ABSTRACT
Objective To isolate, culture and expand SD rat synovial mesenchymal stem cells in vitro and identify their potential multilineage differentiation. Methods Traumatic osteoarthritis models were made in SD rats. Synovial tissue was collected under aseptic conditions. The synovial tissue was digested with type I collagenase to isolate synovial mesenchymal stem cells. Cells were purified by limiting dilution of the monoclonal culture and expanded in vitro. After the third passage of culture, growth curve determination, adipogenesis induction, osteoinduction and chondrogenesis induction were performed. Real-time qPCR and Western blot were used to detect the expression of proteoglycan and type Ⅱ collagen after chondrogenic differentiation. Results In the present study, rat synovial mesenchymal stem cells exhibited a homogeneous, fibroblast-like, spindle-shaped morphology after passage in vitro. Synovial mesenchymal stem cells were subjected to adipogenesis induction, osteoinduction and chondrogenesis induction and being identified. Under the influence of inflammatory factors, mRNA of type Ⅱ collagen and proteoglycans was positively expressed in differentiated cells induced by chondrocytes using real-time qPCR and Western blot. Conclusion Rat-derived synovial mesenchymal stem cells still have good chondrogenic potential in inflammatory environment.
ABSTRACT
Objective To establish a doxycycline-controlled immortalized pre-cartilaginons stem cells (IPCSCs) strains, clone parathroid hormone-related peptide[PTHrP(1-36)] gene and construct re- sponsive plasmid, pTRE-PTHrP (1-36). Methods Plasmid pTet-on was transfected into IPCSCs by using LipoinfectaminTM 2000 and then the stable clones were obtained by G418 screening. The doxycyc- line was added into the medium of monoclonal cells that were transiently transfected with plasmid pTRE- 2Hyg-Lue. The total RNA was extracted from PCSCs and the PTHrP(1-36) gene obtained by RT-PCR method. Then, the PTHrP (1-36) gene was subcloned to plasmids of Tet-responsive element with the se- lection marker of hygromycin pTRE-2Hyg to construct recombinant eukaryotic expression plasmid pTRE- PTHrP(1-36). After transferred into E. coli-DH5α, the clone was amplified, the recombinant plasm0ids were purified and identified by double-enzyme digestion. Results The doxycycline induced IPCSCs line was obtained, with 50 times higher than the non-induced cell line. Double enzyme digestion analysis and sequencing showed that the target gene was cloned into recombinant plasmid. Conclusions The induced IPCSCs line can be used to highly express alien genes. The responsive plasmid containing PTHrP (1-36) gene may be premising for rigorous control of PTHrp (1-36) gene expression.