Biological characteristics of newborn rabbit tracheal chondrocytes

AIM：To investigate the biological characteristics of newborn rabbit tracheal chondrocytes in vitro. METHODS：Newborn rabbit tracheal chondrocytes were obtained by the method of enzyme digestion, and then cultured in monolayer in vitro. Morphological and growth observations were performed under inverted phase contrast microscope. The ultrastructures of the cells were observed under scanning electron microscope and transmission electron microscope. The biological characteristics of secreted extracellular matrix components were detected by real-time PCR, immunocytochemistry staining and toluidine blue staining. RESULTS：Newborn rabbit tracheal chondrocytes isolated and cultured in vitro showed short triangular or irregular shapes, and adherent growth very well. The ultrastructures of the cells showed pore and abundant cytoplasm and organelles, with a lot of protein secretions in the cells. The chondrocytes expressed the mRNA of collagen I, collagen II and proteoglycans, mainly collagen II and proteoglycans. Immunocytochemistry staining showed collagen II and SOX9 positive, and collagen I weakly positive. Toluidine blue staining was also positive. CONCLUSION：Enzyme digestion and monolayer culture are suitable method to obtain newborn rabbit tracheal chondrocytes. These cells， secreting extracellular matrix components, are able to be selected as seed cells for tissue engineering of trachea in vitro, and used to study the therapeutic method for neonatal rabbit tracheal stenosis.     

白果内酯对IL-1β诱导的ATDC5软骨细胞自噬、增殖和凋亡的影响

旨在探讨白果内酯对白介素1β(IL-1β)诱导的ATDC5软骨细胞自噬、增殖和凋亡的影响。采用10 ng·mL^-1 IL-1β诱导ATDC5软骨细胞构建体外炎症模型,随机分为对照组、IL-1β组、白果内酯组(低、中、高剂量)。利用EdU检测细胞新合成的DNA,并结合细胞核标记物(Hoechest)进行双重标记检测细胞增殖速度。使用膜Annexin-V/PI通过流式细胞仪分析ATDC5软骨细胞凋亡情况。通过mRFP-GFP-LC3双荧光系统的组合测量方法检测自噬流。蛋白免疫印迹法(Western blot)和实时荧光定量PCR(qRT-PCR)方法检测各组软骨细胞中LC3-II、Beclin1、BAX、Caspase-3和Bcl2的蛋白和mRNA表达情况。结果显示,IL-1β诱导ATDC5软骨细胞后,细胞增殖减弱,下调自噬促进凋亡,而白果内酯干预能够过促进自噬和细胞增殖,抑制凋亡。白果内酯促进ATDC5软骨细胞的增殖(P<0.05),与IL-1β组相比,抑制了LC3-II、Beclin1、BAX和Caspase-3的蛋白和mRNA表达(P<0.05),促...     

Polyunsaturated fatty acids influence inflammatory markers in a cellular model for canine osteoarthritis

Although it is well recognized that dietary supplementation with fish oil improves clinical symptoms in dogs suffering from osteoarthritis, the molecular basis for the dietary benefit is not yet completely resolved in dogs. This study was designed to further clarify how polyunsaturated fatty acids (PUFA) affect key factors of cartilage degeneration in a canine cell culture system mimicking osteoarthritis. Canine chondrocytes were incubated either without or with 10 μm of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (AA) or 3.6 μm ibuprofen (Ibu) as positive control for 6 days. After the supplementation, cells were stimulated with 10 ng/ml interleukin‐1β (IL‐1β) for another 48 hr to induce osteoarthritic changes, or left unstimulated. We analysed fatty acid uptake via gas–liquid chromatography, nitric oxide (NO) production via Griess assay, prostaglandin E (PGE) production via ELISA and relative gene expression of several cartilage matrix proteinases, inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 via RT‐qPCR. After supplementation, the chondrocytes rapidly incorporated the PUFA into their fatty acid pools. The stimulation with IL‐1β caused a marked increase of most of the inflammatory markers measured. N‐3 PUFA EPA reduced IL‐induced gene expression of iNOS and corresponding production of NO. N‐6 PUFA AA also decreased iNOS and NO, but furthermore lowered gene expression of matrix metalloproteinase‐3. On the other hand, AA upregulated the aggrecanase ADAMTS‐5 and augmented the release of PGE. The effect of n‐3 PUFA DHA turned out to be negligible. Our results reveal molecular mechanisms by which PUFA affect degenerative joint disease in dogs. Of particular importance is that not only EPA but also AA decreased several inflammatory markers in our model. Thus, we conclude that an appropriate balance of both n‐3 and n‐6 fatty acids deserves more attention in dietary interventions.