Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology最新文献

Objective Screening the differentially expressed proteins related to apoptosis in cold-dampness syndrome of rheumatoid arthritis (RA). Methods Peripheral blood mononuclear cells (PBMCs) were collected from healthy people and RA patients with cold-dampness syndrome. 43 apoptosis-related proteins were detected by antibody chip, which was verified by ELISA. Results In 43 apoptosis-related proteins, 10 of them were up-regulated and 3 were down-regulated. The most differentially expressed were tumor necrosis factor receptor 5 (CD40) and soluble tumor necrosis factor receptor 2 (sTNFR2). Compared with the normal group, the expression of CD40 and sTNFR2 in RA patients with cold-dampness syndrome increased significantly. The results of receiver operating characteristic curve (ROC) showed that CD40(AUC=0.8133) and sTNFR2(AUC=0.8117) could be used as diagnostic markers of RA patients with cold-dampness syndrome. The results of Spearman correlation analysis showed that CD40 was negatively correlated with Fas and Fas ligand (FasL), while sTNFR2 was positively correlated with erythrocyte sedimentation rate and negatively correlated with mental health score (MH). Logistic regression analysis showed that rheumatoid factor (RF), 28 joint disease activity scores (DAS28) and vitality (VT) were risk factors for CD40. ESR, anti-cyclic citrullinated peptide (CCP) antibody, self-rating depression scale (SAS) and MH were the risk factors of sTNFR2. Conclusion CD40 and sTNFR2 are proteins related to apoptosis in RA patients with cold-dampness syndrome, which are closely related to clinical indexes and apoptosis indexes.

Objective To establish a Flp-In^TM CHO cell line stably expressing human cytochrome P450 oxidoreductase (POR) to lay a solid foundation for the further construction of cell lines stably co-expressing human POR and human cytochrome P450 (CYP). Methods POR recombinant lentivirus was established and infected with Flp-In^TM CHO cells, and the expression of green fluorescent protein was observed by fluorescence microscope for monoclonal screening. Mitomycin C (MMC) cytotoxic assay, Western blot analysis and quantitative real-time PCR (qRT-PCR) were employed to detect the activity and expression of POR, and eventually obtained a cell line stably expressing POR (Flp-In^TM CHO-POR). Flp-In^TM CHO-POR cells (Flp-In^TM CHO-POR-2C19) stably co-expressing POR and CYP2C19, and Flp-In^TM CHO cells stably expressing CYP2C19 (Flp-In^TM CHO-2C19) were constructed, and the activity of CYP2C19 was measured by cyclophosphamide (CPA). Results The consequences of MMC cytotoxic assay, Western blot and qRT-PCR illuminated that Flp-In^TM CHO cells infected with POR recombinant lentivirus had elevated MMC metabolic activity and boosted the expression of POR mRNA and protein, compared with Flp-In^TM CHO cells infected with negative control virus, indicating that Flp-In^TM CHO-POR cells stably expressing POR were obtained. No significant disparity existed in the metabolic activity of CPA between Flp-In^TM CHO-2C19 and Flp-In^TM CHO cells, whereas the metabolic activity enhanced in Flp-In^TM CHO-POR-2C19 and was significantly higher than in Flp-In^TM CHO-2C19 cells. Conclusion The stable expression of Flp-In^TM CHO-POR cell line is successfully established and can be further applied to construct CYP transgenic cells.

Objective To investigate the effect and mechanism of Mongolian medicine Heisuga-25 on Alzheimer's disease (AD) mice. Methods Six month old SAMP8 mice were divided into model group, Heisuga-25 [360 mg/(kg.d), 90 mg/(kg.d)] treatment group, and donepezil control group[0.92 mg/(kg.d)], with 15 mice in each group. Another 15 6-month-old normal aging SAMR1 mice were selected as blank control group. The mice in the model group and blank control group were fed with normal saline, and the other groups were gavaged according to the dosage. All groups were gavaged once a day for 15 days. From Day 1 to Day 5 after administration, three mice in each group were taken and Morris water maze test was been used to detect the escape latency, times for crossing the platform and the residence time were detected. Nissl staining was used to observe the number of Nissl bodies. Western blot analysis and immunohistochemistry were used to detect the expression of microtubule associated protein 2 (MAP-2) and low molecular weight neurofilament protein (NF-L). ELISA was used to detect the contents of acetylcholine (ACh), 5-hydroxytryptamine (5-HT), norepinephrine (NE) and dopamine (DA) in cortex and hippocampus of mice. Results Compared with the blank control group, the escape latency was significantly prolonged, while the model group showed a decrease in the number of crossing the platform, residence time, Nissl bodies, and the protein expression of MAP-2 and NF-L. Compared with the model group, Heisuga-25 administration group exhibited an increase in the number of crossing the platform and residence time, Nissl bodies, and the protein expression of MAP-2 and NF-L, but a shortened escape latency. The effect of high-dose groupHeisuga-25 [360 mg /(kg.d)] on the above indexes was more obvious. Compared with the blank control group, the contents of ACh, NE, DA and 5-HT in hippocampus and cortex were decreased in the model group. Compared with the model group, the low-dose group, high-dose group and donepezil control group all observed an increase in the contents of ACh, NE, DA and 5-HT. Conclusion Mongolian medicine Heisuga-25 can improve learning and memory by protecting the neural function of AD model mice, which may be accounted for up-regulation of neuronal skeleton protein expression and increased content of neurotransmitters.

