The Effect Of ALK2 On The Proliferation And Differentiation Of Mouse Condylar Chondrocytes

Objectives: To investigate the effect of activin receptor-like kinase-2(ALK2)on the proliferation and differentiation of mouse mandibular condylar chondrocytes in vitro.Methods:(1)The mandibular condylar chondrocytes(MCC)were obtained by enzyme digestion using 1-month-old male C57 mice(body weight: 12-15g).The morphology of MCC were observed by inverted microscope.(2)The levels of COL I,aggrecan,COL II and their distributions in cells were detected by immunohistochemical staining.Pellet culture was performed and cells were induced differentiation into chondrocytes.Total RNA was extracted and the expressions of chondrocyte marker genes(Col I,Col II,Col X)were analyzed by real-time RT-PCR.The pellet was embedded,sectioned,and stained by HE and toluidine blue.(3)Si-RNA was used to silence the expression of Alk2 gene in MCC in vitro.Transfection efficiency was detected by flow cytometry.Silence efficiency was detected by real-time RT-PCR,and the optimal silencing condition was determined.(4)The viability of condylar chondrocytes was detected by MTT assay at1 d and 2 d after transfection.The expressions of chondrocyte marker genes were analyzed by real-time RT-PCR at 3 d and 7 d after transfection..Results:(1)The morphology of MCC were observed by inverted microscope,which present a polygon shape.(2)Immunocytochemical staining showed strong positive signal of COL?,and weak positive signal of COL II and AGGRECAN.Seven days after pellet culture,the expressions of Col II,Col? and Col?was up-regulated detected by reatl time RT-PCR assay.HE staining showed that the surface of the pellet were fibroblast-like cells,which were flat-shaped.Round-shaped cells,like mature chondrocytes,were under the surface.The center of the pellet showed a large number of dead cells,possibly being lack of hypoxia and nutrition.Toluidine blue showed metachrome on those mature chondrocytes-like cells.SO the MCCs were identified by morphology,immunophenotype and marker gene expression.(3)Lipo-si RNA was transfected into MCC and the transfection efficiency was 77.27% detected by flow cytometry.Lipo-Alk2-si RNA was transfected into MCC and the silencing efficiency was 83% detected by real-time RT-PCR assay.(4)MTT results showed that cell number was increased after Alk2 was silenced.Real-time RT-PCR results showed that the expressions of chondrocyte marker genes in Alk2 silencing group were significantly downregulated.Conclusions: ALK2 inhibits the proliferation of mouse mandibular condylar chondrocytes,and promotes the chondrogenic differentiation of mouse MCCs.