Objective The primary culture of synovial fibroblasts is a convenient tool to study the pathology and physiology of synovial tissues .An improved method was constructed in this study by C57BL /6 mice to study the mechanism of rheumatoid ar-thritis(RA) .Methods The synovium around the hip joints were collected .Attention should be paid to eliminate the egg-yolk like yellow oval substance in the middle of the synovium .The synovium was transferred into a 1 .5 mL Eppendorf tube containing 0 .5%type Ⅳ collagenase and cut into 1 mm3 blocks or so .The Eppendorf tube was placed in 37 ℃ Constant temperature orbital shaker incubator for 60 min .After digestion ,the tube was placed on the Vortex for a high-speed oscillation for 1 .5 minutes to guarantee the separation of cells .Results Within about 1 week ,the first passage was performed by the trypsin digestion method .On day 10 , the number of synovial macrophages reached the maximum and then decreased gradually .After the third generation (day 15 to 20) , the synovial macrophages generally disappeared .Vimentin was suitable for the immunofluorescence cytochemical staining for the synovial fibroblasts .The cell purity was indicated as > 95% .The cytometric analysis indicated that purity of Vimentin and CD90 .2-labelled cells was over 95% ;the purity of CD54-labelled cells was 80% approximately .Conclusion It is a simple and effective method for primary culture of synovial fibroblasts in mice .

OBJECTIVETo investigate the effect of tumor necrosis factor-α (TNF-α) on the release of matrix metalloproteinase-3 (MMP-3), MMP-9, and interleukin-17 (IL-17) in cultured mouse bone marrow-derived mast cells (BMMCs) in vitro.

METHODSPrimarily cultured mouse BMMCs at 8 weeks were exposed PBS (control) or TNF-α at the concentrations of 2, 10, or 50 ng/mL for 12 or 24 h. Real-time PCR was performed to detect the mRNA expressions of MMP-3, MMP-9, and IL-17 in the exposed cells.

RESULTSA 12-hour exposure of the BMMCs to TNF-α caused significantly increased expressions of MMP-3, MMP-9, and IL-17 in a concentration-dependent manner (P<0.05). Prolonged exposures of the cells to 2 and 10 TNF-α for 24 h further increased MMP-3, MMP-9, and IL-17 mRNA expressions, but exposure to 50 ng/mL TNF-α for 24 h increased only MMP-3 and MMP-9 expressions but not IL-17 mRNA expression.

CONCLUSIONSTNF-α treatment of primarily cultured BMMCs can significantly increase the cellular expressions of MMP-3, MMP-9, and IL-17 mRNA in a time- and dose-dependent manner.

Animals ; Bone Marrow Cells ; metabolism ; Cells, Cultured ; Dose-Response Relationship, Drug ; Interleukin-17 ; metabolism ; Mast Cells ; metabolism ; Matrix Metalloproteinase 3 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo investigate the influence of the bone marrow-derived mast cells (BMMCs) on the expression of type II collagen and glycosaminoglycan (GAG) in chondrocytes co-cultured with BMMCs.

METHODSPrimarily cultured mouse BMMCs at 4 weeks and the second passage of chondrocytes were plated in a Transwell co-cultured system at a ratio of 1:10 in the presence or absence of sodium cromoglycate (DSCG) or compound 48/80 (C48/80). The chondrocytes were harvested and lysed for detecting type II collagen expression with ELISA and Western blotting and GAG expression using 1,9 dimethylmethylene blue (DBM).

RESULTSAfter a 24-hour culture, the chondrocytes co-cultured with BMMCs showed similar expression levels of type II collagen and GAG to the control group regardless of the presence of DSCG (P>0.05). Compared with chondrocytes cultured alone or with BMMCs, the co-cultured chondrocytes in the presence of C48/80 showed significantly lower expressions of type II collagen and GAG (P<0.01). Such results did not vary significantly as the culture time was extended to 48 h.

CONCLUSIONC48/80-activated BMMCs can reduce the expression of type II collagen and GAG in chondrocytes in the co-culture system.

Animals ; Bone Marrow Cells ; cytology ; Cells, Cultured ; Chondrocytes ; cytology ; Coculture Techniques ; Collagen Type II ; metabolism ; Glycosaminoglycans ; metabolism ; Mast Cells ; cytology ; Mice

ObjectiveTo explore the metabolic changes during the development of Tupaia belangeri breast tumors, to investigate the close relationship between the changes of serum metabolic substances and the occurrence and progression of tumors, and to screen for biomarkers reflecting the progression of breast tumors. MethodsBreast tumors in Tupaia belangeri were induced by orally administering 7,12-dimethylbenzoanthracene (DMBA) three times, with a 15-day interval between each administration, along with a high-fat and high-sugar diet. The DMBA-induced breast cancer group and the DMBA-inducedwithout breast cancer group were compared with the control group. Untargeted determination of serum metabolites was performed using gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) in DMBA-induced Tupaia belangeri with breast cancer, DMBA-induced without breast cancer and the control group. Multidimensional statistical analysis including unsupervised principal component analysis (PCA), and orthogonal partial least squares analysis (OPLS-DA) were conducted. Furthermore, t-test was used for intergroup differential comparison. Differential metabolites were screened under VIP>1 and P<0.05 conditions, and significantly changing differential metabolites were identified using the HMDB online database. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database was utilized to enrich metabolic-related gene regulatory pathways. ResultsThe incidence of breast tumors was 40% in DMBA-induced Tupaia belangeri. Compared with the control group, 30 metabolic differential products were detected in the serum of the group with breast cancer, with 18 down-regulated and 12 up-regulated (VIP>1, P<0.05). KEGG pathway analysis revealed significant changes in four metabolic pathways: glutamate metabolism, glyceride metabolism, citric acid cycle, and alanine metabolism. Compared with the group without breast cancer, 18 metabolic differential products were detected, with 7 down-regulated and 11 up-regulated (VIP>1, P<0.05). KEGG pathway analysis revealed significant changes in the citric acid cycle and glutamate metabolism. Compared with the control group, 31 metabolic differential products were detected in the serum of the groups without breast cancer, with 14 down-regulated and 17 up-regulated (VIP>1, P<0.05). KEGG pathway analysis revealed significant changes in three metabolic pathways: glutamate metabolism, glyceride metabolism, and citric acid cycle. ConclusionMetabolomics analysis can reveal the characteristics of changes in metabolites in the serum of breast tumors. The results suggest that glutamate metabolism, glyceride metabolism, citric acid cycle, and alanine metabolism pathways are associated with the occurrence and development of DMBA-induced breast tumors in Tupaia belangeri. It provides a foundation for further research into the biological mechanism of breast cancer.

A patient with thymoma associated immunodeficiency syndrome (Good's syndrome) and bronchiectasis was retrospectively analyzed. Good's syndrome is a rare condition of immunodeficiency that is characterized by thymoma and hypogammaglobulinemia. It is important to bear in mind that Good's syndrome should be included in the differential diagnosis When patients repeatedly visited for bronchiectasis or infection, we should alert to their immune state and history of thymoma. Early screening of immunological status and aggressive correction of immune deficiency are beneficial to improving the prognosis to patients with Good's syndrome.
Agammaglobulinemia/complications* ; Bronchiectasis/complications* ; Humans ; Retrospective Studies ; Thymoma/complications* ; Thymus Neoplasms/complications*