Identification of the glycosylation sites of Opsin3 and its glycosylation modification function / 生物工程学报

Opsin3 (OPN3) is a photoreceptor membrane protein with a typical seven-alpha helical transmembrane structure that belongs to the G-protein-coupled receptor (GPCR) superfamily and is widely expressed in brain. In recent years, it has been reported that OPN3 is also highly expressed in adipose tissue, and the protein is associated with the production of skin melanin. We found that the N82 site is the glycosylation site of OPN3. SNAP-tagTM has diverse functions and can be applied to a variety of different studies. By constructing a SNAP-tagged OPN3 recombinant protein, the distribution position of SNAP-OPN3 in cells can be clearly observed by fluorescence confocal microscopy using SNAP-Surface® 549 and SNAP-Cell® OregonGreen®, which provides a new method for studying the function of OPN3. It also shows that SNAP-tag does not affect the function of OPN3. Using the SNAP tag we found that OPN3 cannot be taken up to the cell membrane after glycosylation site mutation.

Non-contact Coculture Reveals a Comprehensive Response of Chondrocytes Induced by Mesenchymal Stem Cells Through Trophic Secretion

Coculture between mesenchymal stem cells (MSCs) and chondrocytes has significant implications in cartilage regeneration. However, a conclusive understanding remains elusive. Previously, we reported that rabbit bone marrow-derived MSCs (rbBMSCs) could downregulate the differentiated phenotype of rabbit articular chondrocytes (rbACs) in a non-contact coculture system for the first time. In the present study, a systemic investigation was performed to understand the biological characteristics of chondrocytes in coculture with MSCs. Firstly, cells (MSCs and chondrocytes) from different origins were cocultured in transwell system. Different chondrocytes, when cocultured with different MSCs respectively, consistently demonstrated stimulated proliferation, transformed morphology and declined glycosaminoglycan secretion of chondrocytes. Next, cell surface molecules and the global gene expression of rbACs were characterized. It was found that cocultured rbACs showed a distinct surface molecule profile and global gene expression compared to both dedifferentiated rbACs and rbBMSCs. In the end, cocultured rbACs were passaged and induced to undergo the chondrogenic redifferentiation. Better growth and chondrogenesis ability were confirmed compared with control cells without coculture. Together, chondrocytes display comprehensive changes in coculture with MSCs and the cocultured rbACs are beneficial for cartilage repair.

Biological behaviors of articular chondrocytes on the polydimethylsiloxane with differential surface modifications and stiffnesses / 中国组织工程研究

BACKGROUND: Polydimethylsiloxane (PDMS) is widely used in the basic research on cell biology because of its good biocompatibility and ability to be processed at the micro/nano level. However, cell culture on PDMS has been generally compromised by its strong hydrophobicity.OBJECTIVE: To perform a comparative investigation on the influences of different surface modifications as well as stiffness of PDMS on cellular behaviors.METHODS: PDMS films with varying stiffnesses were subjected to various surface modifications, including serum incubation, type I collagen deposition and air plasma treatment. Bovine articular chondrocytes were seeded on PDMS films and cell adhesion, proliferation and matrix production were characterized using F-actin staining, cell counting kit-8,microscopic examination, Sirius red/Safranin-O staining and quantitative determination of glycosaminoglycans,respectively.RESULTS AND CONCLUSION: Serum incubation, type I collagen deposition and air plasma treatment were all found to promote adhesion and proliferation of bovine articular chondrocytes from the results of F-actin staining and cell prolifereration curve, with air plasma treatment the best. Total amount of glycosaminoglycans (GAG) secretion was only increased by air plasma treatment and GAG/DNA was decreased by all modifications. Stiffness also played roles in cell adhesion, proliferation and GAG production, which was found to be dependent on surface modifications. This study would provide guidance for applying PDMS in cell culture.