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Objective:To investigate the inhibitory effect of RMT1-10-induced tolerogenic dendritic cells (Tol-DCs) in vitro on high-risk corneal allograft rejection in mice and its mechanism. Methods:One hundred SPF male BALB/c mice and fifty SPF male C57BL/6 mice were selected.Bone marrow-derived immature dendritic cells (imDCs) obtained from C57BL/6 mice were divided into imDCs group, mature dentritic cells (mDCs) group, RMT1-10 group, and IgG isotype control group.The imDCs in the four groups were cultured with no intervention, lipopolysaccharide, RMT1-10 and lipopolysaccharide, or IgG isotype antibody and lipopolysaccharide for 7 days according to grouping.The expression levels of different phenotypes of DCs including CD11c, CD80, CD86, major histocompatibility complex (MHC)-Ⅱ, T cell immunoglobulin and mucin domain containing molecule (Tim)-4 and CD103 in the four groups were detected by flow cytometry.The concentrations of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) in the DCs supernatants were determined by enzyme-linked immunosorbent assay.A mixed lymphocyte culture system was established, and the stimulation index (SI) of CD4 + T cell proliferation stimulated with DCs was detected by cell counting kit 8 method.Corneal neovascularization was induced by corneal stromal suture in BALB/c mice, and the 80 mice with neovascularization in 4 quadrants growing into the middle and peripheral cornea were used as recipients.The recipient mice were randomized into imDCs group, mDCs group, RMT1-10 group, and IgG isotype control group using the random number table method, with 20 mice in each group.An injection of corresponding DCs (1×10 6 cells/100 μl) was administered to the recipient mice via the tail vein according to grouping.At 7 days following the injection, C57BL/6 mice were used as donors and penetrating keratoplasty was performed.Within one month after the operation, signs of corneal grafts rejection, including opacity, edema and neovascularization, were observed by slit lamp biomicroscopy and scored every day.At 21 days after the operation, 5 recipients selected from each group were subcutaneously injected with naive C57BL/6 splenocytes (1×10 6 cells/100 μl) behind the ear.The delayed type hypersensitivity (DTH) was evaluated by ear swelling at 24 hours after the subcutaneous injection.The use and care of experimental animals complied with the Regulations on the Management of Experimental Animals promulgated by the State Science and Technology Commission.This study protocol was approved by an Ethics Committee of the Affiliated Hospital of Chengde Medical University (No.CYFYLL2020055). Results:Compared with mDCs group, the expressions of CD80, CD86 and MHC-Ⅱ, and the percentage of Tim-4-positive cells in CD11c-positive cells were significantly decreased in RMT1-10 group, showing statistically significant differences (all at P<0.001). The percentage of Tim-4-positive cells were significantly decreased in RMT1-10 group than imDCs group, and the percentage of CD103-positive cells in RMT1-10 group was significantly higher than imDCs group, mDCs group and IgG isotype control group (all at P<0.001). The concentrations of IL-10 and TGF-β in the cell culture supernatant of RMT1-10 group were significantly higher than those of the other three groups, with statistically significant differences (all at P<0.001). There were statistically significant differences in the SI of CD4 + T cell proliferation simulated by DCs ( Fgroup=1 833.00, P<0.001; Fratio=230.40, P<0.001; Finteraction=3.06, P=0.01). The SI of DCs/CD4 + T cells ratio at 1∶5, 1∶10, 1∶20 and 1∶40 were all significantly lower in imDCs group than mDCs group, and were all significantly lower in RMT1-10 group than imDCs group (all at P<0.05). There was a statistically significant difference in corneal graft survival curve among various groups ( χ2=77.69, P<0.001). The survival rate of RMT1-10 group was significantly higher than that of imDCs group ( χ2=9.74, P=0.002), and the survival rate of imDCs group was significantly higher than that of mDCs group ( χ2=31.02, P<0.001). The ear swelling of recipient mice of positive control group, mDCs group, IgG isotype control group, imDCs group and RMT1-10 group was (503.6±17.2), (475.7±17.6), (456.2±18.8), (225.2±39.4), (118.1±12.6), and (106.4±7.4) μm, with a statistically significant difference among them ( F=377.10, P<0.001). The mice ear swelling was more serious in positive control group than mDCs group, more serious in IgG isotype control group than imDCs group, and more serious in imDCs group than RMT1-10 group (all at P<0.05). Conclusions:RMT1-10 can inhibit the rejection of high-risk corneal transplantation in mice, the mechanism of which may be attributed to inducing imDCs to transform into Tol-DCs in vitro and up-regulating the expression of TGF-β and IL-10, which promotes antigen-specific immune tolerance after adoptive transfer, thereby indirectly prolongs the survival of corneal grafts.
