Experimental Study Of The Differential Effect Of Estrogen On The Metabolism Of Axial And Appendicular Growth Plate

Objective:To study the characteristics of epiphyseal plate-related genes,the histological differences of epiphyseal plate,and the effects of estradiol supplementary therapy on the length of spine and lower limbs in ovariectomized rats.To explore the isolation and culture of primary chondrocytes from epiphyseal plates of limb and spine in mice,and the effect of low concentration serum culture on the proliferation and collagen synthesis of epiphyseal plate chondrocytes.To explore the effects of different estradiol concentrations,time-effect,and the effect of different estrogen receptor inhibitors on the proliferation,differentiation,collagen formation and region-specific differences of epiphyseal plate chondrocytes from limb and spine.Methods:（1）1-week old female rats with similar weight were randomly divided into sham group,ovariectomized group（OVX）and ovariectomized+estradiol group（OVX+E2）.The levels of serum estradiol,the examination of Col II,Col X,Sox-9 and Runx-2,the height of the growth plate,and the corresponding lengths of limb and spine were determined ELISA,PCR,Alcian Blue staining and the X-ray,respectively.（2）Chondrocytes were isolated and cultured by modified sequential digestion method with trypsin and collagenase.CCK-8,PCR and Western Blot were used to detect the proliferative curves of epiphyseal plate chondrocytes,and the expression of Col II and Col X under the condition of low concentration serum culture.（3）Chondrocytes stimulated with different estradiol concentrations and estrogen receptor inhibtors were made to test the growth curve of epiphyseal plate chondrocytes,and the expression of Col II and Col XResults:（1）The serum estradiol concentration,the height of epiphyseal plate,the levels of Col II,Col X,Sox-9 and Runx-2 in OVX group were significantly lower than those in sham group and OVX+E2 group.The length of the spine was significantly shorter than that of the sham group and the OVX+E2 group,while the length of the lower limb was not significantly different.（2）The results demonstrated that the growth curve of the epiphyseal plate chondrocytes of limb and spine was approximately"inverted bell"parabola under the low concentration serum culture.（3）The results of CCK-8,PCR,and Western Blot showed that estradiol with different concentrations had a positive effect on the cell viability and the expression of Col II and Col X of epiphyseal plate chondrocytes from limb and spine,obviously at 10^-7M level.While the epiphyseal plate chondrocytes from limb responded more sensitive to estrogen stimuli than these from spine.Estrogen-mediated with different estrogen receptors on the expression of Col II and Col X in epiphyseal plate chondrocytes,mainly mediated by ERαreceptorConclusions:Estrogen has the important effect on the proliferation,differentiation,collagen formation and epiphyseal plate height of epiphyseal plate from limb and spine for juvenile rats.The growth curve of primary chondrocytes of epiphyseal plate showed a"ascending-descending"morphology under the condition of low concentration serum culture.Estrogen can promote the cell viability and collagen formation of epiphyseal plate chondrocytes from limb and spine,obviously at 10^-7M level.The epiphyseal plate chondrocytes from limb responded more sensitive to estrogen stimuli than these from spine.The effect of estrogen-mediated with different estrogen receptors on epiphyseal plate chondrocytes in collagen formation,mainly mediated by the ERαreceptor,rather than the ERβ receptor.