Background Retinal neovascularization varies with the change of microRNA (miRNA) expression level.Quantitative real-time PCR (qRT-PCR) is a common method for the analysis of miRNA expression profiling.However,the housekeeping genes selected as references may exhibit differential expression levels under the distinctive experimental conditions.The accuracy of the levels of target gene expression often is affected if the selected housekeeping genes with significantly fluctuating expression as references.Determining a reference gene with stable expression level is the premise to consecutive studies.Objective This study was to compare the expression levels and stability of the frequently used reference genes for miRNA expression analysis in normally developing eyes and in eyes of a mouse model of oxygen-induced retinopathy (OIR),and select the optimal reference gene (s) exhibiting stable ocular expression under both hypoxia and normal development conditions.Methods P7,P12,P17,P37 and 8-week-old clean C57BL/6J mice were selected randomly.q-PCR was used to detect the dynamic changes of relative expressions of 5 kinds of reference genes in different ages of mice,including snRNAU6 (RNU6),5S ribosomal RNA (5s rRNA),snoRNA U68 (U68),snoRNA U70 (U70),snoRNA U49A (U49A).Other 40 P7 mice were randomized into the normal control group and the OIR group.The mice of the normal control group were fed in the normal oxygen environment,and those in the OIR group were raised in the (75-±2)％ oxygen environment for 10 days.Fluorescine was injected via ritrobulbar vein for retinal stretched preparation,and the histopathological examination of mouse retinas was performed to identified the models.The expressing difference of 5 kinds of reference genes in the retinas were detected to compare the differences of 5 kinds of genes expression between the two groups.GeNorm program was employed to compare the stability of the expressing genes.This study were approved and granted by Ethical Committee of Tianjin Medical University and met the requirements stipulated in the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.Results Across the developmental ages,the expression levels of U68 in the eye exhibited the greatest variability,and then coming with U49A,U70,RNU6 and 5s rRNA.Significant differences were seen across the developmental ages for the expression levels of U68,U49A,U70,and RNU6 (U70:F =7.005,P =0.000 ; U68:F =10.189,P =0.000 ; U49A:F =21.134,P=0.000;RNU6:F=4.968,P=0.004).Expression of 5s rRNA showed the least variability across the developmental ages (F=2.099,P =0.107).In P17 mice of the OIR group,tortuous access vessels and non-perfusion area were found in the retinal section.Hematoxylin-eosin stain showed the neovascular sprout through across the inner limiting membrane.The relative expression of U70 exhibited the greatest variability and then were U49A,U68 and 5s rRNA in turn in P17 OIR mice,and significantly elevated expressions were found in U70,U49A,U68 and 5s rRNA genes between the OIR group and the normal control group (t =5.174,P =0.000;t =3.376,P =0.012;t =4.802,P =0.000 ;t=2.856,P=0.029).Expression of RNU6 showed the least variability between the two groups (t =2.104,P=0.065).As analyzed by GeNorm program,the stability for the five reference genes across developmental ages was 5s rRNA/RNU6＞ U70 ＞ U49A ＞ U68,and the optimal reference combination was 5s rRNA and RNU6.Whereas the stability for the OIR model was 5s rRNA/RNU6＞U68＞ U49A＞U70,and the optimal reference combination was 5s rRNA and RNU6.Conclusions Reference genes should be selected based on specific subjects and experimental conditions when qRT-PCR is used to analyze miRNA expression levels.In the OIR model,both developmental and hypoxic factors need to be considered.5s rRNA and RNU6 reference combination is the preferred option for the qRT-PCR analysis of ocular miRNA expression if available.

BACKGROUND:Kartogenin can induce chondrogenic differentiation of mesenchymal stem cel s as reported in in vitro experiments. The discovery of Kartogenin finds a novel path to cartilage repair, and it is expected to develop into a new drug to treat osteoarthritis. OBJECTIVE:To observe the inductive role of Kartogenin in the process of human bone marrow mesenchymal stem cel s differentiating into chondrocytes in vitro. METHODS:In vitro cultured human bone marrow mesenchymal stem cel s were grown to the logarithmic phase, and then divided into control group (0μmol/L Kartogenin), 1μmol/L Kartogenin group, and 10μmol/L Kartogenin group. After 72 hours of culture, cel proliferation and differentiation were observed microscopical y. Matrix metal oproteinase 2 and type II col agen levels in the cel supernatant were detected by enzyme linked immunosorbent assay and immunofluorescence staining, respectively. RESULTS AND CONCLUSION:Under the microscope, Kartogenin was shown to significantly promote the proliferation and differentiation of human bone marrow mesenchymal stem cel s. With the increase of Kartogenin concentrations, the level of type II col agen was increased, while the level of matrix metal oproteinase 2 was decreased. These findings indicate that Kartogenin can induce human bone marrow mesenchymal stem cel s to differentiate into chondrocytes, and with the increase of Kartogenin concentration, destruction of the cartilage extracel ular matrix may be inhibited.

