaging showed that the fluorescence intensity distribution of VEGFR2-GFP on the surface of RF/6A cell membrane without ligand stimulation was bimodal, in which monomer and dimer were 86.0% and 14.0% respectively. By counting the steps of GFP fluorescence bleaching, the proportions of receptor monomer, dimer, trimer, and tetramer were 81.4%, 12.9%, 5.5%, and 0.3% respectively.ConclusionIn the absence of ligands, VEGFR2 coexists in the form of monomers and dimers on the surface of RF/6A cell membrane, and monomers are dominant.
ObjectiveTo observe the MiSeq sequencing analysis results of fulvic acid (FA) intervention in hypoxia-induced human retinal microvascular endothelial cell (hRMEC) gene expression profile.MethodshRMEC were cultured in vitro and divided into the hypoxia group (hypoxia treatment) and the FA intervention group (FA intervention after hypoxia). The MTT colorimetric method was used to detect the influence of different concentrations and different modes of FA on hRMEC activity. The optimal concentration of FA was chosen. RT-PCR was used to investigated the effect of FA on hypoxia-induced intercellular adhesion molecule-1 (ICAM-1), IL-1β, IL-4, IL-6, IL-6, IL-8, IL-10, MMP-2, TNF-α, TNF-β, other inflammatory factors in hRMEC, and inflammation-related factors mRNA expression. Cells in the hypoxia group and FA intervention group in the logarithmic growth phase were collected. MiSeq sequencing technology was applyed to complete the whole transcriptome sequencing of the two groups of cells, biological data were obtained, and the differentially expressed miRNA were analyzed on this basis. Gene annotation (GO) functionally significant enrichment analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway significant enrichment analysis were used to analyze the functions and signal pathways of differential miRNAs. The expression of inflammatory factors and inflammation-related factors were compared between groups. The expression level of the corresponding miRNA in the cell was regulated by miRNA mimic, and its effect on cell function was observed, so as to judge the effect of the miRNA.ResultsDifferent concentrations and different modes of action of FA had no effect on the cell viability of hRMEC. The mRNA expression of ICAM-1, IL-1β, IL-6 and TNF-β in the hypoxia group hRMEC were significantly up-regulated compared with the normal group, and the difference was statistically significant (t=3.426, 6.011, 5.282, 6.500; P=0.027, 0.004, 0.006, 0.003); the mRNA expression of ICAM-1, IL-6, TNF-α and TNF-β in the FA intervention group hRMEC was significantly lower than that of the hypoxia group, and the difference was statistically significant (t=9.961, 3.676, 3.613, 3.387; P=0.001, 0.021, 0.023, 0.028). There were 14 differentially expressed miRNAs between the hypoxia group and the FA intervention group, of which 9 were up-regulated genes and 5 were down-regulated genes. The predicted target genes of 4 differential miRNAs (hsa-miR-1285-3p, hsa-miR-30d-3p, hsa-miR-3170, hsa-miR-7976) were all ICAM-1. The results of significant enrichment analysis of GO function showed that the functions of differential genes were mainly enriched in the process of cell development, cell differentiation and single organism development. Significant enrichment analysis of the KEGG pathway showed that the differential miRNA expression was highly enriched in the proteoglycan pathway and the cytokine-cytokine receptor interaction pathway in cancer, and the arachidonic acid metabolism pathway and the amphetamine pathway were the more obvious differential expressions.ConclusionFA may affect the expression level of downstream ICAM-1 mRNA by regulating the expression of multiple miRNAs, thereby affecting the inflammatory state of cells after hypoxia-stimulated hRMEC.