Osteocalcin is a major non-collagenous component of the bone extracellular matrix and is considered to be an indicative factor of osteoblast differentiation. In the present study, we detected osteocalcin expression in different antler areas and growth phases by immunohisto- chemistry. Osteocalcin was highly expressed in all areas during the mineralization period and in mesenchymal cell and chondrocyte areas during the rapid growth period. The nucleotide sequence of the osteocalcin gene in sika deer antler was determined. The open reading frame was 303 bp encoding a protein of 100 amino acids. The estimated molecular mass of osteocalcin was 10.38 kDa and the theoretical isoelectric point was 5.37. The osteocalcin gene with a 6× His-tag at the C-terminus was cloned into the pGEX-4T1 vector and expressed in Escherichia coli under optimal conditions. The recombinant soluble protein fused with GST was purified with Ni-NTA resin. The purified osteocalcin protein exhibited a significant increase in HA adhesion and promoted antler chondrocyte proliferation. Osteocalcin is an important factor in regulating the rapid growth and differentiation of deer antlers.
To explore the role of Toll-like receptors (TLRs) and interferon (IFN) in the innate immunity against porcine epidemic diarrhea virus (PEDV), we detected the expression of TLR genes in PEDV-infected IPEC-J2 cells by real-time PCR. We also detected the level of interferon α (IFN-α) and interferon γ (IFN-γ) by enzyme-linked immunosorbent assay (ELISA). Results showed that IPEC-J2 cells exhibited a clear pathological change after PEDV infection at 24 h. In addition, TLR7, TLR9 and TLR10 expressions were significantly upregulated in PEDV-infected IPEC-J2 cells at 24 h. Interestingly, the expression patterns of TLR2 and TLR4 were consistent at different stages of PEDV infection. The expression level of TLR3 decreased significantly with the increase of infection time, but the expression levels of TLR5 and TLR8 genes at 6 h and 12 h were significantly lower than those in the control group (p<0.01). There were significant correlations among the expression levels of TLR genes (p<0.05). Cytokine detection showed that the secretion level of IFN-α in the PEDV-infected group was significantly higher than that in the control group (p<0.01), and IFN-γ at 6 h and 12 h after PEDV infection was significantly higher than that in control group (p<0.01). Therefore, our results suggest that PEDV infection can induce innate immune responses in intestinal porcine jejunum epithelial cells, leading to changes in the expression of Toll-like receptors, and can regulate the resistance to virus infection by affecting the release levels of downstream cytokines.
The interface characteristics, bending and impact behavior, as well as fracture characteristics of stainless steel clad plates fabricated by vacuum hot rolling at different rolling temperatures of 1100°C, 1200°C and 1300°C are investigated in detail. The interface bonding strength is gradually increased with the increasing rolling temperature due to the sufficient diffusion behavior of alloy element. The bending toughness and impact toughness are gradually decreased, while the bending strength increase with the increase of the rolling temperature, which is attributed to mechanisms of matrix softening and interface strengthening at high rolling temperature. Due to the weak interface at 1100°C, the bending and impact crack propagation path was displaced by delamination cracks, which in turn lead to reduction in stress intensity of the main crack, playing an effective role in toughening the stainless steel clad plates. Moreover, the impact fracture morphologies of clad plates show a typical ductile-brittle transition phenomenon, which is attributed to the matrix softening behavior with the increasing rolling temperature.