2022, Vol. 26 ›› Issue (24): 3785-3789

Osteoblast differentiation after conditional knockout of 3-phosphoinositide-dependent protein kinase-1 gene from bone marrow mesenchymal stem cells

Chen Qiaoling1, Bai Yiguang2, Liu Kang3, Lin Tao2, Luo Xuwei2   

Abstract: BACKGROUND: The research on 3-phosphoinositide-dependent protein kinase-1 (PDK-1) in and outside China is mainly concentrated in endocrinology and oncology. There are no systematic studies and reports on its influence on osteogenic differentiation in orthopedics.
OBJECTIVE: To observe the role of PDK-1 in osteoblast differentiation by transfecting PDK-1flox/flox mouse bone marrow mesenchymal stem cells with adenovirus stably expressing cre enzyme (pHBAd-cre-EGFP).
METHODS: Bone marrow mesenchymal stem cells from homozygous PDK-1flox/flox mice were obtained and cultured in vitro. A control group, an empty virus group (pHBAd-EGFP) and a pHBAd-cre-EGFP group were set. In the control group, osteoblast induction medium was used to induce bone marrow mesenchymal stem cells. In pHBAd-EGFP group, pHBAd-EGFP was used to transfect bone marrow mesenchymal stem cells. In the pHBAd-cre-EGFP group, the recombinant adenovirus containing Cre recombinant enzyme (pHBAd-cre-EGFP) was used to transfect with the PDK-1 gene in bone marrow mesenchymal stem cells followed by osteogenic induction. Alkaline phosphatase staining, alkaline phosphatase activity assay, alizarin red staining, and qPCR were used to detect osteogenesis-related gene expression and to evaluate the differentiation and maturation of osteoblasts.
RESULTS AND CONCLUSION: The alkaline phosphatase secretion, alkaline phosphatase activity, and the ability of cell mineralization in the pHBAd-cre-EGFP group were significantly lower than those in the other two groups (P < 0.05, P < 0.01), and the expression levels of Runx2, osteocalcin, and collagen I were also significantly lower than those in the other two groups (P < 0.01). The results have confirmed that interference with PDK-1 gene in vitro can significantly inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells.
Key words: metastatic bone tumor, osteoporosis, bone destruction, osteoblasts, PDK-1, signaling pathway