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Objective:To investigate the role and underlying mechanisms of inhibiting high mobility group box-1 (HMGB1) in the expression of matrix metalloproteinase-9 (MMP-9) in spinal cord astrocytes (AS) in rats after spinal cord injury (SCI).Methods:After an SCI model was established in Sprague-Dawley (SD) rats using a modified Allen's Weight-Dropping method and ethyl pyruvate (EP) or glycyrrhizin (GL) was used to inhibit the effect of HMGB1, the rats were divided into a sham group, an SCI group, an SCI+EP (50 mg/kg) group, and an SCI+GL (100 mg/kg) group. The expression levels of glial fibrillary acid protein (GFAP) and MMP-9 in spinal cord AS were observed. After the spinal cord AS in SD rats was cultured and incubated by the oxygen-glucose deprivation/reoxygenation (OGD/R) procedure, the expression of MMP-9 protein was detected at 6 h/R 6 h, 12 h, 24 h, and 48 h after OGD. The time point with the highest expression was chosen in the subsequent experiments as an OGD/R group. HMGB1 was inhibited by HMGB1 shRNA or EP to observe the effect of HMGB1 on the expression of MMP-9 protein in AS treated with OGD/R. Then, toll-like receptor 4 (TLR4) inhibitor, TIR-domain-containing adaptor inducing interferon- β (TRIF) inhibitor, and nuclear factor-kappa B (NF- κB) inhibitor were used to investigate the effects of TLR4/TRIF/NF- κB signaling pathway during the regulation of HMGB1 on MMP-9 in vitro. Results:Western blot showed that the expression of MMP-9 protein in the spinal cord was significantly increased in rats at 1 d after SCI, and the expression of MMP-9 protein in the SCI+EP group and the SCI+GL group was significantly lower than that in the SCI group ( P<0.001). Immunofluorescence showed that GFAP and MMP-9 proteins were co-localized in the spinal cord after SCI, and the expression of GFAP and MMP-9 proteins in the SCI+EP and SCI+GL groups was significantly lower than that in the SCI group ( P<0.05). Since the expression of MMP-9 protein in the spinal cord AS cultured in vitro was significantly higher in the OGD 6h/R 12h group than that in the normal group and the OGD 6h/R 6h, 24, and 48 h groups, the OGD 6h/R 12h was taken as the OGD/R group. The MMP-9 protein expression in AS in the OGD/R+HMGB1 shRNA group and the OGD/R+EP group was significantly lower than that in the OGD/R group ( P<0.001). In the cultured AS, moreover, inhibiting TLR4, TRIF, and NF- κB reduced MMP-9 protein expression after OGD 6 h/R 12 h when compared with that in the OGD/R group ( P<0.001). Conclusions:HMGB1 inhibition may result in a reduction in MMP-9 expression both in the spinal cord AS in SCI rats and in AS after OGD/R treatment in vitro. HMGB1 may regulate MMP-9 protein expression in AS after OGD/R treatment via the TLR4/TRIF/NF- κB signal pathway.
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Objective To optimize the method of the directional differentiation of adipose-derived stem cells into keratinocytes.Methods Adipose-derived stem cells (ADSCs),separated by collagenase digestion method,were isolated and cultured.Then the expression of surface specific markers CD34,CD44 and CD90 were detected by flow cytometer.The effect of different induced mediums cultured for two weeks on the differentiation of ADSCs into KCs was demonstrated:Group 1,the DMEM supplied with 2％ FBS and 49％ supertant of KCs;group 2,KSFM medium;group 3,DMEM medium supplied with 10％ FBS and 5 μM ATRA;10％ FBS DMEM as the control group.Immunofluorescene staining was applied to detect the expression of keratin CK14 and F-actin.Results A flattened fibroblast-like morphology was observed in cells,the positive expression rate of CD34 was 0.08％,while those of CD44 and CD90 were 99％.The cells that could differentiate into osteoblasts and chondrocytes,indicated that the cells were ADSCs.There was no significant change in the cell morphology in the group 1 under the induction medium;about 10％ of the cells in group 2 were altered;the morphological changes were obvious in group 3,and approximately 20％ of the cells showed irregular polygon.The immunofluorescene staining of the cells in group 3 indicated that the cells showed cobblestone-like phenotype and an organized cytoskeletal network with dense actin fibers at the edges;some cells were positive for CK14.Conclusions ADSCs show higher induction rate under ATRA stimulation.
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Objective:In order to apply the index system for clinical evaluation of implementation effect in hospitals.Methods:A total of 862 patients with vaginal delivery from 9 hospitals were randomly divided into an clinical pathway group (n=496) and a control group (n=366).The patients in the control group received traditional treatment procedure while the patients in the clinical pathway group experienced procedure of the clinical treatment.The index system was used for clinical evaluation of implementation effect.Results:There were obvious advantages in 15 indicators in the clinical pathway group than those in the control group (P＜0.05).The comprehensive score of the clinical pathway group was higher than the control group of the corresponding grade and nature of the hospital.The comprehensive score for secondary hospitals (Ci=0.7967) were higher than that for the tertiary hospitals (Ci=0.2033).The comprehensive score for the general hospitals (Ci=0.8948) were higher than that for the specialized hospitals (Ci=0.1052).As for clinical implementation effect,the secondary hospitals were better than the tertiary hospital,and the general hospitals were better than the specialized hospitals.Conclusion:The index system for clinical evaluation could quantify the implementation effect,and compare the implementation effect in different hospitals,which provides reference for the management of clinical pathway.