Background Retinal neovascularization (RNV) is one of the major causes of blindness worldwide,but the pathogenic mechanism of this disease remains unclear, and therapeutic modalities need to be improved.Therefore,it is necessary to identify ocular protein markers with significant expression changes during RNV, thereby providing novel therapeutic targets for neovascular retinopathies.Objective This study was to investigate retinal vessel morphological characteristics and protein expression profiling in a mouse model of oxygen-induced retinopathy (OIR).Methods Forty four C57BL/6J mouse pups were randomly divided into normal control group and OIR group at postnatal day 7 (P7).The mice in the normal control group were raised under the normal air for 10 days.The mice of the OIR group were exposed to (75±2)％ oxygen for 5 days.The mother mice were alternated between the two groups every day.The mice of the OIR group returned to normal air at P12.Fluorescein isothiocyanate-dextran (FITC-dextran) was retrobulbarly injected at P17 mice from both groups, and the retinal flatmounts were prepared after fixation.The FITC-dextran-labeled retinal vessels were observed and quantified;the paraffin sections of eyeballs were processed for hematoxylin and eosin staining,and the number of pre-retinal vascular cell nuclei was quantified.The total proteins were extracted from the eyes, and the expression profiling was analyzed by a customized protein array and verified by Western blot and ELISA.Results The retinal flatmounts labeled with FITC-dextran showed that the peripheral retinal microvessels in the OIR group were tortuous, disorganized with neovascular buds,and the vascular obliteration was prominent in the center of retina.Contrastly,the vessels were smooth,organized, and evenly distributed in the normal control group.The percentage of vascular obliteration area in the OIR group was (25.53±2.16)％ ,which was significantly higher than (0.66±0.36)％ in the normal control group (t=-27.61 ,P＜ 0.01).The number of pre-retinal vascular cell nuclei,as revealed by hematoxylin and eosin staining, was (28.41 ± 3.97)/slide in the OIR group, which was substantially higher than (0.16±0.31)/slide in the normal control group (t =-54.42,P＜0.001).Protein array showed that 10 out of the 62 examined pro-inflammatory, pro-angiogenic cytokines exhibited more than 1.5-fold expression changes,including 3 up-regulated cytokines and 7 down-regulated cytokines;4 cytokines showed more than 2-fold expression changes,in which 3 cytokines were down-regulated and 1 cytokine was up-regulated.The differential expressions were verified by Western blot and ELISA.The expression trends of platelet factor 4 (PF-4), vascular endothelial growth factor A (VEGF-A), selectin P (SELP), vascular cell adhesion molecule 1 (VCAM-1) ,soluble tumor necrosis factor receptor Ⅱ (sTNF-RⅡ) and chemokine C-X-C motif ligand 16 (CXCL16) were consistent with those revealed by protein array.PF-4,VEGF-A and SELP were up-regulated,and the other 3 were significantly down-regulated (all at P＜0.05).Conclusions Differential expression patterns of the cytokines,including PF-4,VEGF-A, SELP, VCAM-1, sTNF-RⅡ and CXCL16, are identified between normal and OIR mouse eyes.These differential expression patterns suggest that under the condition of OIR,the platelet system is activated,and proinflammatory factors are down-regulated.PF-4 might become a new target for VEGF-independent therapeutic strategy against RNV.

