Objective To investigate the inhibitory effects of IBI302 on experimental choroidal neovascularization (CNV). Methods Affinity of IBI302 to vascular endothelial growth factor (VEGF) family cytokines (including VEGF-A165, VEGF-A121 and placental growth factor PlGF) and complements (C3b, C4b) was determined by enzyme-linked immunosorbent assay (ELISA). The antagonist effect of IBI302 on VEGF was measured by proliferation, migration and tube formation tests of human umbilical vein endothelial cells (HUVEC). The anti-complement activity of IBI302 was measured by hemolysis test mediated by complement classical pathway and alternative pathway. Rhesus laser-induced CNV model was divided into 5 groups including model control group, bevacizumab group, IBI302 0.25 mg group, IBI302 0.50 mg group and IBI302 1.25 mg group. Fluorescein angiography and optical coherence tomography were performed on these monkeys at 14 and 28 days after drug delivery to observe the fluorescein leakage area and retinal thickness. The aqueous VEGF concentration was measured at 29 days after drug delivery. Results IBI302 showed good affinity to VEGF-A165, VEGF-A121 and PlGF, as well as C3b and C4b. IBI302 significantly inhibited the proliferation, migration and tube formation of HUVEC induced by VEGF-A165. IBI302 inhibited the hemolysis induced by complements obviously. At 14 and 28 days after drug delivery, the area of fluorescein leakage and retinal thickness in IBI302 0.25 mg group, IBI302 0.50 mg group, IBI302 1.25 mg group were reduced. The differences of the area of fluorescein leakage and retinal thickness in three IBI302 groups were not significant (P > 0.05). At 29 days after drug delivery, the VEGF concentration in the aqueous of rhesus monkey in bevacizumab group [(38.644±6.521) pg/ml] was decreased than that in model control group [(94.203±17.360) pg/ml], the difference was significant (P < 0.05). The VEGF concentration in the aqueous of rhesus monkey in three IBI302 groups were less than 31.300 pg/ml. Conclusion IBI302 inhibited experimental CNV through blocking the activity of VEGF and complement.
ObjectiveTo investigate the association of high density lipoprotein cholesterol (HDL-C) and cholesterol ester transfer protein (CETP) TaqIB mutation with non-arteritic anterior ischemic optic neuropathy (NA-AION) in the Shaanxi Han ethnic population. MethodsThe study cohort consisted of 45 individuals that had been diagnosed with NA-AION and 45 healthy controls (matched for age, gender). None of the cases or controls had a history of diabetes, serious cardio-cerebral vascular diseases, liver and kidney dysfunction that might influence plasma lipid levels. Plasma HDL-C was detected by enzyme-linked immunosorbent one-step, through the Toshiba TBA-40FR automatic biochemical analyzer. CETP TaqIB gene polymorphism was determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques for analysis. B2B2 genotype was only a fluorescence band with 535 bp; B1B1 genotype was 2 fluorescence bands with 361, 174 bp; B1B2 genotype was 3 fluorescence bands with 535, 361, 174 bp. The relative risk of genotype, HDL-C and disease occurrence was analyzed by logistics regression analysis. ResultsThere have no significant difference between NA-AION patients and controls about plasma total cholesterol level and triglyceride level (t=1.907, 1.877; P > 0.05). The plasma HDL-C levels were significantly lower in NA-AION patients than in controls (t=2.367, P=0.022). Compared with controls, the prevalence of B1B1 genotype and B1 allele was higher (χ2=17.289, P=0.001), the prevalence of B2 allele (χ2=15.648, P=0.000) was lower in NA-AION patients. The lower concentration of HDL-C was risk factor of NA-AION (odds ratio=6.143, 95% confidence interval 1.262-29.895, χ2=27.676;P=0.013). The proportion of B1B1 genotype was significantly higher in NA-AION patients than in controls (odds ratio=2.24, 95% confidence interval 2.427-36.323, χ2=10.526; P=0.001). ConclusionsThe low plasma HDL-C is independent risk factor for NA-AION and is associated with the development of NA-AION in the Shaanxi Han ethnic population. CETP TaqIB mutation is associated with low plasma HDL-C in NA-AION in the Shaanxi Han ethnic population.
