This article introduced a new method to repairthe mandibular micrognathia of mandible. Thehalf-split clavicle bone with bilateralsternocleidomastoid muscular pediclcs was used.Based on the results of our clinical data, it hadthe advantages of bone graft with vascularpodicle, no functional ill- effect in the donorsite, shortens the operative time, and might beaccomplished reliability in a one stage.
ObjectiveTo investigate the effects of interferon gene stimulating protein (STING) inhibitor (C176) on human retinal microvascular endothelial cells (hRMEC) under oxidative stress. MethodsAn animal experimental study. In vivo experiment: 48 healthy male C57BL/6J mice were randomly divided into wild type mice group (WT group) and diabetes (DM) group, with 24 mice in each group. DM mice were induced by streptozotocin to establish DM model. After successful modeling, DM group was divided into DM+dimethyl sulfoxide (DMSO) group and DM+C176 group, with 12 mice in each group. The mice in the DM+DMSO group were intraperitoneally injected with DMSO at the dose of 50 mg/kg. Mice in DM+C176 group were intraperitoneally injected with STING inhibitor C176 750 nmol at the dose of 50 mg/kg. Four weeks after modeling, immunohistochemical staining, Western blot and real-time fluorescence quantitative polymerase chain reaction were used to detect the expression of STING in the retina of WT and DM mice. The leukocyte adhesion test was used to detect the number of leukocytes adhering to hRMEC in mice with WT, DM+DMSO and DM+C176 groups. In vitro experiment: hRMEC was randomly divided into conventional culture cell group (N group), dimethyl sulfoxide (DMSO) group (with DMSO intervention) and C176 group (with C176 intervention). The cells were induced by 150 μg/ml glycation end products for each group. In vitro leukocyte adhesion test combined with 4', 6-diamino-2-phenylindole staining was used to detect the number of leukocytes adhering to hRMEC. The adherent leukocytes were quantitatively analyzed by flow cytometry; H2DCFDA/reactive oxygen species (ROS) fluorescence probe was used to detect ROS expression in cells; Seahorse XFe96 cell energy metabolism analyzer was used to measure the level of intracellular glycolysis. t-test was used to compare the two groups; single factor analysis of variance was used to compare the three groups. ResultsIn vivo experiment: compared with WT group, the expression level of STING (t=73.248) and the relative expression amount of mRNA (t=67.385) in the retina of DM group mice increased significantly (P＜0.05). Compared with WT group, the number of leukocytes adhering to the retinal vessels of mice in DM+DMSO group was significantly increased, while that in DM+C176 group was significantly decreased (F=84.352, P＜0.01). In vitro: compared with N group and DMSO group, the number of leukocyte adhesion on hRMEC in C176 group decreased significantly (F=35.251, P＜0.01). Compared with N group, the number of leukocytes adhering to hRMEC in DMSO group and C176 group decreased significantly (F=26.374, P＜0.01). The ROS level in hRMEC in C176 group was significantly lower than that in N group and C176 group (F=41.362, P＜0.01). Compared with N group and DMSO group, the glycolysis level of hRMEC in C176 group was significantly reduced, with a statistically significant difference (F=68.741, P＜0.01). ConclusionInhibiting the expression of STING in retinal vascular endothelial cells can improve the progress of DM by inhibiting leukocyte adhesion, ROS production and glycolysis level.
ObjectiveTo investigate the effects of targeted regulation of SMAD9 expression by bone morphogenetic protein 4 (BMP4) on Müller cell migration, reactive oxygen species (ROS) generation and vascular endothelial growth factor (VEGF) expression. MethodsMüller cells cultured in vitro were divided into normal control group, BMP4 group, BMP4+ no-load plasmid group (BMP4+NC group) and BMP4+SMAD9 small interference plasmid group (BMP4+siSMAD9). Cells in BMP4 group, BMP4+NC group and BMP4+siSMAD9 group were induced by adding 100 ng/ml BMP4 into cell medium for 24 h. Subsequently, BMP4+NC group was transfected with empty plasmid. BMP4+siSMAD9 group was transfected with SMAD9 small interference plasmid for 48 h. The effect of BMP4 on Müller cell migration was determined by cell scratch test. The effect of BMP4 on the production of ROS in Müller cells was detected by flow cytometry. Western blots and real-time quantitative fluorescence polymerase chain reaction (qPCR) were used to detect the relative mRNA expression levels of glutamine synthetase (GS) and glial fibrinoacidic protein (GFAP) in Müller cells. VEGF expression in Müller cells was detected by immunofluorescence. One-way analysis of variance was used to compare groups. ResultsThe results of cell scratch test showed that the cell mobility of BMP4+siSMAD9 group was significantly lower than that of BMP4 and BMP4+NC group, and the difference was statistically significant (F=68.319, P＜0.001). Flow cytomethods showed that the level of ROS in BMP4+siSMAD9 group was significantly lower than that in BMP4 and BMP4+NC group, and the difference was statistically significant (F=52.158, P＜0.001). Western blot and qPCR results showed that the protein levels of GS and GFAP (F=42.715, 36.618) and mRNA relative expression levels (F=45.164, 43.165) in BMP4+siSMAD9 group were significantly lower than those in BMP4 and BMP4+NC group. The difference was statistically significant (P＜0.01). The results of immunofluorescence detection showed that the intracellular VEGF fluorescence intensity in BMP4 group and BMP4+NC group was significantly higher than that in BMP4+siSMAD9 group, and the difference was statistically significant (F=46.384, P＜0.05). ConclusionTargeted regulation of SMAD9 expression by BMP4 can up-regulate VEGF expression and promote the migration and ROS production of Müller cells.
