Higher expression of AdPLA in orbital adipose tissue of patients with thyroid associated ophthalmopathy of Ⅲ level and stationary phase / 眼科新进展

Objective To detect the expression levels of adipose-specific phospholipase A2 (AdPLA) mRNA in orbital adipose tissue of thepatients with thyroid associated ophthalmopathy and the normals.Methods Sixteen patients with TAO of m level and stationary phase underwent orbital decompression,and 29 normals underwent ocular plastic surgery in Shenzhen Eye Hospital between August,2015 and October,2016.Orbital fat samples were collected from one eyes of these patients during surgery.The age,gender,height,weight,body mass index (BMI),exophthalmos degree,orbital fat of the patients with TAO and the normals were recorded and calculated.Using real time PCR,the AdPLA mRNA were detected from these orbital fat samples.Results There was no significant difference between the patients with TAO and the normals in age,gender,and BMI (all P ＞ 0.05).TAO group had more exophthalmos degree (20.406 ± 1.369)mm than the normals (14.207 ± 1.146) mm.TAO group had more orbital fat (32.162 ± 1.923) mL than the normals (24.279 ± 1.070) mL.The average expression level of AdPLA in patients with TAO was 0.039 42 ± 0.009 85,and 0.004 42 ± 0.001 36 in the normal.There was significant difference between two groups (P ＜ 0.05).Conclusion The patients with TAO of Ⅲ level and stationary phase have more exophthalmos degree and orbital fat than the normals.AdPLA mRNA is higher expressed in orbital adipose tissue of the patients with TAO of Ⅲ level and stationary phase than the normals.The high expression of AdPLA may reduce lipolysis in the orbital adipose tissue,lead to fat accumulation in orbits,and aggravate exophthalmos of patients with TAO.

Detection and analysis on serum antibodies for five common pathogenic microbes in patients with Posner-Schlossmarm syndrome / 中华实验眼科杂志

Background Posner-Schlossman syndrome (PSS) is often recurrent and is a cause of blindness.The etiology of PSS remains to be elucidated.It is reported that there is a certain association between pathogenic microorganisms and PSS in rather small samples.Objective This study was to analyze the related serum antibody levels of cytornegalovirus (CMV),herpes simplex virus (HSV),rubella virus (RV),helicobacter pylori (HP) and anti-streptolysin O (ASO) and provide a clue for the study on pathogenesis and therapy of PSS.Methods A prospective cases-controlled study was carried out in Shenzhen Eye Hospital from December,2014 to December,2016 under the approval of Ethic Committee of this hospital and informed consent of each subject prior to initial of any medical examination.Peripheral blood samples were collected from 82 PSS patients as the PSS group and 100 age-and gender-matched healthy blood donors as the normal control group.The positive rates of serum CMV IgG,CMV IgM,HSV IgG,HSV IgM,RV IgG,RV IgM,HP IgG and HP IgM in the subjects were detected by indirect ELISA,and the positive rate of serum ASO antibody was determined by immuno-scatter turbidmetry.Results The positive rates of serum CMV-IgG,CMV-IgM,HP-IgG,HP-IgM and ASO antibody were 22.0％,17.1％,22.0％,17.1％ and 17.1％ in the PSS group,which were significantly higher than 5.0％,0.0％,10.0％,2.0％ and 7.0％ in the normal control group (x2 =11.726,18.496,4.943,12.766,4.479,all at P＜0.05).The positive rates of serum HSV-IgG,HSV-IgM,RV-IgG and RV-IgM in the PSS group were not significantly different from those in the normal control group (x2 =3.305,0.986,0.898,0.503,all at P ＞ 0.05).Conclusions CMV,HP and hemolytic streptococcal infection may participate in the occurrence and development of PSS.

Pedigree and clinical characteristics in a Chinese family with lattice corneal dystrophy / 中华实验眼科杂志

Background Lattice corneal dystrophy (LCD) is a progressive disease,whose clinical features are varied in different stages.It is rarely be reported that clinical findings of different stages and factors of promoting the occurrence and development on LCD in a family.Objective The aim of this study was to identify the characteristics of the pedigree and clinical features of different stages in a LCD family,and further to discuss its influence factors.Methods A cross-sectional study was performed in this study.A Chinese family with LCD was enrolled in Shenzhen Eye Hospital from 2015 to 2016.Questionnaires for disease-related history,visual acuity measurement,ocular anterior segment examination and color photography were carried out for all the members of the family.In addition,anterior segment OCT (AS-OCT),laser scanning confocal microscope and corneal endothelium microscope were used to observe the morphology of corneal stroma and changes of corneal endothelial cells.The pedigree chart was drawn by Cyrillic2.1 software and analyzed based on Mendel law.Results This family included 5 generations of 73 members.Patients with LCD were found in each generation with similar morbidity in different gender,which followed the law of autosomal dominant inheritance.Eleven patients were found in 49 members related with Ⅲ1 of this family with the prevalence rate of 22.45％ and onset age at 21-50 years old,and the course of disease was 3-34 years.All of the members had no systemic disease except for two patients (Ⅲ 1 and Ⅲ 5) with hypertension.In the early stage of LCD,some bifurcate striolae appeared in the patients' corneal stroma without symptoms for many years.In the progressive stage,there was corneal irritation symptom accompanying with vision's decrease in the eyes with LCD.The bifurcate striolae were increased,widened and interwoven into lattice lines that the boundaries gradually became fuzzy,then corneal macula was formed because of recurrent corneal infiltration,and eventually resulted in corneal leucoma.High reflection corresponding to the pathologic region was showed by laser scanning confocal microscope and AS-OCT.No significant differences were found in corneal endothelial cell density and the percentage of hexagonal cells between LCD patients and normal phenotype families (t =1.887,P=0.075;t=-0.719,P =0.481).Penetrating keratoplasty was performed in a patient with corneal opacity and serious corneal opacity occurred near the surgical incision one year after the surgery.One patient was diagnosed as LCD 2 years after laser assisted in-situ keratomileusis.One patient was a welder.Conclusions LCD is autosomal dominant inheritance in the family.The clinical manifestations of LCD in the early,progressive and late stage can be seen in the pedigree,which offers a reference for ophthahnologists.Corneal surgery and lesion may induce the onset or aggravation of LCD.

