Retinitis pigmentosa (RP) is a genetic disorder of photoreceptor cell apoptosis and retinal pigment epithelium (RPE) cell atrophy caused by gene mutation. The clinical manifestations are night blindness, peripheral visual field loss and progressive vision loss. RPE cell apoptosis plays an important role in the progression of RP, and exogenous implantation of RPE cells as an alternative therapy has shown certain efficacy in animal experiments and clinical trials. With the diversification of cell sources, the update of surgical techniques and the continuous emergence of biological materials, more possibilities and hopes are provided for cell therapy. To further promote the development of this field in the future, it is still necessary to strengthen the cooperation between medicine, bioengineering and other disciplines in the future to jointly promote the innovation and development of therapeutic methods. It is believed that RPE cell transplantation therapy will show a brighter prospect in the future
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 changes in refractive status of eyes with idiopathic macular hole (IMH) after vitrectomy and phacoemulsification and IOL implantation (combined surgery).MethodsA retrospective clinical study. From January 2016 to June 2019, 51patients (56 eyes) of IMH who underwent combined surgery at the Tianjin Medical University Eye Hospital. were included in the study. Among them, there were 17 males and 34 females with the average age of 66.79±4.33 years. All the affected eyes underwent BCVA, retinoscopy and axial length (AL) measurement. The IOL power was calculated according to the SRK-T formula and the refractive power (predicted value) was predicted. The average BCVA of the affected eye was 0.20±0.13. The average anterior chamber depth was 2.89±0.28 mm. The average △corneal astigmatism was 0.73±0.43 D, the average AL was 22.92±0.70 mm, the average predicted refractive power was 0.10±0.66 D. All the affected eyes underwent standard transciliary flat part three-channel 25G combined surgery. Six months after the operation, the actual value (actual value) of the diopter after the operation was measured with the same equipment and method before the operation. Paired t test was used to compare the difference between the predicted value and the actual value.ResultsSix months after the operation, the actual value of the refractive power was -0.19±0.64 D. Compared with the pre-operative refractive power, the difference was not statistically significant (t=1.665, P=0.102). The difference between the actual value and the predicted value was －0.33±0.81 D.ConclusionsThe refractive status of the IMH eye undergoes myopia drift after combined surgery. The preoperative IOL power budget can be appropriately reserved for +0.3 D hyperopia.
ObjectiveTo observe the inhibitory effect of lentivirus (LV)-mediated miR-191 on the proliferation and angiogenesis of human retinal vascular endothelial cells (hREC) cultured in vitro.MethodsThe hREC cell lines were cultured in vitro and divided into control group, hypoxia group, LV-empty vector (LV-vector) group, and LV-miR-191 (LV-191) group. The LV-vector group and LV-191 group were transferred to the corresponding lentiviral vector respectively. Flow cytometry was used to detect cell transfection efficiency. Cell Counting Kit-8 (CCK-8) test was used to detect cell proliferation ability. Scarification test and invasion chamber (Transwell) test were used to detect cell migration ability. Matrigel test was used to detect cell lumen formation ability. Real-time quantitative polymerase chain reaction (qPCR) was used to detect the relative expression of miR-191 and relative mRNA expression of its downstream target genes p21, vascular endothelial growth factor (VEGF), cell division protein kinase (CDK) 6, cyclin-D1 (Cyclin D1). Independent sample t test was used for pairwise comparison. ResultsThe results of flow cytometry showed that the transfection efficiency of cells in the control group and the LV-191 group were 0.615% and 99.400%, respectively. The results of CCK-8, scarification, Transwell and Matrigel test showed that, compared with the control group, the number of cell proliferation (t=6.130, 4.606), the cell mobility (t=4.910, 6.702), the number of stained cells on the microporous membrane (t=7.244, 6.724) and the lumen formation ability cells (t=8.345, 9.859) were significantly increased in the hypoxia group and the LV-vector group (P<0.01), while the LV-191 group showed completely opposite performance (t=14.710, 6.245, 5.333, 5.892; P≤0.01). The qPCR test results showed that, compared with the control group and the LV-vector group, the relative expression of miR-191 mRNA in the cells of the LV-191 group was significantly up-regulated (t=44.110, 42.680), the relative expression of Cyclin D1 mRNA (t=29.940, 14.010) and CDK6 mRNA (t=15.200, 7.645) decreased significantly, and the difference were statistically significant (P<0.01); the relative expression of p21 mRNA increased, however, the difference was not statistically significant (t=2.013, 2.755; P>0.05). There was no significant difference in the relative expression of VEGF mRNA in the 4 groups of cells (F=0.966, P>0.05). ConclusionsLV-191 can inhibit the proliferation, migration and tubing of hREC by up-regulating p21 and down-regulating CDK6 and Cyclin D1.