Microglia are the resident immune cells of the central nervous system (CNS). Microglia are usually in the surveillant or quiescent state which is tightly regulated by several mechanisms called microglial immune checkpoints. Microglial immune checkpoint mechanism consists of four dimensions: soluble restraining factors, cell-to-cell interactions, seclusion from the circulation, and transcriptional regulators. Stress may lead to a more potent activation state of microglia when a subsequent immune challenge arrives, which is known as the microglial priming. Stress can prime microglia by affecting microglial checkpoints.

Objective To identify the key targets and molecular mechanisms of Sangbaipi decoction in the treatment of acute exacerbations of chronic obstructive pulmonary disease (AECOPD) by using network pharmacology. Methods The active components of Sangbaipi Decoction were searched in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database, with the corresponding targets predicted. The related targets of AECOPD were searched within gene banks, OMIM and Drugbank.The names of prediction targets and disease targets were standardized by UniProt, and the intersection targets were selected. TCM component target network diagram was drawn and analyzed by Cytoscape 3.6.0. The common targets were imported into the metascape database for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and molecular docking was carried out by using auto dock tools software. Results A total of 126 active ingredients in Sangbaipi decoction, 1351 predicted corresponding targets and 2296 disease-related targets were detected. The main active ingredients include quercetin, luteolin, kaempferol, wogonin β. The core targets of sitosterol involve tumor necrosis factor (TNF), interleukin-6 (IL-6), tumor protein p53 (TP53), mitogen activated protein kinase 8 (MAPK8) and MAPK14. A total of 2720 signals were obtained from GO enrichment analysis and 334 signal pathways were obtained from KEGG enrichment analysis. The molecular docking results showed that the main active components can bind to the core target, at a stable the binding conformation. Conclusion Sangbaipi decoction may have anti-inflammatory, anti-oxidant and other biological effects through multiple active ingredients, multiple targets and signal pathways, thus effectively treating AECOPD.

Objective To investigate how the human GLIS family zinc finger protein 2 (GLIS2) regulate the Wnt/β-catenin pathway and its influence on the differentiation of human bone marrow mesenchymal stem cells (BMMSCs). Methods Human BMMSCs were randomly divided into blank group, osteogenic induction group, GLIS2 gene overexpression (ad-GLIS2) group, ad-GLIS2 negative control group, gene knockdown (si-GLIS2) group, and si-GLIS2 negative control (si-NC) group. The expression of GLIS2 mRNA in each group was detected by reverse transcription-PCR to determine the transfection status; alkaline phosphatase (ALP) activity was detected by phenyl-p-nitrophenyl phosphate (PNPP), and calcified nodule formation was tested by alizarin red staining to determine its osteogenic properties; the activation of intracellular Wnt/β-catenin pathway was detected by T cell factor/lymphoid enhancer factor (TCF/LEF) reporter kit; the expression of GLIS2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osterix was detected by Western blot analysis. The interaction between GLIS2 and β-catenin was verified by GST-pulldown. Results Compared with the blank group, the ALP activity and calcified nodule formation of BMMSCs in the osteogenic induction group increased, and the Wnt/β-catenin pathway activity and the expression of osteogenic differentiation-related proteins increased, the osteogenic ability increased, while the expression of GLIS2 decreased. Up-regulating the expression of GLIS2 could inhibit the osteogenic differentiation of BMMSCs, while suppress the activity of the Wnt/β-catenin pathway and the expression of osteogenic differentiation-related proteins on the contrary. Down-regulating the expression of GLIS2 could promote the osteogenic differentiation of BMMSCs, and improve the activity of the Wnt/β-catenin pathway and the expression of osteogenic differentiation-related proteins. There was an interaction between β-catenin and GLIS2. Conclusion GLIS2 may negatively regulate the activation of Wnt/β-catenin pathway and affect the osteogenic differentiation of BMMSCs.

Objective To Clone, express, and purify the focal adhesion kinase (FAK) gene C-terminal focal adhesion location sequence (aa798-aa1041), and to prepare and identify the rabbit anti-FAK polyclonal antibodies. Methods The C-terminal (2671 bp-3402 bp) gene of the FAK gene was amplified by PCR in vitro and cloned into pCZN1 vector to construct a pCZN1-FAK recombinant expression vector. The recombinant expression vector was transformed into E. coli expression strain BL21 (DE3) competent cells, and then induced by isopropy-β-D-thiogalactoside (IPTG). The protein was purified by affinity chromatography resin Ni-NTA and immunized with New Zealand white Rabbit to prepare polyclonal antibodies. The antibody titer was detected by indirect ELISA and the specificity was identified by Western blot analysis. Results The pCZN1-FAK recombinant expression vector was successfully constructed. The FAK protein was mainly expressed in the form of inclusion bodies. After purification of the target protein, the prepared rabbit anti-FAK polyclonal antibody showed a titer of 1:512 000, and could specifically react with exogenous and endogenous FAK proteins. Conclusion The FAK protein is successfully cloned, expressed and purified, and a rabbit anti-FAK polyclonal antibody is prepared, which can be used for the specific detection of endogenous FAK protein.