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Objective:To observe and analyze the changes of macular retinal thickness and related factors in children with occult myopia.Methods:A prospective longitudinal control study. From February 2021 to February 2022, 120 eyes of 60 children who first visited Department of Ophthalmology of The Affiliated Hospital of Chengde Medical College without any corresponding myopia correction treatment were included in the study. There were 32 males (64 eyes) and 28 females (56 eyes), with the age of 4-6 years. Visual acuity, medical optometry, corneal topography, spectral-domain optical coherence tomography (OCT) and axial length (AL) were measured at the first visit and 3, 6, 9 and 12 months. The children were divided into occult myopia group with 60 eyes of 30 cases and control group with 60 eyes of 30 cases according to visual acuity, equivalent spherical refraction (SE), AL and corneal curvature (CC). The macular fovea retinal thickness was measured by spectral-domain OCT enhanced deep imaging technique. According to the treatment and study of diabetic retinopathy, the retina within the 6 mm of the macular center was divided into three concentric circles with the macular fovea as the center, the central fovea of 1 mm, the inner ring of 1-3 mm and the outer ring of 3-6 mm. In the inner ring and outer ring, the retina was divided into 4 areas, upper and lower, left and right, with a total of 9 regions. The four regions of the inner ring and the outer ring were superior, inferior, nasal and temporal, respectively. The differences of AL, CC, SE, macular retinal thickness and the changes of related factors with time were compared between the occult myopia group and the control group. SE, AL, CC and retinal thickness in different macular regions of the two groups were compared by repeated measurement analysis of variance. In pairwise comparison, the minimum significant difference t test was used in different measurement time points, and the independent sample t test was used between groups at the same measurement time points. Results:At first diagnosis and after diagnosis, there were significant differences in SE ( F=783.710), AL ( F=742.192), macular fovea ( F=330.292), inferior and temporal ( F=158.250, 108.292) side of the outer ring retinal thickness in occult myopia group ( P <0.01). In the control group, there were significant differences in the retinal thickness of SE ( F=1 793.976), AL ( F=457.362), macular fovea ( F=31.029), inferior and temporal ( F=7.405, 77.245) side of the outer ring retinal thickness ( P<0.01). In both groups, with the prolongation of the time after the first diagnosis, the SE gradually increased, the AL gradually lengthened, and the thickness of the macular fovea, inferior and temporal side of the outer ring retinal thickness gradually thinned. Independent sample t test was performed on the data with differences between groups at the same time, and the results showed that at 3, 6, 9 and 12 months after first diagnosis, there were significant differences in SE, AL and CC between the occult myopia group and the control group ( t=-4.801,-11.532, 16.276, -17.145), AL ( t=24.203, 26.353, 27.057, 25.552); CC ( t=-23.362, -25.382, -25.890, -24.350; P<0.01). There were significant differences in macular fovea, inferior and temporal side of the outer ring retinal thickness at 6, 9 and 12 months, macular fovea ( t=-2.596, -2.542, -2.941; P<0.05), outer ring temporal ( t =-2.285, -2.610, -2.506; P<0.05). Conclusion:The SE and AL of the occult myopia group and the control group increase with time, and the former increase more rapidly than the latter; the macular fovea, inferior and temporal side of the outer ring retinal become thinner, and the former become thinner than the latter.