Activation of serum complement in Posner-Schlossman syndrome patients / 中华实验眼科杂志

Background Activation of serum complement system is involved in the pathological process of uveitis and open angle glaucoma.Pathogenesis and pathological characteristics of Posner-Schlossman syndrome (PSS) are similar to uveitis and open angle glaucoma.However,etiology of PSS remains unelucidated.The activation complement in PSS patients' serum is rarely reported.Objective The aim of this study was to investigate the activation of serum complement in PSS patients for PSS pathogenesis.Methods A prospective case-controlled study was designed.The peripheral blood simples of 79 PSS patients were collected from Shenzhen Eye Hospital during December 2013 to December 2015,and the peripheral blood simples were obtained from 83 unrelated healthy blood donors as healthy control group.Immuno-scatter turbidmetry was adopted to detect the common activated components in complement pathway in each group including complement C3 (a vital intersection molecule in the three pathways),C4 (the vital molecule both the complement classical and lectin pathways),split products C3a,soluble membrane attack complex (sC5b-9),C 1q (complement classical pathway),L-ficolin (complement lectin pathway),complement factor Bb (complement alternative pathway),IgG,IgA and IgM.The correlation between serum C3a content and sC5b-9 content in PSS group was analyzed.The serum contents of fabric binding protein 2 (FCN2) (a marker of serum classical pathway),factor Bb (a marker of complement alternative pathway),C3a (the common activation products of three complement activation pathways),and sC5b-9 were assayed by ELISA.This research protocal was approved by Shenzhen Eye Hospital and written informed consent was obtained from each PSS patient prior to any medical examination.Results Compared with normal control group,the serum levels of C3,C4,C3a,sC5b-9,C1q,FCN2,IgG,IgA and IgM were significantly higher in PSS group (Z =-4.743,-2.913,-1.985,-2.620,-2.062,-2.500,-7.010,-6.327,-3.652,all at P ＜ 0.05).The serum complement factor Bb level was 13.87 (9.24,32.00) μg/ml in PSS group,which was significantly lower than 20.51 (12.90,33.50) μg/ml in normal control group (Z =-2.515,P =0.012).Serum C3a content was positively correlated with the serum sC5b-9 content in PSS group (rs =0.832,P＜0.001).Conclusions The serum complement system is activated in PSS patients.Complement alternative pathway,classical pathway and lectin pathway might all be involved in the activative process of complement system.

Association of HLA-DPA1 and -DPB1 polymorphisms with Posner-Schlossman syndrome among southern Chinese Han population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assess the association of HLA-DPA1 and -DPB1 polymorphisms with Posner-Schlossman syndrome (PSS) in southern Chinese Han population.</p><p><b>METHODS</b>A total of 100 randomly selected PSS patients of southern Chinese Han origin were served as the experimental group, while 128 unrelated healthy blood donors of the same origin were served as the control group. All samples were subjected to sequencing-based typing (SBT) for exon 2 of HLA-DPA1 and -DPB1 loci in both directions. HLA genotype was assigned using an Assign 3.5 HLA SBT software. The allele frequencies and haplotype frequencies of HLA-DPA1 and -DPB1 of the two groups were compared. x² test, P value and odds ratio (OR) value were calculated.</p><p><b>RESULTS</b>Six HLA-DPA1 alleles in the experimental group and 4 HLA-DPA1 alleles in the healthy control group were identified. The allelic frequency for HLA-DPA1*02:01 in the experimental group was significantly lower than the control group (4.50% vs. 12.109%; x²=8.124, P=0.004). Sixteen HLA-DPB1 alleles were identified in both the experimental and control groups. The allelic frequencies for HLA-DPB1*14:01 and - DPB1*17:01 in the experimental group were significantly lower than those of the control group ( DPB1*14:01: 1.00% vs. 4.688%, x²=5.130, P=0.024; DPB1*17:01: 0% vs. 2.344%, x²=3.897, P=0.048). The DPA1-DPB1 haplotypes for the experimental and control groups were 23 and 25, respectively. The haplotype frequencies for both DPA1*02:01- DPB1*14:01 and DPA1*02:01- DPB1*17:01 were significantly lower than those of the control group.</p><p><b>CONCLUSION</b>DPA1*02:01- DPB1*14:01 and DPA1*02:01- DPB1*17:01 haplotypes may provide considerable protection effect against PSS in the southern Chinese Han population.</p>

