Objective:To observe the expression of S100A8 in plasma exosomes, microvesicles (MV), plasma and vitreous in patients with diabetic retinopathy (DR), and verify it in a diabetic rat model, and to preliminarily explore its role in the occurrence and development of DR.Methods:A case-control study. From September 2018 to December 2019, a total of 73 patients with type 2 diabetes, hospitalized patients undergoing vitrectomy, and healthy physical examinations in the Tianjin Medical University Eye Hospital were included in the study. Among them, plasma were collected from 32 patients and vitreous fluid were collected from 41 patients, which were divided into plasma sample research cohort and vitreous sample research cohort. The subjects were divided into simple diabetes group (DM group), non-proliferative DR group (NPDR group) and proliferative DR group (PDR group) without fundus changes; healthy subjects were regarded as normal control group (NC group). In the study cohort of vitreous samples, the control group was the vitreous humor of patients with epimacular membrane or macular hole. Plasma exosomes and microvesicles (MVs) were separated using ultracentrifugation. Transmission electron microscopy, nanometer particle size analyzer and Western blot (WB) were used to characterize exosomes and MVs. The mass concentration of S100A8 was determined by enzymelinked immunosorbent assay. Eighteen healthy male Brown Norway rats were divided into normal control group and diabetic group with 9 rats in each group by random number table method. The rats of diabetes group were induced by streptozotocin to establish diabetic model. Five months after modeling, immunohistochemical staining and WB were used to detect the expression of S100A8 in the retina of rats in the normal control group and the diabetes group. t test was used for the comparison of measurement data between the two groups. Single-factor analysis of variance were used for the comparison of multiple groups of measurement data.parison of measurement data between the two groups. Single-factor analysis of variance were used for the comparison of multiple groups of measurement data. Results:Exosomes and MVs with their own characteristics were successfully separated from plasma. The concentrations of plasma exosomes and vitreous S100A8 in the PDR group were higher than those in the NPDR group, DM group, NC group, and the difference was statistically significant ( P=0.039, 0.020, 0.002, 0.002, P<0.000,<0.000). In the plasma sample cohort study, It was not statistically significant that the overall comparison of the S100A8 mass concentrations of plasma and plasma MV in the four groups of subjects ( F=0.283, 0.015; P=0.836, 0.996). Immunohistochemical staining showed that retinal ganglion cells, bipolar cells, cone rod cells and vascular endothelial cells in the diabetic group all expressed S100A8 protein. Compared with the normal control group, the expression level of S100A8 in the retina of the diabetic group increased, and the difference was statistically significant ( t=8.028, P=0.001). Conclusions:The level of S100A8 protein in circulating exosomes increases significantly with the severity of DR in patients with type 2 diabetes. S100A8 may be an influential factor in the inflammatory environment of DR and a potential anti-inflammatory therapeutic target.

Objective:To analyze the protein expression changes in the retina of non-arteritic anterior ischemic optic neuropathy (NAION) in rats.Methods:The rat NAION (rNAION) model was established by Rose Bengal and laser. Twenty Sprague-Dawley rats were randomly divided into 4 groups, the normal control group, the laser control group, the RB injection control group, and the rNAION model group, with 5 rats in each group. The right eye was used as the experimental eye. The retina was dissected at the third day after modeling. Enzyme digestion method was used for sample preparation and data collection was performed in a non-dependent collection mode. The data were quantitatively analyzed by SWATH quantitative mass spectrometry, searching for differential proteins and performing function and pathway analysis.Results:Compared with the other three control groups, a total of 184 differential proteins were detected in the rNAION group (expression fold greater than 1.5 times and P<0.05), including 99 up-regulated proteins and 85 down-regulated proteins. The expressions of glial fibrillary acidic protein, guanine nucleotide binding protein 4, laminin 1, 14-3-3γ protein YWHAG were increased. Whereas the expressions of Leucine-rich glioma-inactivated protein 1, secretory carrier-associated membrane protein 5, and Clathrin coat assembly protein AP180 were decreased. The differential proteins are mainly involved in biological processes such as nerve growth, energy metabolism, vesicle-mediated transport, the regulation of synaptic plasticity, apoptosis and inflammation. Pathway enrichment analysis showed that PI3K-Akt signaling pathway and complement and thrombin reaction pathway was related to the disease. Conclusion:The protein expressions of energy metabolism, nerve growth, synaptic vesicle transport and PI3K-Akt signaling pathway can regulate the neuronal regeneration and apoptosis in NAION.