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Objective:To study the effects of resveratrol on the expressions of Caspase-3 and Bcl-2 in the retina tissue of the rats with retinal ischemia-reperfusion injury(RIRI),and to investigate the therapeutic effect of resveratrol on the RIRI and its mechanism.Methods:A total of 90 SD rats were randomly divided into sham operation group,model group and treatment group,30 rats in each group.The RIRI models were established by pressing the anterior chamber of the rats.The rats in model and treatment groups received ischemia-reperfusion for 1,6,12,24,and 48 h;the rats in treatment group were treated with micro-syringe intravitreal injection of 0.5 nmol·L-1of resveratrol 5 μL.The retinal tissue structure was observed by inverted microscope.The expression levels of Caspase-3 and Bcl-2 in retina tissue were detected by immunohistochemistry and Western blotting methods.Results:The retinal tissue edema of the rats in model group was found with vacuolar degeneration of the ganglion cells,and the arrangement of the cell layer was loose;the number of retinal ganglion cells was decreased,the boundary was blurred,and the nerve fiber layer thinned obviously.The degree of retinal tissue structure,the degree of injury and the degeneration of ganglion cells in treatment group were lighter than those in model group.The results of immunohistochemistry showed that the expressions of Caspase-3 and Bcl-2 in the retina tissue of the rats in treatment group were significantly increased compared with model group.The results of Western blotting method showed that the expression levels of Bcl-2 protein in the retina tissue of the rats in treatment group at different time points were increased compared with model group;and there were significant differences at 24 and 48 h(P<0.05);the expression levels of Caspase-3 protein in treatment group at different time points were lower than those in model group(P<0.05).Conclusion:Resveratrol can improve the retinal tissue structure of the RIRI rats,and its mechanism may be related to decreasing the expression level of Caspase-3 and increasing the expression level of Bcl-2 in the retinal tissue.
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Objective To evaluate the therapeutic effects of ultrasound biomicroscopy (UBM) assisted intraocular lens suspension surgery with one loop suturing for residual lens capsule aphakia.Methods Fifty aphakia patients (50 eyes) with residual lens capsular equal or greater than 180 degrees were chosen.The patients were randomly divided into 2 groups and underwent intraocular lens suspension surgery with one loop suturing:Trial group (25 cases,25 eyes) selected suture site according to the preoperative UBM results,and control group (25 cases,25 eyes) selected the suture site according to the clinical experience.The uncorrected visual acuity (UCVA),astigmatism at preoperative and postoperative 1 week,1 month and 3 months were observed,the eccentric and tilt value of the lens were calculated by UBM measurement at 3 month after operation,and the postoperative complications of the two groups were observed.Results UCVA at each time point after operation were higher than that before operation,and gradually increased with time.There was no significant difference between the two groups at 1 week before and after the operation of UCVA (P ＞ 0.05).At 1 month and 3 months after operation,UCVA in trial group were significantly higher than those of control group (all P ＜ 0.05).The astigmatism value at 1 week after operation in two groups was higher than that before operation and gradually decreased with time.The astigmatism value in trial group at 1 month and 3 months after operation had no significant difference with that of preoperative (P ＞ 0.05),while the astigmatism value in control group increased (all P ＜ 0.05).The astigmatism values had no significant difference between the two groups before and 1 week after operation (all P ＞0.05),which were significantly lower in trial group at 1 month and 3 months after operation (all P ＜ 0.05).Three months after surgery,the proportion of lens accurately implanted in the ciliary sulcus was significantly higher than control group (P ＜ 0.01),the eccentric and tilt values of the lens in trial group were significantly less than control group (P ＜ 0.01).No serious complication was observed in all patients.Conclusion UBM assisted intraocular lens suspension surgery with one loop suturing can significantly improve the postoperative visual quality,safety and lens stability,which can be used as a routine preoperative examination of intraocular lens suspension surgery.In addition,UBM provides an alternative method for the calculation of the eccentricity and tile value of the intraocular lens after the implantation of the intraocular lens.