Background Retinal neovascularization (RNV) occurs in multiple eye diseases,which can lead to bleeding and retinal detachment.Therefore,inhibition of pathological RNV is becoming crucial to the treatment of ocular diseases.Research has shown that α-melanocyte-stimulating hormone (α-MSH) inhibits retinal angiogenesis during physiological development;however,the effects of α-MSH on pathological RNV remain unknown.Objective This study was to investigate the effects of intravitreal injection of α-MSH at different concentrations on pathological RNV in a mouse model of oxygen-induced retinopathy (OIR).Methods Forty healthy clean C57BL/6J mice were randomly divided into OIR+0.33 μg/μl α-MSH,OIR+ 1.67 μg/μl α-MSH,OIR+3.30 μg/μl α-MSH,OIR,and normal control groups at postnatal day 7 (P7),with 8 pups in each group.The α-MSH intervention groups and OIR group were exposed to high oxygen (75 ±2)％ for 5 days,then maintained under normal air condition for another 5 days;whereas the normal control group was raised under normoxia for 10 days.Retro-orbital injection of high molecular weight fluorescein isothiocyanate-dextran (FITC-dextran) was performed on P17 mice.The retina whole mounts were prepared to reveal retinal vasculature and quantify relative area of vessel obliteration.The mouse eyeballs were subjected to paraffin sections and hematoxylin-eosin staining,and the average number of pre-retinal nuclei per section was quantified.Results FITC-dextran labeled retinal whole mounts showed that the relative vessel obliteration area in normal control,OIR,OIR+0.33 μg/μl α-MSH,OIR+ 1.67 μg/μl α-MSH,and OIR+3.30 μg/μl α-MSH groups were (0.00±0.00) ％,(23.01 ±3.39) ％,(18.14±7.20) ％,(15.64±7.07) ％,and (7.62±6.52) ％,respectively.There was a statistical significance in the relative avascular area among the groups (F=19.635,P＜0.05).The relative avascular area in the OIR group was significantly higher than that in the OIR+3.30 μg/μl α-MSH group (t=4.293,P＜0.01).The results of histopathological examinations showed that the average number of pre-retinal nuclei per section in normal control,OIR,OIR+0.33 μg/μl α-MSH,OIR+1.67 μg/μl α-MSH,and OIR+3.30 μg/μl α-MSH groups were 0.00±0.00,11.45 ±4.26,6.35 ±2.34,4.96 ± 1.79 and 1.03 ± 1.25,respectively.There was a statistical significance in the average number of pre-retinal nuclei per section among the groups (F =147.87,P＜0.05).The average number of pre-retinal nuclei per section in the OIR group was significantly higher than that in the OIR+0.33 μg/μl α-MSH,OIR+ 1.67 μg/μl α-MSH,and OIR+3.30 μg/μl α-MSH groups,the differences between the groups had statistical significances (all at P＜0.001).Conclusions α-MSH reduces the relative area of vessel obliteration and the average number of pre-retinal nuclei in the retinas of OIR mouse model.The inhibitory effects of α-MSH on the pathological RNV are dose-dependent.

The pathogenesis of neovascular age-related macular degeneration (nAMD) is complex and macular neovascularization (MNV), a key pathogenic factor in nAMD, is prone to recurrence.Vitreous injection of anti-VEGF drugs is the main therapy of nAMD.In recent years, a lot of progress has been made in fundus imaging techniques and optical coherence tomography angiography (OCTA) with non-invasive, rapid, stratified and high-definition functions has shown strong advantages in diagnosis, differential diagnosis, disease dynamic monitoring and follow-up of nAMD.Clinicians have had a certain understanding of the important role of OCTA in the diagnosis of nAMD and other diseases, and its clinical application value has been recognized gradually.However, its application value in follow-up of patients with nAMD and polypoid choroidal vasculopathy (PCV) is still not well understood.By reviewing a large number of recent relevant literature on OCTA, and combining the clinical practice of our research team in monitoring the course of AMD and PCV disease by OCTA, we have gained new knowledge and understanding of the pathological mechanism of AMD and PCV.In this paper, we elucidated the latest understanding of the diagnostic value of OCTA in AMD based on long-term series of OCTA studies, the new findings of OCTA in AMD management of our team, as well as its impact on ophthalmology clinical practice.Then we forecasted the role of OCTA in the prediction of recurrence and anti-VEGF treatment response, as well as the clinical value of OCTA in the optimization of nAMD treatment and follow-up plan.It is recommended that clinicians pay more attention to the clinical value and guiding role of OCTA in long-term treatment monitoring and follow-up of AMD.