[Abstract]Objective To observe the clinical and imaging features of non-arteriotic central retinal artery occlusion (NA-CRAO) with internal boundary membrane detachment (ILMD), and to analyze its relationship with visual prognosis. MethodsA retrospective clinical study. A total of 88 patients with NA-CRAO hospitalized in Department of Ophtalmology, Xi'an People's Hospital (Xi'an Fourth Hospital) from January 2014 to June 2023 were included in the study. Best corrected visual acuity (BCVA), optical coherence tomography (OCT) and fluorescein fundus angiography (FFA) were performed. The BCVA test used the international standard visual acuity chart, which was statistically converted to the logMAR visual acuity. OCT observed the presence of ILMD and the thickening of the inner retina and the disappearance of anatomical stratification. FFA recorded arm-retinal circulation time (A-Rct) and retinal arterion-distal filling time (FT), and observed ciliary retinal artery, fluorescein retrograde filling, cotton spots, luciferin nodal filling, macular non-perfusion, capillary fluorescein leakage, optic disc strong fluorescence, choroidal background weak fluorescence and other characteristics. According to whether there was ILMD, the patients were divided into ILMD group and non-ILMD group, with 44 cases and 44 eyes respectively. The two groups received the same treatment. The follow-up time was 30 days after treatment. The clinical, FFA characteristics and BCVA before and after treatment were compared between the two groups. t-test was used for comparison between groups. ResultsIn ILMD group and non-ILMD group, there were 43 cases of male and 1 case of female, respectively, and the proportion of male was significantly higher than that of female. Before and after treatment, the logMAR BCVA of ILMD group and non-ILMD group were 2.35±0.42, 2.01±0.46, 1.47±0.60, 0.77±0.49, respectively. There were significant differences in logMAR BCVA between the two groups before and after treatment (t=8.025, 12.358; P＜0.001). Before treatment, A-Rct and FT in ILMD group were longer than those in non-ILMD group, and the difference was statistically significant (t=3.052, 3.385; P＜0.05). After treatment, there was no significant difference (t=1.040, 1.447; P＞0.05). The proportion of ciliary retinal artery and cotton plaque in ILMD group was lower than that in non-ILMD group. There was no significant difference in ciliary retinal artery between the two groups (χ2=－0.961, P＞0.05), but there was a significant difference in cotton wool plaque between the two groups (χ2=－3.364, P＜0.05). Compared to the non-ILMD group, The proportion of retrograde fluorescein filling in retinal artery (χ2=－2.846), segment filling (χ2=－3.907), macular non-perfusion (χ2=－6.656), capillary fluorescein leakage (χ2=－4.367), optic disc strong fluorescence (χ2=－3.525) and choroidal background weak fluorescence (χ2=－2.276) increased, the difference was statistically significant (P＜0.05). ConclusionsIn patients with NA-CRAO, compared with those without ILMD, those with ILMD have more severe retinal ischemia and worse BCVA before and after treatment. ILMD is one of the poor prognostic markers of NA-CRAO vision.