OBJECTIVESTo evaluate the relationship of vascular endothelial growth factor expression with estrogen receptor (ER) subtypes in fresh human breast cancer samples, and study the potential effect of ER subtypes on tumor angiogenesis.

METHODSWestern blot was used to detect the VEGF and ERbeta protein expression in 86 fresh samples of human breast cancer. ERalpha was analyzed by immunohistochemistry routinely.

RESULTSAmong 86 samples, 42 (48.8%) showed low expressed VEGF and 44 (51.2%) high expressed VEGF. The level of VEGF protein was correlated with ERbeta. In high expressed VEGF group, ERbeta was also highly expressed (chi(2) = 7.36, P < 0.01). But there was no significant difference between VEGF protein level and ERalpha (P > 0.05).

CONCLUSIONIn human breast cancer samples, VEGF may be related to ERbeta protein expression.

Adult ; Aged ; Blotting, Western ; Breast Neoplasms ; metabolism ; Endothelial Growth Factors ; metabolism ; Estrogen Receptor beta ; Female ; Humans ; Lymphokines ; metabolism ; Middle Aged ; Receptors, Estrogen ; classification ; metabolism ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

Objective To evaluate the feasibility of low-tube-voltage, low injection rate, low contrast agent dosage in combination with iterative model reconstruction ( IMR) algorithm in CT angiography ( CTA) of the head-and-neck vessels .Methods Sixty patients who underwent CT angiography of the head-and-neck vessels were randomly divided into groups A and B with 30 cases in each group .Patients in group A received a conventional scan with 120 kVp and filterback projected ( FBP) reconstruction .Patients in group B received a low-dose scan with 80 kVp, and image reconstruction with FBP ( group B1) and IMR (group B2)algorithm.The contrast agent protocol were as follows: the injection time in all patients was 10s, the injection rate was 4.5-5.5 ml/s in group A while 3.5-4.0 ml/s in group B.The CT values of artery, image noise, signal to noise ratio ( SNR) and contrast to noise ratio ( CNR) were measured and compared among three groups with One-way ANOVA analysis . Image quality was evaluated by two radiologists with five scale method , and compared with Kruskal-Wallis test .The CT dose index volume (CTDIvol) and dose length product ( DLP) were recorded and compared between groups with two independent samples t-test.Results The image quality scores of groups A , B1and B2 were 3-5, 2-4 and 3 -5, respectively .Image quality of twelve patients in group B 1 couldn′t meet the diagnostic requirements but none in group A and B 2.The objective image parameters SNR and CNR for group B 2 were equal to group A ( P >0.05), while those for group B1 were lower than group A (t=13.39, 9.45, P<0.05) and group B2 (t=-12.14, -9.96,P<0.05).CTDIvol and DLP for group B were separately 80.9%, 81.3%lower than those of group A(t=39.1, 32.2,P<0.05).The injection rate and contrast agent volume for group B were separately 22.0%, 22.1% lower than those of group A ( t=20.8, 20.8, P<0.01) .Conclusions It is feasible in CT angiography of the head-and-neck vessels with lower tube-voltage, lower injection rate, lower contrast agent dose and combining with iterative model reconstruction algorithm.This protocol can reduce the radiation dose by 81.3% while maintaining image quality .Trial registration Chinese clinical trial registry ,ChiCTR-BOC-16010060.

Alzheimer' s disease (AD) is a neurodegenerative disease that gradually impairs cognitive functions. Recently, there has been a conceptual shift toward AD to view the disease as a continuum. Since AD is currently incurable, effective intervention to delay or prevent pathological cognitive decline may best target the early stages of symptomatic disease, such as subjective cognitive decline (SCD), in which cognitive function remains relatively intact. Diagnostic methods for identifying AD, such as cerebrospinal fluid biomarkers and positron emission tomography, are invasive and expensive. Therefore, it is imperative to develop blood biomarkers that are sensitive, less invasive, easier to access, and more cost effective for AD diagnosis. This review aimed to summarize the current data on whether individuals with SCD differ reliably and effectively in subjective and objective performances compared to cognitively normal elderly individuals, and to find one or more convenient and accessible blood biomarkers so that researchers can identify SCD patients with preclinical AD in the population as soon as possible. Owing to the heterogeneity and complicated pathogenesis of AD, it is difficult to make reliable diagnoses using only a single blood marker. This review provides an overview of the progress achieved to date with the use of SCD blood biomarkers in patients with preclinical AD, highlighting the key areas of application and current challenges.

