Abstract:

Objective To investigate the effect of the NIPBL gene knockdown on osteogenic differentiation of the mouse bone marrow mesenchymal stem cells （BMSCs）， and to elucidate the underlying mechanism through the sonic hedgehog （Shh） signaling pathway. Methods The BMSCs isolated from C57 mice were cultured in vitro to the third generation. Flow cytometry was used to identify the expressions of cell surface markers on the BMSCs. Lentivirus carrying green fluorescence protein （GFP） was used to construct the BMSC model with stable NIPBL knockdown. The cells were divided into control group（routine culture）， sh-NC group （transfected with Scramble shRNA lentivirus）， and sh-NIPBL group（transfected with NIPBL shRNA lentivirus）. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of NIPBL mRNA in the BMSCs in various groups. The BMSCs in various groups were subjected to osteogenic induction， and the morphological changes were dynamically observed. The cells were collected on days 7， 14， and 21 after induction. Western blotting method was used to detect the expression levels of Shh signaling pathway proteins， including Shh， patched homolog 1 （PTCH1）， and glioma-associated oncogene homolog 1 （GLI1）， as well as osteogenic markers， including alkaline phosphatase （ALP）， osteocalcin （OCN）， and collagen type Ⅰ alpha 1 chain （COL1A1） proteins in the cells in various groups. Alizarin red staining was used to assess the mineralization capacities of the BMSCs in various groups on day 21 after induction. Results Third-generation BMSCs exhibited good growth status， showing a long-spindle or fusiform morphology and the typical CD105?CD45? phenotype. The stable GFP expression rates in the BMSCs of the mice in sh-NIPBL group and sh-NC group exceeded 90%， while no fluorescence signal was observed in control group. Compared with control group， the expression level of NIPBL mRNA in the BMSCs in sh-NIPBL group was significantly decreased （P<0.001）， indicating successful establishment of the NIPBL-knockdown model. On day 21 after osteogenic induction， dense mineralized nodules were observed in control group， while only a small number of scattered， tiny， sparse， and non-fused mineralized nodules were seen in sh-NIPBL group， suggesting delayed osteogenic differentiation. On day 21 after induction， compared with sh-NC group， the expression levels of Shh， PTCH1， GLI1， COL1A1， ALP， and OCN proteins in the BMSCs in sh-NIPBL group were significantly decreased （P<0.05）. On days 7， 14， and 21 after induction， the expression levels of aforementioned proteins in the BMSCs in sh-NIPBL group were consistently lower than those in control group and sh-NC group. The Alizarin red staining results showed a large number of dense， fused orange-red mineralized nodules in the BMSCs in control group and sh-NC group， while only scattered orange-red spots in the BMSCs were observed in sh-NIPBL group. Compared with control group， the mineralization capacity of cells in sh-NIPBL group was significantly decreased （P<0.01）. Conclusion Knockdown of the NIPBL gene may reduce the osteogenic differentiation capacity and mineralization capacity of mouse BMSCs， and its mechanism is related to inhibiting the activity of the Shh signaling pathway and downregulating the expression of osteogenesis-related proteins.

Key words: NIPBL gene, Bone marrow mesenchymal stem cells, Sonic hedgehog signaling pathway, Osteogenic differentiation, Cornelia de Lange syndrome