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Objective To investigate long-term data of dry eye after LASIK in young myopia patients.Methods Young myopia patients were divided into treated group (group A) and control (group B),who were interviewed before surgery and on 1 year,3 years and 5 years after surgery.Their diopters,uncorrected visual acuity (UCVA),tear breakup time test,Schirmer I test and corneal fluorescent staining were taken,then dry eye was diagnosed and OSDI questionnaire were scored.Results The postoperative incidence of dry eye in group A was higher than that before surgery (35.6％) and decreased with time (51.7％,44.9％ and 40.7％,respectively),and there was no significant difference in group B at each time point (P ＞ 0.05).The preoperative incidence of dry eye was equal between two groups (P ＞ 0.05),which were much higher in group A after surgery (all P ＜ 0.05).In group A,the postoperative constituent ratio of mild dry eye was the majority and decreased with time (65.6％,52.8％,41.7％,respectively),while moderate and severe dry eye was increased,especially the latter (6.6％,11.3％ and 16.7％,respectively;P ＜ 0.05).At 5 years after surgery,the UCVA and visual function score in dry eye patients were lower than those of non-dry eye patients (all P ＜ 0.05),while the residual refraction and ocular symptom score were higher than those of dry eye patients (all P ＜ 0.05).Conclusion Dry eye disease may last for a long time and may getting worse after LASIK.Therefore,we should pay more attention to the development of dry eye long after LASIK in young myopic patients.
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Objective:To establish rat diabetes and eye disease models by injection of STZ and explore the therapeutic effect of panaxnotoginseng polysaccharides (PNP) on the diabetes and eye diseases of the model rats,and to clarify their mechanisms.Methods:Seventy SD male rats were randomly divided into blank control (n=10) and model groups (n=60), and the rats in model group were fed with high fat diet. 2 weeks later, the rats in model group were intraperitoneally injected with 35 mg·kg-1 STZ to establish the models.And 3 d later, the rats were treated with fasting and water deprivation for 12 h,the fasting blood glucose (FBG)was tested, and the models were assessed to be successful as the FBG>11.1 mmol·L-1.The rats with hyperglycemia were selected and divided into model, melbine(150 mg·kg-1), and low, middle and high doses (75,150 and 300 mg·kg-1) of PNP groups.After orally administration for 5 and 8 weeks, the FBG levels of rats were recorded.And 8 weeks later, the sugar tolerance, hepatic glycogen levels,serum glutathione(GSH) and nitric oxide(NO) levels of the rats were tested.The rat retinas were removed to analyze the expression levels of vascular endothelial growth factor(VEGF) and inducible nitric oxide synthase(iNOS) by using Q-PCR.The pathological changes of retinas were observed by HE staining method.Results:Compared with model group,the FBG level in middle dose of PNP group was decreased 5 weeks after treatment(P<0.05).Eight weeks later, compared with model group, the levels of FBG, sugar tolerance and hepatic glycogen in different doses of PNP groups were all decreased (P<0.05 or P<0.01).Compared with model group, the level of serum GSH in high dose of PNP group was remarkably increased(P<0.01), and the NO level was significantly decreased (P<0.05 or P<0.01).Compared with model group, the expression levels of VEGF and iNOS in high dose of PNP group were significantly decreased (P<0.05).The results of HE staining showed that the neurodeatrophia of the rats in low and middle doses of PNP groups were improved;and the vascular proliferation and neurodeatrophia of the rats in high dose of PNP group were significantly improved.Conclusion:PNP could decrease the blood sugar, increase the levels of GSH and NO, and up-regulate the gene expression levels of VEGF and iNOS, resulting the treatment of diabetes and its related retinopathy.
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Objective:To establish the rat diabetic models by introperitoneal injection of streptozotocin (STZ) to observe the expressions of MMP-2 in retina tissue of the diabetic rats at different periods,and to clarify the effect of MMP-2 in the diabetic retiropathy (DR) of the diabetic rats.Methods:The femal SD rats were divded into normal control group (n=24),4-week model group (n=30),6-week model group (n=30) and 8-week model group (n=30).The rats in model groups were intraperitoneally injected with STZ for consecutive 5 d.The rats in normal control group were injected with sodium citrate solution at the same volume.The body weights and blood glucose levels of the rats in each group were measured at 4,6,and 8 weeks.The retina of each rat was removed;RT-PCR and Western blotting methods were used to detect the expression levels of MMP-2 mRNA and protein,and the immunohistochemistry was conducted to observe the morphology.Results:The body weights and blood glucose levels of the rats in each group had no differences (P＞0.05) before modeling.Three rats died in 4-week model group,5 rats died at 6 weeks and 7 rats died at 8 weeks.The body weights of the rats in model group were significantly lower than those in normal control group at the same time (P＜0.05 or P＜0.01),and the fasting blood glucose levels were signifieantly increased (P＜0.05 or P＜0.01).The expression levels of MMP-2 mRNA and protein in the retina tissue of the rats in model group were significantly higher than those in normal control group (P＜0.05).In normal control group,the retinal structure was clear and the cells were arranged in order,and the ganglion cells were arranged in a single layer;in model group,the retinal tissue structure was loose,the number of ganglion cells were significantly reduced,the inner nuclear layer and rod cell layer cell membrane outside were fuzzy.The MMP-2 positive cells were found to be brown yellow granules,especially in the cytoplasm of ganglion cells and vascular endothelial cells.The positive expression levels of MMP-2 in model group were higher than those in normal control group at 4,6,and 8 weeks.Conclusion:MMP-2 can express in the retina tissue of the diabetic rats.