OBJECTIVES: This study evaluated the association between obesity and glaucoma in middle-aged and older people. A population-based retrospective cohort study was conducted using data from the China Health and Retirement Longitudinal Study. METHODS: Glaucoma was assessed via self-reports. Multivariate logistic regression analysis and a Cox proportional hazards model were used to assess the relationship between obesity and glaucoma risk. RESULTS: Older males living in urban areas who were single, smokers, and non-drinkers were found to have a significantly higher incidence of glaucoma (all p<0.05). Diabetes, hypertension, and kidney disease were also associated with higher glaucoma risk, while dyslipidemia was associated with lower risk (all p<0.05). After the model was adjusted for demographic, socioeconomic, and health-related variables, obesity was significantly associated with a 10.2% decrease in glaucoma risk according to the Cox proportional hazards model (hazard ratio, 0.90; 95% confidence interval [CI], 0.83 to 0.97) and an 11.8% risk reduction in the multivariate logistic regression analysis (odds ratio, 0.88; 95% CI, 0.80 to 0.97). A further subgroup analysis showed that obesity was associated with a reduced risk of glaucoma in people living in rural areas, in smokers, and in those with kidney disease (all p<0.05). Obesity also reduced glaucoma risk in people with diabetes, hypertension, or dyslipidemia more than in healthy controls (all p<0.05). CONCLUSIONS This cohort study suggests that obesity was associated with a reduced risk of glaucoma, especially in rural residents, smokers, and people with kidney disease. Obesity exerted a stronger protective effect in people with diabetes, hypertension, or dyslipidemia than in healthy people.

OBJECTIVETo study the severe acute respiratory syndrome (SARS)-associated coronavirus genotype and its characteristics.

METHODSA SARS-associated coronavirus isolate named ZJ01 was obtained from throat swab samples taken from a patient in Hangzhou, Zhejing province. The complete genome sequence of ZJ01 consisted of 29,715 bp (GenBank accession: AY297028, version: gi: 30910859). Seventeen SARS-associated coronavirus genome sequences in GenBank were compared to analyze the common sequence variations and the probability of co-occurrence of multiple polymorphisms or mutations. Phylogenetic analysis of those sequences was done.

RESULTSBy bioinformatics processing and analysis, the 5 loci nucleotides at ZJ01 genome were found being T, T, G, T and T, respectively. Compared with other SARS-associated coronavirus genomes in the GenBank database, an A/G mutation was detected besides the other 4 mutation loci (C:G:C:C/T:T:T:T) involved in this genetic signature. Therefore a new definition was put forward according to the 5 mutation loci. SARS-associated coronavirus strains would be grouped into two genotypes (C:G:A:C:C/T:T:G:T:T), and abbreviated as SARS coronavirus C genotype and T genotype. On the basis of this new definition, the ZJ01 isolate belongs to SARS-associated coronavirus T genotype, first discovered and reported in mainland China. Phylogenetic analysis of the spike protein gene fragments of these SARS-associated coronavirus strains showed that the GZ01 isolate was phylogenetically distinct from other isolates, and compared with groups F1 and F2 of the T genotype, the isolates of BJ01 and CUHK-W1 were more closely related to the GZ01 isolate. It was interesting to find that two (A/G and C/T) of the five mutation loci occurred in the spike protein gene, which caused changes of Asp to Gly and Thr to Ile in the protein, respectively.

CONCLUSIONAttention should be paid to whether these genotype and mutation patterns are related to the virus's biological activities,epidemic characteristics and host clinical symptoms.

Chronic inflammation is critical in the onset and progression of atherosclerosis (AS). The lipopolysaccharide (LPS) level in the circulation system is elevated in AS patients and animal models, which is correlated with the severity of AS. Inspired by the underlying mechanism that LPS could drive the polarization of macrophages toward the M1 phenotype, aggravate inflammation, and ultimately contribute to the exacerbation of AS, LPS in the circulation system was supposed to be the therapeutic target for AS treatment. In the present study, polymyxin (PMB) covalently conjugated to PEGylated liposomes (PLPs) were formulated to adsorb LPS through specific interactions between PMB and LPS. In vitro, the experiments demonstrated that PLPs could adsorb LPS, reduce the polarization of macrophages to M1 phenotype and inhibit the formation of foam cells. In vivo, the study revealed that PLPs treatment reduced the serum levels of LPS and pro-inflammatory cytokines, decreased the proportion of M1-type macrophages in AS plaque, stabilized AS plaque, and downsized the plaque burdens in arteries, which eventually attenuated the progression of AS. Our study highlighted LPS in the circulation system as the therapeutic target for AS and provided an alternative strategy for AS treatment.