Objective To analyze structural features for spike (S) protein of the SARS-CoV-2 and to predict potential B cell and T cell epitopes using bioinformatics. Methods The amino acid sequence of S protein from the NCBI GenBank database was retrieved. Its physicochemical properties were analyzed using ProtParam online program. The secondary structure of S protein was analyzed using Lasergene software and SOPMA online service. The tertiary structure model of S protein was established by Phyre2 and Rasmol software. Finally, B cell epitopes were predicted using ABCpred, BepiPred and BcePred; T cell epitopes were predicted using IDBE software. Results S protein is a 1273 amino acid sequence with the isoelectric point at 6.24 and atomic composition as C₆₃₃₆H₉₇₇₀N₁₆₅₆O₁₈₉₄S₅₄, which was classified as a stable and hydrophilic protein. GramierRobson method analysis revealed that the secondary structure of S protein comprised 23.5% α-helixes, 53.7% β-sheets, 14.9% β-turns and 8.33% random coils. Chou-Fasman method analysis revealed that the secondary structure of S protein comprised 20.9% α-helixes, 35.5% β-sheets, 35.2% β-turns. Online service SOPMA analysis revealed that the secondary structure of S protein comprised 28.59% α-helixes, 23.25% β-sheets, 3.38% β-turns and 44.78% random coils. The numbers of B cell epitopes according to ABCpred, BepiPred and BcePred databases were 5,11 and 6. Five epitopes for CD8⁺ T cell and CD4⁺ T cell were chosen as potential epitopes. Conclusion Bioinformatics can predict B cell and T cell epitopes in the S protein of the SARS-CoV-2, which lays a foundation for developing vaccines.

Objective To investigate the possible off-target effects of dynamin (DNM) inhibitor Dyngo-4a in dynamin-dependent endocytic pathways. Methods Bone marrow mesenchymal stem cells (BMSCs) obtained from SD rats were isolated and cultured, and identified by flow cytometry. The cells were divided into inhibitor control group, Dyngo-4a-treated group, negative control siRNA (si-NC) transfection group, DNM2 siRNA transfection (si-DNM2) group, si-DNM2 and Dyngo-4a co-treated group. Real time quantitative PCR and Western blot analysis were used to verify the silencing efficiencies of DNM2 gene and CCK-8 assay were used to detect the cell viability after Dyngo-4a treatment. Confocal microscopy was used to detect the number and mean fluorescence intensity (MFI) of transferrin-Dylight649-positive and dextran-TMR-positive vesicles. Results The mRNA and protein expression levels of DNM2 were down-regulated using small interfering RNA. The number of transferrin-Dylight649-positive vesicles significantly decreased in si-DNM2 group compared with si-NC group. For the number and MFI of dextran-TMR-positive vesicles, no significant change was observed between the si-DNM2 group and the si-NC group, but there was a significant reduction in the si-DNM2 and Dyngo-4a co-treated group compared with the si-DNM2 group. A significant decrease was also found in the Dyngo-4a-treated group compared with the inhibitor control group. Conclusion The off-target effects of dynamin inhibitor Dyngo-4a presents in the internalization of dextran by BMSCs.

Objective To construct and validate a prognostic model for lung adenocarcinoma based on bioinformatics of metabolic genes. Methods Lung adenocarcinoma-related data from The Cancer Genome Atlas (TCGA) database and gene expression omnibus (GEO) were acquired, and LASSO regression was used to construct multi-gene prognostic models and calculate risk-score (RS). Univariate and multivariate Cox independent prognostic analysis was performed. The area under receiver operating characteristic (ROC) curve (AUC) of the model was evaluated by ROC curve and survival analysis was performed. Nomogram were constructed to evaluate the feasibility of the model, and metabolic gene functional enrichment analysis was performed by GSEA. Tumor immune estimation resource (TIMER) database was used to analyze the correlation of patients RS with immune cell infiltration and with the expression of immune checkpoint molecules. Results The TCGA database was used to construct a prognostic model for lung adenocarcinoma based on 18 metabolism-related genes, and RS was used as an independent prognostic factor. The area under the ROC curve was 0.713. Survival analysis showed that overall survival was higher in the low-risk group compared to the high-risk group, and the prognostic model was associated with infiltration of immune cells and with the expression of immune checkpoint molecules. Conclusion RS is an independent prognostic factor in the prognostic model of lung adenocarcinoma with metabolic genes, suggesting a high prognostic value of this model.