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Objective:To investigate the effect of astaxanthin on the refractive status and pathological morphology of the guinea pig myopia models induced by concave lens,and to elucidate the improvement effect of astaxanthin on the refractive status of myopia.Methods:A total of 48 healthy guinea pigs were randomly divided into blank control group (n=12) and model group (n=36);the guinea pig myopia models (right eyes) were established with far vision optical method (left eyes were used as control).The guinea pig models were divided into model control group,high of astaxanthin (50 ig · kg-1) group and low dose of astaxanthin (25 ig · kg 1) group.After 2 and 4 weeks treatment,the diopters and the lengths of optic axis of both eyes of each guinea pig in various groups were tested,and at 4 weeks the right eyes of the pigs were removed and the pathomorphology was observed by HE staining.Results:The diopters of the model eyes in model group were lower than those of left eyes used as control (P＜0.05) and those of right eyes in blank control group after 2 and 4 weeks (P＜0.05 or P＜0.01).The length of optic axis of the model eye in model group was increased compared with left eye used as controls and blank control group after 2 weeks (P＜0.05) and especially at 4 weeks (P＜0.01).Compared with model group,the axial oculi and diopters of the guinea pigs in astaxanthin groups were increased (P＜0.05);the pathohistological changes were improved,the collagen fracture was obviously decreased,and the collagen fibers were orderly arranged.Conclusion:Astaxanthin can recover the length of optic axis,diopter and histomorphology of sclera of guinea pig myopia models in some extents.
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Objective:To establish the rat diabetic models by introperitoneal injection of streptozotocin (STZ) to observe the expressions of MMP-2 in retina tissue of the diabetic rats at different periods,and to clarify the effect of MMP-2 in the diabetic retiropathy (DR) of the diabetic rats.Methods:The femal SD rats were divded into normal control group (n=24),4-week model group (n=30),6-week model group (n=30) and 8-week model group (n=30).The rats in model groups were intraperitoneally injected with STZ for consecutive 5 d.The rats in normal control group were injected with sodium citrate solution at the same volume.The body weights and blood glucose levels of the rats in each group were measured at 4,6,and 8 weeks.The retina of each rat was removed;RT-PCR and Western blotting methods were used to detect the expression levels of MMP-2 mRNA and protein,and the immunohistochemistry was conducted to observe the morphology.Results:The body weights and blood glucose levels of the rats in each group had no differences (P＞0.05) before modeling.Three rats died in 4-week model group,5 rats died at 6 weeks and 7 rats died at 8 weeks.The body weights of the rats in model group were significantly lower than those in normal control group at the same time (P＜0.05 or P＜0.01),and the fasting blood glucose levels were signifieantly increased (P＜0.05 or P＜0.01).The expression levels of MMP-2 mRNA and protein in the retina tissue of the rats in model group were significantly higher than those in normal control group (P＜0.05).In normal control group,the retinal structure was clear and the cells were arranged in order,and the ganglion cells were arranged in a single layer;in model group,the retinal tissue structure was loose,the number of ganglion cells were significantly reduced,the inner nuclear layer and rod cell layer cell membrane outside were fuzzy.The MMP-2 positive cells were found to be brown yellow granules,especially in the cytoplasm of ganglion cells and vascular endothelial cells.The positive expression levels of MMP-2 in model group were higher than those in normal control group at 4,6,and 8 weeks.Conclusion:MMP-2 can express in the retina tissue of the diabetic rats.