Objective: To determine the changes of protein expressions in human retinal microvascular pericytes (HRMPCs) stimulated with high glucose by using quantitative proteomics, which provides new clues for future investigation of diabetic retinopathy (DR). Methods: HRMPCs were divided into two groups.The cells in control group were cultured in DMEM basic medium with 25 mmol/L glucose, while the cells in high glucose group were cultured in DMEM medium with 35 mmol/L glucose.The amount of living cells was measured by cell counting kit-8(CCK-8). The proteins were collected from the two groups and then were digested with trypsin.Peptides of 2 μg were injected into the time of flight-mass spectrometer and the acquisition mode was DDA.The results were further analyzed using bioinformatics software. Results: CCK-8 results showed that the absorbance (A₄₅₀) of HRMPCs in high glucose group was 0.75±0.04, which was significantly lower than 0.91±0.05 in control group (t=5.784, P=0.000 2). In total, 1 972 proteins were identified and 54 of them were significantly different between the two groups (fold change >1.5). Among them, 13 proteins were up-regulated, including CTNNB1 and CTBP2; while 41 proteins were down-regulated, including SQSTM1 and HMGCS1.The differentially expressed proteins were mainly involved in citric acid cycle and aerobic respiration. Conclusions The expressions of many proteins in HRMPCs change under the stimulation of high glucose, which may influence the respiration and the ATP production of cells and eventually induce the loss of pericyte.

Objective: To observe the expression of retinal proteins in experimental autoimmune uveitis (EAU) mice and to explore the possible molecular mechanism of autoimmune uveitis. Methods: Twelve female C57BL/6J mice were randomly divided into model group and normal control group, 6 mice in each group.In the model group, the EAU model was established by subcutaneous injection of human interphotoreceptor retinoid-binding protein (IRBP) 651-670.The fundal change of EAV mice was assessed by direct ophthalmoscope, OCT and histopathological staining.At 18 days after immunization, the retinas of the two groups were taken for retinal protein extraction, protein restriction enzyme digestion, mass spectrometry detection, data analysis, and bioinformatics analysis.This study was approved by the experimental animal Ethics Committee of Tianjin Medical University Eye Hospital (TJYY2018070113). The feeding and use of experimental animals follow the ARVO statement. Results: The EAU mouse model was successfully established.At 10 days after immunitation, the retina of EAV mouse was damaged.At 18 days after immunization, retinal edema and infiltration of inflammatory cells into vitreous were observed.Proteomic results showed that a total of 4 458 proteins were identified in this study, of which 522 were differentially-expressed proteins (fold change>1.5, P<0.05). Among these differentially-expressed proteins, 147 proteins including Stat1 and Stat3 were up-regulated and 375 proteins including Gucy2f and Rho were down-regulated.These differentially-expressed proteins were closely related to platelet activation, integrin signaling, T cell activation, the phototransduction cascade, Toll-like receptor and so on. Conclusions EAU is related to the abnormal expression of Stat1, Stat3 and other proteins, as well as the abnormal regulation of platelet activation and integrin signal pathway.

