| Objective:To investigate the feasibility of isolation, purification, expansion rabbit synovial mesenchymal stem cells(SMSCs) with the method of explant culture, and study the multi- lineage potential in vitro; establish the enhanced Green Fluorescent Protein(e GFP) and the superparamagnetic iron oxide nanoparticles(SPIO) double labeling technique to provide an efficient tracer methods for imaging SMSCs repair articular cartilage in the intra-articular.Methods:SMSCs were isolated from rabbit knee fat pad synovial tissue through the method of explant culture, and purified by the limiting dilution method in sterile, then proliferation in vitro; Morphology and growth characteristics was examined by electron microscopy; Best Multiplicity O f Infection(MOI) of e GFP- lentiviral infected the cells in logarithmic growth phase, the infection efficiency were analyzed by both fluorescent microscope and flow cytometry; e GFP labeled cells fully screened by puromycin, t hen labeled with superparamagnetic iron oxide nanoparticles(SPIO), and the mark result were observed by Prussian blue staining and transmission electron microscope. Double- labeled SMSCs differentiated into chondrocytes, osteoblast and dipocytes. And the d ifferentiated cells were identified by alcian blue staining, alkaline phosphatase staining, oil red O staining, respectively. Lastly, the cell activity and proliferation ability of the dual- labeled SMSCs were evaluated by cck-8. Results:1. Rabbit primitive synovial cells began to appear around the synovial tissue with the method of explant culture 2-3 days, and after 10-12 days later large scale we will harvest, cell morphology became more homogenized by the limiting dilution method in vitro.2. Visible green fluorescence could see after SMSCs GFP labeled under the fluorescent microscope, and fluorescence expression rate gradually strengthen with the increases MOI; Over(38.4±2.7)% SMSCs were e GFP-positive at 3days after transfection by lentivirus at MOI of 100, and could get above 95% e GFP-positive by screening with puromycin;After 30 d, fluorescent labeled cells still remained stable expression.3. The Prussian blue staining showed that the e GFP-positive purified SMSCs got above(98.4±1.2)% marker efficiency after SPIO labeled by electron microscope, and considerable high electron density particles could be observed in the cytoplasm and Pinopodes through the transmission electron microscope, but the cells of the control group were negative.4. After chondrocytes induction 21 days later, alcian blue staining showed that extracellular matrix stained blue; Alkaline phosphatase staining showed that alkaline phosphatase was positive after osteogenetic induction; O il red O staining showed that red lipid droplet existed in cells after dipocytes induction.5.CCK-8 test result:e GFP/SPIO double- labeled SMSCs still remained good activity and proliferative state, showed no significant difference with unlabeled group(P> 0.05), cell macker did not produce significant cytotoxicity. Conclusions:1. the explant culture method can efficiently obtain primary SMSCs with simple operation and little probability of cell contamination.2. SMSCs which we separated and purified in this study have good potentiality of proliferation.3. e GFP / SPIO double labeling technique will provide an high efficient and feasible tracer method for imaging SMSCs repair articular cartilage in the intra-articular.And the labeled cells still has potential to induce differentiation, did not appear significant cytotoxicity,... |
PDF Full Text Request