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Objective:To investigate the effect of astaxanthin on the refractive status and pathological morphology of the guinea pig myopia models induced by concave lens,and to elucidate the improvement effect of astaxanthin on the refractive status of myopia.Methods:A total of 48 healthy guinea pigs were randomly divided into blank control group (n=12) and model group (n=36);the guinea pig myopia models (right eyes) were established with far vision optical method (left eyes were used as control).The guinea pig models were divided into model control group,high of astaxanthin (50 ig · kg-1) group and low dose of astaxanthin (25 ig · kg 1) group.After 2 and 4 weeks treatment,the diopters and the lengths of optic axis of both eyes of each guinea pig in various groups were tested,and at 4 weeks the right eyes of the pigs were removed and the pathomorphology was observed by HE staining.Results:The diopters of the model eyes in model group were lower than those of left eyes used as control (P＜0.05) and those of right eyes in blank control group after 2 and 4 weeks (P＜0.05 or P＜0.01).The length of optic axis of the model eye in model group was increased compared with left eye used as controls and blank control group after 2 weeks (P＜0.05) and especially at 4 weeks (P＜0.01).Compared with model group,the axial oculi and diopters of the guinea pigs in astaxanthin groups were increased (P＜0.05);the pathohistological changes were improved,the collagen fracture was obviously decreased,and the collagen fibers were orderly arranged.Conclusion:Astaxanthin can recover the length of optic axis,diopter and histomorphology of sclera of guinea pig myopia models in some extents.
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Objective To observe whether the bone marrow stromal cells (BMSCs) which carry BMP-2/Tet-on genes and are microcapsulated by alginate polylysine alginate (APA) and seeded onto calcium phosphate cement (CPC) scaffolds can express bone morphogenetic proteins-2(BMP-2) and secrete collagen proteins.Methods The BMSCs from rats were prepared,transfected with adenoviruses carrying BMP-2/Tet-on genes,and then microcapsulated by APA.Eight porous CPC scaffolds were prepared and mounted onto a 12-hole plate.The experiment was conducted in 3 groups,with 4 parallel specimens in each group.Microcapsulated BMSCs with the target gene were seeded onto the CPC scaffolds in the experimental group; the transfected BMSCs were seeded into the plate holes as positive controls; APA microcapsules without cells were seeded onto the CPC scaffolds as blank controls.The culture medium in each group was taken for ELISA examination on 3,6,9 and 12 days to evaluate the BMP-2 concentrations respectively.The samples in the experimental group were fixed,sliced and dyed to observe secretion of collagen.Results On 3,6,9 and 12 days after culture,the BMP-2 concentrations were 4713.98 ± 178.50 μg/mL,3288.85 ± 194.38 μg/mL,1292.25 ± 300.11 μg/mL and 337.19 ± 84.49 μg/mL respectively in the experimental group; 4663.87 ±242.99 μg/mL,3250.67 ±293.72 μg/mL,1276.74 ± 157.10 μg/mL and 293.65 ±92.48 μg/mL respectively in the positive controls; 105.14 ± 10.93 μg/mL,91.42 ± 18.00 μg/mL,89.63 ± 12.99 μg/mLand 108.72 ± 23.90 μg/mL respectively in the blank controls.There were significant differences between the 3 groups at each same time point (P ＜ 0.05).The blank controls were significantly different from both the experimental group and the positive controls (P ＜ 0.05),but there were no significant differences between the experimental group and the positive controls (P ＞ 0.05).Collagen fibrils were observed by optic microscopy in the sections of the experimental group.Conclusion As microcapsulated BMSCs can survive in CPC scaffolds and express a significant amount of BMP-2 and secrete collagen proteins as expected,they can be used for further experiments in vivo.
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BACKGROUND: The reason for the short of cornea donator is the olden donating conception or the limit knowledge of cornea transplantation remains poorly understood. OBJECTIVE: To explore the awareness level of the medical staff to corneal transplantation and corneal donation. METHODS: The study consisted of 820 medical staff from different departments of 5 hospitals, including doctor, nurse, technician, and administrator. Awareness level was evaluated with an interviewer-study. RESULTS AND CONCLUSION: Totally 805 interviewer-studies were effect. The correct answer fill rate in department of ophthalmology was higher than other departments. Different measures should be taken to teach the medical staff to improve the awareness level of the transplantation and corneal donation.