Objective To detect the protein expression change in the proliferation of human retinal microvascular endothelial cells (hRMECs) stimulated with 4-Hydroxynonenal (4-HNE).Methods hRMECs were in a logarithmic growth phase, and then were separated into 4-HNE-stimulated group and negative control group. The concentration of 4-HNE included 5, 10, 20 and 50 μmol/L in 4-HNE-stimulated group, while the negative control group was added in the same volume of ethanol (the solvent of 4-HNE). Then the cells were stimulated with 4-HNE for 24 hours following by CCK-8 kits incubating for 4 hours to detect absorbance. It was found that 10 μmol/L 4-HNE had the most obvious effect on the proliferation of hRMECs. Therefore, the cellular proteins from 10 μmol/L 4-HNE-stimulated group and negative control group were acquired and prepared by FASP sample preparation method. Data independent acquisition was used for data acquisition, and the GO analysis and pathway enrichment were performed for analysis of differentially expressed proteins. Results CCK-8 kits detection results showed that theA value of the 10 and 20 μmol/L 4-HNE-stimulated groups were significantly higher than negative control group and 5 μmol/L 4-HNE-stimulated group (F=25.42, P<0.01), while there were no differences between 10 and 20 μmol/L 4-HNE-stimulated groups, and theA value of 50 μmol/L 4-HNE-stimulated groups was lower than negative control. A total of 2710 quantifiable proteins were identified by peoteomics,and 118 proteins were differentially expressed (fold change>1.5,P<0.05). Seventy-two proteins were up-regulated after 4-HNE stimulation, whereas 46 proteins were down-regulated. Particularly, the expressions of Heme oxygenase-1, Sulfoxdoxin-1, Heat shock protein A1B, Thioredoxin reductase-1, Glutathione reductase, ATPase and prothrombin were increased when cells were added in 4-HNE, whereas the expressions of apolipoprotein A1 and programmed cell death protein 4 were decreased. These differentially expressed proteins were mainly involved in the biological processes such as oxidative stress, cell detoxification, and ATPase-coupled membrane transport.Conclusion After stimulated with 4-HNE, the oxidative stress, cell detoxification, and ATPase-coupled membrane transport protein expression may change in hRMECs in order to regulate oxidative stress and growth situation.

Endogenous peptides, in the form of cytokines, growth hormones and hormone peptides, play an important role in human hormones, nerves, cell growth and reproduction. Neuropeptide is a kind of endogenous peptide, which is related to the physiological activities of pain, sleep, emotion, learning and memory. Neuropeptides exist not only in the nerve cells of the brain, but also in other body fluids and organs. At present, there is still a lack of research on endogenous peptides, especially on neuropeptides. In this study, high-throughput liquid chromatography tandem mass spectrometry was used to identify the distribution of endogenous peptides in the pancreas, heart, liver and kidney as well as the types of neuropeptides. The results showed that the number of endogenous peptides and neuropeptides in the liver was the highest while that of the pancreas was the lowest. The identified endogenous peptides were organ-specific and presented different dynamic distribution in four kinds of organs. The number of LPV (Longest peptide variant) of neuropeptide in the four organs varies greatly, and the distribution of gene family is also different. For example, neuropeptide in pancreas belongs to Glucagon family, while neuropeptide in heart belongs to ACBD7, Granins, PEBP and other families. The identification results will provide reference value for the mechanism study of diseases and the research and development of therapeutic drugs.
Amino Acid Sequence ; Animals ; Carrier Proteins ; Chromatography, Liquid ; Humans ; Mass Spectrometry ; Mice ; Peptides

Objective:To investigate the protein expression changes of human umbilical cord mesenchymal stem cells (hUCMSCs) modified with pigment epithelium-derived factor ( PEDF) gene mediated by lentivirus. Methods:The hUCMSCs were divided into the control group and experimental group.Cells in the control group were normal hUCMSCs and the cells in experimental group were hUCMSCs with PEDF modification.The proteins from the two groups were collected and processed by FASP method.Samples were fractionated by liquid chromatography and analyzed by tandem mass spectrometry, and SWATH mode was applied.Differential protein analysis, Gene Ontology (GO) analysis and Reactome pathway enrichment analysis were performed. Results:A total of 5 361 quantified proteins were detected in this experiment, of which 432 proteins were differentially expressed (fold change>1.5, P<0.05). There were 219 of the 432 proteins up-regulated, including serpin family F member 1 (SERPINF1) (PEDF), DEAD-box helicase 59 (DDX59) and thrombospondin 1 (THBS1), etc., whereas 213 proteins were down-regulated, including collagen type Ⅰ alpha 1 (COL1A1), COL18A1, etc.GO analysis indicated that the differential proteins were mainly involved in fibrinolysis, extracellular structure organization, regulation of transporter activity, biosynthetic process of secondary alcohol and cholesterol, coenzyme metabolic process and regulation of peptidase activity, etc.Reactome pathway analysis showed that the differential proteins were mostly involved in regulation of insulin like growth factor (IGF) transport and uptake by IGF binding protein, post-translational protein phosphorylation, extracellular matrix organization, metabolism of steroids. Conclusions:After gene modification with PEDF mediated by lentivirus, the expression of many proteins in hUCMSCs were changed. PEDF gene modification can alter the structure of extracellular matrix and regulate the expression of proteins associated with cell proliferation, self-renewal and multipotency.