ObjectiveTo analyze the expression of miRNA involved in regulating retinal neovascularizationin in retinal tissue of oxygen-induced retinopathy (OIR) mice.MethodsEighty healthy C57BL/6J mice were randomly divided into control group and OIR group at postnatal day 7(P7). Control group were not received any treatment and then exposed to room air. The OIR group was exposed to (75±2)% oxygen and then under room air at P12. Mice of all groups were euthanized at P17. Retinal neovasculation (RNV) was evaluated by counting the number of pre-retinal neovascular cells and analysing no perfusion area by immunofluorescent staining of the mouse retina.Total RNA was extracted from retinal tissue,and miRNA microarrays was performed to identify differentially expressed miRNA in the two groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed differential microRNA.ResultsCompared with the control group,the retinal neovascular tufts and the no perfusion area were both significantly smaller than those in OIR group. The number of pre-retinal neovascular cell nuclei in retinas from control group were obviously lower than those in the retinas from OIR group (t=9.025, P＜0.05). MiRNA microarray analysis showed that 54 miRNA in OIR group showed statistically different expression in control group, 47 miRNA were up-regulated and 7 miRNA were down-regulated. The results of PCR were consistent with the trend of microarray. In GO analysis, 1112 items were significantly different (P＜0.05), and 65 items were significantly different in KEGG analysis of expression profile (P＜0.05).ConclusionsThe miRNA expression in retinal tissue of OIR mice is different from that of normal mice, and these miRNA may be involved in the development of RNV. There are 54 miRNA expression differences in retinal tissue of OIR compared with normal mouse retinal tissue.
Objective To observe the expression of miRNA in retinal tissue of mice with oxygen-induced retinopathy (OIR), and screen miRNAs related to p21 and retinal neovascularization (RNV) formation. MethodsA experimental study. Forty healthy 7-day-old C57BL/6J mice were randomly divided into normal group and OIR group, with 20 mice in each group. The oxygen induced RNV model was constructed in the OIR group, and no treatment was performed in the normal group. At the age of 17 days, the mice were killed and the RNV of mice was observed by retinal fluorescence; the nuclei of vascular endothelium that broke through the inner limiting membrane of retina were counted under light microscope. The retinal tissues were taken for miRNA chip analysis to detect the differentially expressed miRNAs between the normal group and the OIR group. The resulting differential miRNA target genes were subjected to enrichment analysis based on gene annotation (GO) and Kyoto Encyclopedia of genes and genomes (KEGG); miRNAs and pathways that may be related to p21 were screened through Targetscan, MiRanda and MicroT-CDs database alignment. Independent sample t-test was used for pairwise comparison between groups. ResultsCompared with the normal group, the area of nonperfusion area, RNV and the number of vascular endothelial nuclei that broke through the inner limiting membrane of the retina in the OIR group increased significantly, differences were statistically significant (t=18.800, 9.025; P＜0.05). Compared with the normal group, there were 54 miRNAs that were statistically differentially expressed in the OIR group, of which 47 were up-regulated and 7 were down-regulated. A total of 13 miRNAs related to p21 were screened from the alignment results of the three databases with the obtained differential miRNAs. According to the difference multiples, they were miR-7218-5p, miR-322-5p, miR-224-5p, miR-335-5p, miR-329-3p, miR-362-3p, miR-532-5p, miR-20b-5p, miR-20a-5p, miR-195a-5p, miR-423-5p, miR-497a-5p, and miR-129-5p. Differential miRNA target gene enrichment analysis yielded 1 112 go entries and 50 KEGG pathways, of which 50 go entries and 13 KEGG pathways were related to p21. Conclusion13 miRNAs related to p21 were screened out in the OIR model.
ObjectiveTo observe the efficacy of dexamethasone intravitreal implant (DEX) combined with pars plana vitrectomy (PPV) in eyes with severe idiopathic epimacular membrane (IMEM). MethodsA prospective clinical case study. From December 2018 to May 2021, 24 patients with 25 eyes of severe IMEM diagnosed in Tianjin Medical University Eye Hospital were included in the study. Among them, 7 males had 7 eyes, 17 females had 18 eyes. Age was 57 to 84 years old. The IMEM stage was 3 to 4 examined by spectral domain optical coherence tomography (SD-OCT). All eyes were performed best corrected visual acuity (BCVA) and central macular thickness (CMT) by SD-OCT. The patients were randomly divided into PPV group (11 eyes) and PPV+DEX group (14 eyes). Standard PPV by three-channel 25G was performed. Phacoemulsification, membrane stripping and intraocular lens implantation were combined during the operation. Patients received vitreous injection of 0.7 mg DEX in PPV+DEX group at the end of the operation. At 1 week, 1 month, 3 months and 6 months after operation, the same equipments and methods were used to perform relevant examinations. The changes of BCVA and CMT were compared between the two groups by t test. ResultsCompared with before operation, at 1, 3 and 6 months after operation, the BCVA of the eyes in the PPV+DEX group was significantly improved (t=3.974, 4.639, 4.453), CMT was significantly decreased (t=2.955, 3.722, 4.364), the differences were statistically significant (P＜0.05); at 3 and 6 months after surgery, the BCVA of the eyes in the PPV group was significantly improved (t=2.983, 4.436), CMT was significantly decreased (t=2.983, 3.461), the differences were statistically significant (P＜0.05). ConclusionIn the treatment of severe IMEM, DEX can accelerate the early postoperative visual recovery and reduce CMT.