ObjectiveTo observe the protective effect of polypyrimidine bundle-binding protein-related splicing factor (PSF) over-expression on RPE cell injury induced by advanced glycation end products (AGEs).MethodsThe human RPE cells cultured in vitro were divided into three groups: normal control group (N group), blank control group (N + AGEs group), empty vector control group (Vec + AGEs group), and PSF high expression group (PSF + AGEs). group). RPE cells in N group were routinely cultured; RPE cells in N + AGEs group were only transfected but did not introduce any exogenous genes combined with AGEs induction; Vec +AGEs group and PSF + AGEs group were transfected with pcDNA The empty vector or pcDNA-PSF eukaryotic expression plasmid was introduced into RPE cells and induced by AGEs. Except the N group, the other 3 groups of cells were transfected accordingly, and were stimulated with 150 μg/ml AGEs for 72 h after 24 h. HE staining and Hoechst 33258 staining were used to observe the effect of high PSF expression on the morphological changes of RPE cells; ROS level detection was used to analyze the effect of PSF high expression on the ROS expression of RPE cells induced by AGEs; MTT colorimetric method was used to detect the high PSF expression Effects on the viability of RPE cells; Western blot was used to detect the effects of different time and dose of PSF on the expression of heme oxygenase 1 (HO-1).ResultsHE staining and Hoechst 33258 staining observation showed that the cells in group N were full in shape, the nucleus was round, the cytoplasm was rich, and the staining was uniform; the cells in N + AGEs group and Vec + AGEs group were reduced in size, the eosinophilic staining was enhanced, and the nucleus was densely densely stained. Pyrolysis and even fragmentation; the morphology of cells in the PSF + AGEs group was still full, the cytoplasm staining was more uniform, and the nucleus staining was uniform. The results of MTT colorimetry showed that high expression of PSF can effectively improve the viability of RPE cells, but this effect can be effectively antagonized by ZnPP, and the difference is statistically significant (F=33.26, P＜0.05). DCFH-DA test results showed that compared with the N + AGEs group and Vec + AGEs group, the ROS production in PSF + AGEs group decreased, the difference was statistically significant (F=11.94, P＜0.05). Western blot analysis showed that PSF protein up-regulated HO-1 expression in a time- and dose-dependent manner. The relative expression level of HO-1 at 24, 48, and 72 h after PSF protein was significantly higher than that at 0 h, and the difference was statistically significant (F=164.91, P＜0.05). The relative expression level of HO-1 under the action of 0.1, 0.5, 1.0, 1.5, and 2.0 μg PSF protein was significantly higher than 0.0 μg, and the difference was statistically significant (F=104.82, P＜0.05).ConclusionPSF may inhibit the production of ROS by up-regulating the expression of HO-1, thus protecting the RPE cells induced by AGEs.
ObjectiveTo investigate the inhibitory effect of lentivirus-mediated polypyrimidine bundle binding protein-associated splicing factor (PSF) on retinal neovascularization (RNV) in mice model of oxygen-induced retinopathy (OIR).MethodsOne hundred and twelve 5-day-old C57BL/6J mice were randomly divided into normal control group, simple OIR model group, OIR model + lentivirus empty vector treatment group (Vec group) and OIR model + PSF lentivirus treatment group (PSF group), with 16, 32, 32 and 32 mice, respectively. When the mice were 7 days old, the mice in the normal control group were fed in a routine environment, and the mice in the OIR model group, Vec group and PSF group were established OIR model. The mice in the Vec group and PSF group were given an intravitreal injection of 1 μl of lentiviral vector and PSF lentivirus (titer 1×1011 TU/ml) at the age of 12 days. No injection was performed in the normal control group and simple OIR group. RNV was evaluated by counting the number of pre-retinal neovascular cells and analysis of non-perfusion area by immunofluorescent staining of the mouse retina. Real-time quantitative PCR was applied to detect the mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1). Western blot analysis was applied to detect the protein expression of Nrf2, HO-1 and PSF. Results Of the normal control group, simple OIR model group, Vec group and PSF group, the number of pre-retinal neovascular cell nuclei were 0.00, 14.36±5.50, 15.67±4.96, 8.13±2.09, the non-perfusion area were 0.00%, (35.71±2.81)%, (36.57±4.53)%, (15.33±4.75)%, respectively. The differences of the number of pre-retinal neovascular cell nuclei and non-perfusion area among 4 groups were significant (F=24.87, 165.70; P＜0.05). Compared with the normal control group, there were more pre-retinal neovascular cell nucleis and larger non-perfusion area in the simple OIR model group and Vec group (P＜0.05). Compared with the simple OIR model group and Vec group, there were lower pre-retinal neovascular cell nucleis and smaller non-perfusion area in the PSF group (P＜0.05). Real-time quantitative PCR and Western blot showed that the mRNA expression of Nrf2, HO-1 (F=53.66, 83.54) and protein expression of Nrf2, HO-1 and PSF (F=58.38, 52.69, 24.79) among 4 groups were significant (P＜0.05). The mRNA expression of Nrf2, HO-1 and protein expression of Nrf2, HO-1 and PSF in the simple OIR model group and Vec group decreased significantly than those in the normal control group (P＜0.05). The mRNA expression of Nrf2, HO-1 and protein expression of Nrf2, HO-1 and PSF in the PSF group were increased significantly than those in the simple OIR model group and Vec group (P＜0.05). model group and Vec group (P＜0.05).ConclusionIntravitreal injection of lentivirus-mediated PSF inhibits RNV in mice model of OIR possibly through up-regulating the expression of Nrf2 and HO-1.
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.