细胞因子信号抑制因子3对小鼠颅顶成骨前体细胞成骨分化的影响OA

Objective·To construct a stable suppressor of cytokine signaling 3(Socs3)-knockdown MC3T3-E1 mouse calvarial osteoblast precursor cell line and to investigate the role of SOCS3 in the osteogenic differentiation of calvarial osteoblast precursors.Methods·The expression levels of SOCS3 in MC3T3-E1 cells after osteogenic induction were detected by real-time quantitative PCR(RT-qPCR)and Western blotting.Two pairs of short hairpin RNAs(shRNAs)specifically targeting the Socs3 gene were designed,and lentiviral vectors for Socs3 knockdown were constructed.MC3T3-E1 cells were then infected with the packaged lentiviruses,followed by puromycin screening to obtain MC3T3-E1 cell lines with stable Socs3 knockdown.Knockdown efficiency was verified by RT-qPCR and Western blotting.The effect of SOCS3 on the proliferation capacity of MC3T3-E1 cells was assessed using a CCK-8 assay.After osteogenic differentiation for 7 d,alkaline phosphatase(ALP)staining and RT-qPCR detection of osteogenesis-related genes were performed.After 14 d of osteogenic differentiation,alizarin red S(ARS)staining was performed to assess the osteogenic differentiation capacity of the stable knockdown cell lines.Results·RT-qPCR and Western blotting showed that both Socs3 mRNA and SOCS3 protein levels were elevated at 4 d and 7 d after osteogenic induction compared with those before induction,and the levels at 7 d were higher than those at 4 d(both P<0.001).In MC3T3-E1 stable cell lines infected with sh Socs3-1 and sh Socs3-2 lentivirus,the expression levels of Socs3 mRNA and SOCS3 protein were significantly inhibited(P<0.001),indicating the successful establishment of stable Socs3-knockdown MC3T3-E1 cell lines.The CCK-8 assay showed that Socs3 knockdown inhibited the proliferation capacity of MC3T3-E1 cells at 1 d,4 d,and 7 d of culture(P<0.001).ALP staining demonstrated reduced ALP activity in the Socs3-knockdown MC3T3-E1 cell lines after 7 d of osteogenic induction(P<0.001).ARS staining indicated that Socs3 knockdown inhibited mineralized nodule formation in MC3T3-E1 cells(P<0.001).RT-qPCR detection of osteogenic marker genes indicated that the expression levels of runt-related transcription factor 2(Runx2),alkaline phosphatase(Alp),osterix(Osx),α1 type Ⅰ collagen(Col1a1),secreted phosphoprotein 1(Spp1),and bone γ-carboxyglutamate protein(Bglap)were significantly reduced in the Socs3-knockdown group(all P<0.001).Conclusion·SOCS3 expression increases during the osteogenic differentiation of MC3T3-E1 cells.Knockdown of Socs3 can inhibit both the proliferation and osteogenic differentiation capacities of MC3T3-E1 cells.