| [Objective] To explore the SD rats Condylar cartilage cells (Mandibular Condylar Chondrocytes, MCC) and limbs of articular cartilage cells in vitro culture method and its biological characteristics, determined by MTT method to observe the transformation of different concentrations of exogenous growth factors, transforming growth factor beta 2, TGF-beta 2) effect on two kinds of cartilage cell proliferation, the 0.20 ng/ml of TGF-beta 2 of Condylar cartilage cells and PCNA of limbs articular cartilage cells and II mRNA expression in collagen.[Methods] 10 newborn SD rats of 3 days only, extraction of bilateral condylar cartilage and limbs, articular cartilage combined digestion method for the cartilage cells, with 15% FBS DMEM culture develop.Through morphology, toluidine blue staining and immunohistochemical II type identification of cells;Extend the second generation, the two kinds of cartilage cells are divided into three groups, respectively, to join different concentrations of TGF-beta 2,0.10 ng/ml,0.20 ng/ml,0.40 ng/ml, determined by MTT method to observe the different concentration TGF-beta 2 of two kinds of cartilage cell proliferation differentiation;Screening suitable concentration of TGF-beta 2 to 12 h、24 h、48 h as monitoring, determined by MTT detection of two kinds of cartilage cell proliferation at each time point;Rt-pcr detection in two kinds of cartilage cells join 0.20 ng/ml TGF-beta 2 role after 12 h mRNA expression of PCNA and Ⅱ type collagen differences.[Results] Condylar cartilage cells and limbs of articular cartilage cells of primary cell after 24-48 h began to stick wall,72-96 h can form a single layer,144 h can be covered and extend; After the wedding, condylar cartilage cells adherent time is 4 h, the limbs of articular cartilage cells adherent time for 6 h;Two kinds of the first three generations in cartilage cell phenotype stability, strong ability of proliferation;Began the fourth generation of cell proliferation ability weakened, the formation of monolayer time variable-length phenomenon; Toluidine blue staining and immunohistochemical staining showed two chondrocytes express positive, limbs Ⅱ type collagen protein positive expression of articular cartilage cells than condylar cartilage cells;Different concentrations of TGF-beta 2 condylar cartilage cells,0.2 ng/ml,0.4 ng/ml TGF-beta 2 has obvious proliferation promoting effect than 0.1 ng/ml, the difference is statistically significant,0.2 ng/ml,0.4 ng/ml TGF-beta 2 effect was no significant difference on the condylar cartilage cells;0.2 ng/ml of TGF-beta 2 is the most suitable for limb articular cartilage cells proliferation concentration, while more than 0.2 ng/ml showed some inhibitory effect;Under the action of the same concentration of TGF-beta 2, condylar cartilage is the peak number of living cells in about 12 h, and the limbs of articular cartilage living cells to peak at about 24 h; 0.2 ng/ml of TGF-beta 2 for both Ⅱ type collagen in cartilage cells and PCNA mRNA expression in control group were increased, one of the condylar chondrocytes Ⅱ type collagen and PCNA mRNA expression in relatively weak limbs articular cartilage cells expressed.[Conclusion] Through this experiment, the original generation of condylar chondrocytes and limbs of articular cartilage cells there are differences in cell morphology and growth characteristics, condylar cartilage cell immunohistochemical staining expression Ⅱ collagen type is relatively weak limbs expression of articular cartilage; Exogenous TGF-beta 2 for two kinds of cartilage cell proliferation effect is different, the same concentration of TGF-beta 2 live for two kinds of cartilage cells peak time difference; 0.2 ng/ml of exogenous TGF-beta 2 of two kinds of chondrocytes Ⅱ type collagen protein and mRNA expression of PCNA vary, at the genetic level to discuss two kinds of cells for exogenous TGF-beta 2 boost express different reaction;Further illustrate two kinds of cartilage cells on the growth characteristics and response to growth factors exist differences. |
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