Objective To clarify the relationship between inhibition of proliferation and cxpression of Ki-67 in cultured human retinal pigment epithelial(RPE) cells. Methods The cultured human RPE cells were treated with daunoblastina at a dose of 180 mu;g/L for 12h.Twenty-four hours later,DNA inhibiting rate was studied by using tritium-labelled thymidine deoxyribose(3H-TdR)incorporation assay.The expression of Ki-67 was evaluated by immunocytochemical staining technique and image analysis system.Flow cytometry was used to analyse cell cycle. Results DNA inhibiting rate was directly proportional to the dosage of daunoblastina.The proportion of the cells positive staining to Ki-67 in the control and the daunoblastina-treated group were 89.3% and 45.6%(Plt;0. 01),and the integral optical density values for expression of Ki-67 in the two groups were 68.1plusmn;6.2 and 27.3plusmn;5.5(Plt;0.01),respectively.The percen tage of cells in G2 phase of cell cycle increased from 8.9% to 29.5%. Conclusion G2 block was induced and poliferation was inhibited by daunoblastina in cultured human RPE cells.There is a relatively good correlation between Ki-67 immunostaining and inhibition of RPE cell proliferation. (Chin J Ocul Fundus Dis，2000，16：1-70)
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 effect of TGF-β receptor inhibitor Compound C on the directed differentiation of human embryonic stem cells (hESC) into retinal pigment epithelial (RPE) cells. MethodsH1 hESC were divided into control group and experimental group. When the hESC reached over confluence, the medium was changed to knockout serum replacement medium without bFGF to induce RPE differentiation. The experimental group was supplemented with 1 μmol/L TGF-β receptor inhibitor Compound C at the first six days of induction. Real-time PCR was carried out to examine the expression of paired-box gene 6 (PAX6), microphthalmia-associated transcription factor (MITF), cellular retinaldehyde blinding protein (CRALBP), and RPE65 in both groups at the 1, 3, 5 weeks of the induction process. hESC-derived RPE (hESC-RPE) cells were isolated mechanically and purified. Real-time PCR, Western blot and immunofluorescence were used to characterize the purified hESC-RPE cells. ResultsPigmented colonies were observed in experimental group at the 4 weeks of the induction process, while no pigmented colony could be detected in the control group. All the purified pigmented cells from experimental group showed polygons morphology. Experimental group showed significantly higher expression of RPE marker genes PAX6, MITF, CRALBP and RPE65 than the control group(P<0.05). Compared with the hESC and ARPE-19 cells line, purified hESC-RPE cells showed much higher expression of PAX6, MITF, CRALBP and RPE65(P<0.05).High expression level of PAX6 and RPE65 proteins were observed in hESC-RPE cells. Immunofluorescence verified the expression of PAX6 and ZO-1 in hESC-RPE cells. ConclusionTGF-β receptor inhibitor Compound C significantly improved the differentiation efficiency of hESC into RPE.
Objective To review the recent advances in transforming growth factor-β(TGF-β) super family study and its role in new bone formation. Methods The latest original articles related to this subject were retrieved extensively,especially the effect of TGF-β, bone morphogenetic proteins(BMPs) and activin(ACT) on distractionosteogenesis. Results TGF-β, BMPs and ACT play important roles in prompting new bone formation and each of them has different effects. Among them, TGF-β can stimulate the proliferation of osteoblast and synthesis ofextra cellular medium; BMPs can initiate the differentiation of interstitial cell toosteocyte; then ACT displays the combine effect of above two factors. Conclusion TGF-β superfamily can regulate new bone formation and thus shorten the course of mandibular distraction osteogenesis.
ObjectiveTo explore the role of osteopontin (OPN) in hyperoxia-induced acute lung injury and its relationship with nuclear factor-κB (NF-κB),matrix metalloproteinase 2 and 9 (MMP-2,MMP-9). MethodsNinety-six mice were randomly divided into a phosphate buffer solution intranasal inhalation group (PBS group) and a recombinant OPN intranasal inhalation group. The mice were exposed in sealed cages >95% oxygen for 24-72 hours to induce lung injury or room air as control. The severity of lung injury was evaluated. The expression of NF-κB,MMP-2,MMP-9,TIMP-1 and TIMP-2 mRNA in lung tissue at 24,48 and 72 hours under hyperoxia were examined by reverse transcript-polymerase chain reaction (RT-PCR). Immunohistochemistry (IHC) was performed for detection of NF-κB protein in lung tissue. ResultsPBS group mice developed more severe acute lung injury at 72 hours under hyperoxia.TIMP-1 and TIMP-2 mRNA expressions were significantly increased in r-OPN group than their matched PBS group when exposed to hyperoxia. IHC study showed higher expression of NF-κB protein in lung tissue of PBS group at 72 hours of hyperoxia. ConclusionOPN can protect against hyperoxia-induced lung injury by inhibiting the expressions of NF-κB,MMP-2 and MMP-9.
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.
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.
Objective To explore the relationship between the diabetic retinopathy (DR) and the changes of erythrocyte deformability(ED),erythrocyte membrane phospholipid and spectrin. Methods One hundred and eight patients with non-insulin dependent diabetes mellitus were divided into DR group(55 cases)and nonDR(NDR)group(53 cases).The changes of erythrocyte filtration index(EFI),erythrocyte membrane phospholipid and spectrin dimers(SP-D)and spectrin tetramers (SP-T)were measured in patients of DR and NDR groups and compared with the results of 53 cases of normal control group. Results The EFI,SP-D, SP-D/SP-T,sphingomyelin (SM) /phophatidylcholine(PC)were higher,and SPT,SM,PC,phophatidylserine(PS)and phatidylethanolamine(PE)were lower in patients with DR than those in control and NDR patients (F=8.467～18.925,q=6.845～12.627,Plt;0.001).The changes of all indicators in proliferative DR(PDR) patients were more obvious than those in background DR(BDR) patients(t=5,825-15.443,Plt;0.001).The EFI in DR patients was positively correlated to SM/PC,SP-D and SP-D/SP-T(Plt;0.01)，negatively correlated to SM,PC,PE,PS and SP-T(Plt;0.01). Conclusions The decrease of ED caused by the abnormalities of erythrocyte membrane phospholipid and spectrin might participate in the occurance and development of DR,and correlated to the degree of pathologic changes. (Chin J Ocul Fundus Dis, 1999, 15: 160-162)