| Objective:To investigate the dedifferentiation of the rabbits articularchondrocytes cultured in vitro, and to verify the inhibition todedifferentiation and the promoting to developing the substrate componentof cartilage by transduction with Adenovirus-human insulin like growthfactor-I transgene construct in order to provide a basis for protecting andresurfacing articular cartilage damaged by the means of gene therapy. Methods:Articular cartilage was aseptically dissected from thefemoral condyles, the tibial plateaus and the femoral head of 3-week-oldNew Zealand White rabbit. The excised cartilage was diced intoapproximately 1mm3 segments. Cells were enzymatically digested fromcartilage tissue segments and cultured in DMEM supplemented with 10%Fetal Bovine Serum (FBS). The media was changed every 2-3 days, and thecells are ready for subculturing after reaching 80%-90% confluence. Thefirst generation chondrocytes were assigned to transducted group andnon-transducted group after they were transducted by Ad- hIGF-I of 500multiplicity of infection. The chondrocyte morphology was observed byinverted light microscopy and cells growth curve was drawed by the meansof MTT. The first,third,fifth and seventh generation chondrocytes beingtransducted and non-transducted were assayed by histological examinationof HE, toluidine blue, safranin O staining and immunocytochemistry toobserve their biological characteristical changes. ELISA, alkalinehydrolysed sample assay and alician blue test were performed to these cellsto identify the expression and biosynthesis of IGF-I, type â…¡ collagen,glycosaminoglycans(GAG). The data of this assay was analysized by SPSS10.0 software package. Results:Primary cultured chondrocytes began to anchorage to theculture flask in 48 to 72 hours. The shape of the chondrocytes was triangle,shuttle shape or spindle-shape. Subcultured cells began to anchorage to theculture flask in 12 hours, and non-transducted cells would changemorphologically into long-shuttle-shape cells following the increase ofsubcultures, that is to say, the cells presented the sign of dedifferentiationand lost their cartilage-specific phenotype, but transducted chondrocytesmaintained their phenotype and characteristics of articular cartilage, theirmorphology hadn't significant changes at passage 7. Both primary cells andpassaged cells could recover to round shape cells after they were digestedrecently. However, the development and proliferation of chondrocytescultured in vitro from passage 1 to passage 3 was steady, and theypresented contact inhibition and density inhibition. The growth curveshowed that chondrocytes subcultured went through a typical growth cycle,lag phase,logarithmic growth phase and stationry phase,and at 9th day, thecell prolifertion rose up to the top. The colour of Chondrocyte nucleusstained by HE was blue, and the colour of cytoplasm was pink. The colourof the chondrocytes stained by toluidine blue was blue, and the colour ofcell stained by safranin O was red. toluidine blue staining and safranin Ostaining showed that the colour of every generation transducted cells hadn'tsignificant difference, but the colour of non-transducted chondrocytes wasthiner and thiner. Cytoplasm was stained brown by immunocytochemistryfor type â…¡ collagen, but the cells of the control group remained negative.The respective concentration of IGF- I , hydroxyproline and GAG intransducted chondrocytes culture medium had significant differencecompare to same generation in non-transducted group(P<0.05), and theirconcentrations of transducted group and non-transducted group were alldecreased gradually following the increase of subcultures. Conclusion : The successful culture in vitro of rabbit articularchondrocytes indicated that Rabbit articular chondrocytes in monolayerculture have the tendency of dedifferentiation and the biologicalcharacteristy of chondrocytes cultured in vitro from passage 1 to passage 3was steady. The results suggested that chondrocytes transducted byAd-hIGF-I could produce endogenous IGF-I and increase synthesis...
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