Effects Of Estrogen On Proliferation And Differentiation Of Human Embryo Mandibular Condylar Chondrocytes

Objective:①Preliminary research on the expression of estrogen receptor and type II collagen in mandibular condylar cartilage of human embryo.②To establish a model of the human embryo mandibular condylar chondrocytes (MCCs) in vitro.③Observe the of effect estrogen on the proliferation and differentiation in the level of cell.Methods:①Expression and localization of ERa, ERβand typeⅡcollagen in mandibular condylar cartilage of human embryo were examined by routine HE and immunohistochem-istry staining.②Isolate human embryo MCCs by dissection and improved two-step sequential digestion, observe cellular morphology by cultivated in vitro, take advantage of direct counting method to depict the growth curvature, identify the second generation cells by toluidine blue and typeⅡcollagen immunohistochemical staining, establish the cultivating system of human embryo MCCs in vitro, to supply applicable cells for experiments.③Estradiol was added into the culture medium at a variety of concentrations (10-6mol/L,10-8mol/L,10-10mol/L,10-12mol/L,Omol/L), MCCs growth were measured with methylthiazol tetrazolium (MTT) method, in order to investigate the effects of estradiol on proliferation and differentiation of MCCs in human embryo in vitro.Results:①ERαand ERβwere almost not expressed in the fibrous and proliferative layers of human embryo mandibular condylar cartilage; ERa immunoreactivity was majorly localized in the transitional and hypertrophic cartilaginous layers of condylar cartilage, calcified cartilaginous layer had a small expression; and ERβwas exclusively expressed in the hypertrophic cartilaginous layer, calcified cartilaginous layer was also expressed a little. ERa and ERβwere found in both cytoplasms and nuclei by immunoreactivity. ERa was distributed evenly, while ERβwas localized in nuclei. The expression of ERa was wider than ERβin condylar cartilage.②TypeⅡcollagen immunoreactivity was localized in the transitional and hypertrophic cartilaginous layers of condylar cartilage,was almost not expressed in the fibrous, proliferative cartilaginous layers and calcified cartilage layer had a little expression.③The majority of MCCs in earlier generations (1～3 generation) were polygonal and triangular, cell nucleus were round or oval with 1-2 nucleoli in the same size, flagstone-like appearance; whereas more fusiform and spindle-shaped cells were found after 4th generation, and begin to be a spiral growth. Toluidine blue dyeing was positive, and cells expressed collagenⅡ,which means the cultivated cells are chondral cells;④The experimental results show that the effects of estradiol on MCCs growth exhibited in a dose-and-time-dependent manner:when the concentrations of E2 were 10-12mol/L and 10-6mol/L, the growth of MCCs were remarkable inhibited; while as the concentration was increasing(10-10 mol/L～10-8mol/L), E2 promoted the growth of MCCs significantly, peaking at 10-8 mol/L. And the acceleration or inhibition were more noticeable with the prolonging action of E2.Conclusion:①These results indicated that the distribution of ERa, ERβand Type II collagen in the mandibular condylar chondrocytes were heterogeneous.②Dissection and improved two-step sequential digestion could get homogeneous and high vigour MCCs.③It was concluded the E2 affected human embryo MCCs with both stimulating and inhibiting in cell proliferation and differentiation depending upon its concentration, and these effects were more remarkable with the prolonging action of E2.