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UL48 plays essential role in replication of MDV genome and interacts with UL36 as well as other MDV tegument proteins.To investigate the interaction between UL48 and UL36 during MDV oncogenisis,antibody against UL48 was prepared and characterized in current study.UL48 gene was amplified from MDV-Ⅰ genome and then subcloned into pTYB1 and pGEX-4T3 vectors for UL48 expression with induction of IPTG in BL21(DE3) E..coli cells.Chitin-sepharose and Glutathion-sepharose were,respectively,used to purify fusion protein intein-UL48 and GST-UL48.Four subcutaneous injections of intein-UL48 fusion protein were done on the lower back and the thigh of rabbit and then other three injections with an interval 10 days.The titer of antibody was measured by the sandwich ELISA with UL48 protein isolated from GST-UL48 after cleavage of thrombin.Western blot was carried out for specificity analysis of antibody against UL48 protein.The results suggested that UL48 antibody was succesfully prepared,and its titer was 1 ∶ 512 000.
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Objective We increase the soluble expression of artificial tandem hybrid ubiquitin binding domains ( ThUBD) in prokaryotic cytoplasm of Escherichia coli BL21 ( DE3 ) , which offer an effective and special profiling for ubiquitin conjugates( UbC) .Methods Codon optimization of the ThUBD was performed, followed by analysis of codon relative adaptiveness based on relative frequency of synonymous codon ( RFSC) of E.coli.Further induced expression and yeast ubiquitin conjugate enrichment quantified the soluble ThUBD-S and tested the ability to bind UbC.Results The statistical result showed that the percentage of codon of the highest usage frequency was increased from 48%to 75%, and codon adaptation index( CAI) was increased from 0.63 to 0.88 after codon optimization, which might suggest a higher expression of the ThUBD in E.coli BL21 (DE3).The subsequent SDS-PAGE indicated that the soluble target protein was increased four times, which accounted for 13.06%of total cell lysis.Further ubiquitinated proteome of yeast demonstrated that the ability to bind and enrich UbC of optimized ThUBD-S did not change compared with original ThUBD.Conclusion The expression of ThUBD-S can quadruple after codon optimization.At the same time, codon optimization does not impact its soluble expression and the ability to bind UbC.
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Ubiquitination is one of the most major post-translational modifications playing important role in regulation of intra-cellular proteins' stability, degradation, localization and biological activity. However, these proteins are difficult to be detected due to their low abundance, short half-life. In this study, ubiquitin-binding domains (UBDs) were constructed to purify the ubiquitinated proteins from Hep3B cells. Ubiquitinated proteins and sites were detected by LC-MS/MS. A total of 1 900 potential ubiquitinated proteins were identified. Among them, 158 ubiquitinated sites were identified, belonging to 102 proteins. Bioinformatics analysis revealed that the enriched pathways of ubiquitinated proteins were closely related to tumor occurrence and development. The dysfunction of ubiquitin-proteasome has a high correlation with cell signaling and extracellular matrix changing in tumor cells.
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The differentiation of stem cells into target cells in a particular region is an important prerequisite for the organ construction and tissue engineering. The processes are multi-directionally regulated by the surface properties of biomaterials, and among them the influences of mechanical rigidity and surface morphology of biomaterials on morphological characteristics, focal adhesion assemblies, and cytoskeletal structure of cells are considered to be the most important factors in regulating the differentiation of stem cells into specific cell lineages. This review summarizes the progresses on this topic in the past few years, which may provide a reference to the design of the biomaterials in regenerative medicine and tissue engineering.
Subject(s)
Humans , Biocompatible Materials , Metabolism , Cell Differentiation , Cell Lineage , Musculoskeletal System , Cell Biology , Metabolism , Regenerative Medicine , Stem Cells , Cell Biology , Surface Properties , Tissue EngineeringABSTRACT
The aim of the present study was to explore the differentially expressed genes in the blood vessel endothelial cells (BVECs) between diffuse large B-cell lymphoma (DLBCL) and reac- tive lymph node hyperplasia (RLNH), and to perform an initial bioinformatics analysis on a novel gene, C20orf14, which is highly expressed in lymph node of lymphoma. The mRNA of the tissue from the BVECs of DLBCL and RLNH tissues was labeled with biotin respectively and hybridized with expression profile microarray, and the differentially expressed genes were obtained. Initial bio- informatics analysis was performed on a novel gene named C20orf14. Its gene structure, genomic lo- calization, the physical and chemical characteristics of the putative protein, subcellular localization, functional domain etc. were predicted, and the systematic evolution analysis was performed on the similar proteins among several species. By using expression profile microarray, many differentially expressed genes were uncovered. The efficient bioinformatics analysis have fundamentally identified that C20orfl4 was a nuclear protein, and may be involved in the post-transcription modification of mRNA. Therefore, microarray is an efficient and high throughout strategy for the detection of differ- entially expressed genes, and C20orf14 is thought to be a potential target for tumor metastasis re- searches by bioinformatics analysis.