Stability analysis of CHO cell line expressing heterologous rhBMP2 / 生物工程学报

Bone morphogenetic protein 2 (BMP2), which belongs to the transforming growth factor-beta (TGF-beta) superfamily, is a multifunctional molecule with distinct abilities to induce bone formation. BMP2 has been identified to have eminent pharmaceutical importance for clinical application. We previously constructed stable cell line in Chinese hamster ovary cells (CHO) that highly expressed recombinant human BMP2 (rhBMP2). For large-scale production of the recombinant protein used in clinical application, it is critical to have both high expression and stability of the protein. In the present study, the stability of the cell line (rCHO(hBMP2)-C8) with the highest expression, as well as the stability of rhBMP2 protein were investigated systematically. We cultured the rCHO (hBMP2)-C8 cell line in the presence or absence of MTX for two months, the cell growth and rhBMP2 production characteristics were examined during the culture; we found the duration that the rCHO(hBMP2)-C8 cell line could secret rhBMP2 continually into the serum-free medium. Moreover, we detected the temperature sensitivity of rhBMP2 in culture medium. This study will contribute to our understanding for further producing rhBMP2 by large-scale culture technology.

Effect of different tags on pulldown assays implemented by LMO2 fusion protein / 生物工程学报

Pulldown assay is an in vitro method for studies of protein-protein interactions, in which tagged proteins are usually expressed as the bait to enrich other proteins that could bind to them. In this technology, the GST tag is broadest used for its modest size and hydrophilic property. In most cases, the GST tag could increase the hydrophility of the fusion protein and help to avoid the formation of inclusion bodies. However, in the other few cases, the target protein may be strongly hydrophobic or have complicated structures that were hard to fold and assemble in correct conformations without champerons, and even the existence of GST tag could not make them soluble. These proteins were always expressed as inclusion bodies and had no functions. LMO2 was a small molecular weight and insoluble protein, in this study, GST system and MBP system were used to express GST-LMO2 and MBP-LMO2 fusion proteins, respectively. We found that GST-LMO2 fusion protein was expressed as inclusion bodies whereas MBP-LMO2 fusion protein was expressed in soluble form. Moreover, the production rate of MBP-LMO2 was also much higher than GST-LMO2. Then MBP-LMO2 fusion proteins and renatured GST-LMO2 fusion proteins were used as bait in pulldown assay to study the interaction between LMO2 and endogenous GATA1 in K562 cells. Western blot analyses showed that both of these proteins could bind to endogenous GATA1 in K562 cells, but recovered GATA1 protein by MBP-LMO2 fusion protein was much more than GST-LMO2 fusion protein. These results suggest that using of MBP system is a helpful attempt in the case of studying small molecular weight, strong hydrophobic proteins.

Soluble expression of recombinant human BMP6 in Escherichia coli and its purification and bioassay in vitro / 生物工程学报

BMP6 is a potent protein for future treatment strategies of bone regeneration as it is a very important regulator of bone homeostasis. Active BMP6 is a dimer containing multidisulfide bonds and is a highly hydrophobic protein prone to aggregation. To obtain soluble and active BMP6 in Escherichia coli, we compared the effects of four N-terminal fusion tags (TRX, GST, MBP and CBD) and N-terminal His6-tag. The expression and solubility were tested under the different conditions (expression hosts, temperatures and inductor concentrations). A series of experiments leads to the finding that the placement of MBP before the BMP6 is best in availing the soluble expression of the protein. Our study alsodemonstrates that in E. coli BL21trxB(DE3) cytoplasm, which is a thioredoxin reductase mutant strain, soluble homodimeric BMP6 can be formed. The overexpressed MBP-BMP6 fusion protein is purified by chromatography, and shown to be functionally active.

The Role of E3 Ubiquitin Ligase Smurfs in BMP and TGF-bata Signaling in Bone Cells / 生物化学与生物物理进展

The ubiquitin-proteasome system is composed by multiple enzymes which are ubiquitously expressed in mammalian cells and plays an essential role in a variety of biological processes. As key members of the protein degradation enzymatic system, the function of E3 ubiquitin ligases has been extensively investigated. BMP and TGF-? are critical molecules that regulate proliferation, differentiation and apoptosis of osteoblasts and chondrocytes through different signaling pathways. Resent findings indicate that the ubiquitin-proteasome system functions as a key regulator in bone cells and the E3 ligase mediates the proteolytic degradation of critical molecules in BMP and TGF-? signaling pathways. Recent progress on studies of HECT domain E3 ligase, Smurf, in osteoblasts and chondrocytes were summarized. The regulatory role of Smurf1 and Smurf2 in BMP and TGF-? signaling and osteoblast and chondrocyte function has been reviewed.