ObjectiveTo observe the inhibitory effect of lentiviral vector miR-191 (LV-191) on retinal neovascularization (RNV) in mice model of oxygen-induced retinopathy (OIR).MethodsEighty healthy 7-day-old C57BL/6J mice were randomly divided into 5 groups including normal group, non-intervention group, normal saline (NS) group, LV-191 group and LV-green fluorescent protein (GFP) group, 16 mice in each group. The OIR model was established in the non-intervention group, NS group, LV-191 group and LV-GFP group. NS group, LV-191 group and LV-GFP group were given an intravitreal injection of 1 μl of NS, LV-191 and LV-GFP at the age of 12 days. No injection was performed in the non-intervention group. In normal group,newborn mouse were maintained in room air form P0 to P17, and no treatment was performed. Mice in all five groups were euthanized at P17. Retinal neovasculation (RNV) was evaluated by counting the number of pre-retinal neovascular cells and analysis of non-perfusion area area by immunofluorescent staining of the mouse retina. Real-time quantitative PCR (RT-PCR) to detect miR -191 and P21 expression of retinal tissue.ResultsIn the LV-191 group, the non-perfusion area were both significantly smaller than those in non-intervention group, NS group and LV-GFP group (F=127.20, P＜0.001). The number of pre-retinal neovascular cell nuclei in retinas from LV-191 group were obviously lower than those in the retinas from non-intervention group, NS group and LV-GFP group (F=31.71, P＜0.05). RT-PCR showed that the LV-191 and P21 level of LV-191 group increased significantly than other groups (F=10.95, 15.60; P＜0.05).ConclusionIntravitreal injection of LV-191 inhibits RNV in mice model of OIR possibly through up-regulating p21.
ObjectiveTo evaluate changes in operational effectiveness after the implementation of ambulatory surgical management in pars plana vitrectomy (PPV). MethodsA retrospective clinical study. 17 528 surgeries in 10 895 eyes of 10 895 patients who underwent minimally invasive PPV on an ambulatory and/or inpatient basis at Tianjin Medical University Eye Hospital from August 2015 to June 2023 were included in this study. Among them, 5 346 eyes in 5 346 cases were male; 5 549 eyes in 5 549 cases were female. The age ranged from 0 to 95 years, with the mean age of (57.74±13.15) years. 6 381 surgeries in 3 615 eyes from August 2015 to December 2018 (the initial period of day surgery) were used as the control group; 11 147 surgeries in 7 280 eyes from January 2019 to June 2023 (the expanded period of day surgery) were used as the observation group. According to the management mode of ambulatory surgery, the observation group was subdivided into the decentralized management group (January 2019 to December 2020) and the centralized management group (January 2021 to June 2023), with 2 905 and 4 375 eyes and 4 646 and 6 501 surgeries, respectively. Changes in the percentage of day surgery, average hospitalization days, and average unplanned reoperation rate were compared. The Mann-Whitney U test was used to compare numerical variables between groups; the chi-square test or Fisher's exact test was used to compare categorical variables. ResultsThe number of cases of daytime PPV performed in the observation group and control group was 7 852 (70.44%, 7 852/11 147) and 24 (0.38%, 24/6 381) cases, respectively, and the average hospitalization days were 1 (1) and 5 (3) d. Compared with the control group, the observation group had a significantly higher percentage of day surgery (χ2=8 051.01) and a considerably lower mean hospitalization day (Z=4 536 844.50), and the differences were statistically significant (P＜0.000 1). The mean hospitalization days in the decentralized and centralized management groups were 2 (3) and 1 (0) d, respectively, and unplanned reoperations were 34 (0.73%, 34/4 646) and 171 (2.63%, 171/6 501) eyes, respectively. Compared with the decentralized management group, average hospitalization days was significantly lower (Z=1 436.94) and unplanned reoperation rate was significantly higher (χ2=54.10) were significantly lower in the centralized management group, both of which were statistically significant (P＜0.000 1). ConclusionPPV ambulatory management model can significantly reduce the average hospitalization day, but also results in higher rates of unplanned reoperations.