2023, Vol. 27 ›› Issue (10): 1477-1483

Knockdown of NIPBL gene regulates chondrogenic differentiation of mouse bone marrow mesenchymal stem cells

Ma Wenqing1, Zhang Huirong2, Liu Hui1, Dong Lili1, Yang Juandi1   

Abstract: BACKGROUND: At present, NIPBL gene mutation has been used as the preferred indicator for the diagnosis of Cornelia de Lange syndrome. However, due to the genetic heterogeneity of the disease, clinical diagnosis and treatment are more difficult; especially, the incidence of skeletal dysplasia in children is high, and its pathogenesis is still unclear. There is currently no targeted therapy program, and the average life expectancy of children is significantly shorter than that of the general population.  
OBJECTIVE: To investigate the effect of knockdown of NIPBL gene on chondrogenic differentiation ability of mouse bone marrow mesenchymal stem cells and its possible molecular regulation mechanism.
METHODS: Mouse bone marrow mesenchymal stem cells transfected with NIPBL shRNA by lentivirus were used as sh-NIPBL group, and bone marrow mesenchymal stem cells transfected with lentivirus empty vector were used as sh-NC group. Bone marrow mesenchymal stem cells without lentivirus interference were used as the blank control group. Next, the chondrogenic induction culture was carried out in the three groups. After 21 days of induction, the perimeter of the largest cross section of cartilage microspheres was measured and Alicia blue staining was used to identify the induced differentiation. The expression level of Collagen II was detected by immunofluorescence method. The expression levels of Sox-9, TGF-β1, Smad2, and Smad4 mRNA in chondrocytes were detected by real-time quantitative PCR.
RESULTS AND CONCLUSION: (1) On day 21 of chondrogenic induction, the perimeter of the largest cross section of cartilage microspheres in the sh-NIPBL group was significantly smaller than that in the control group (P < 0.05). More blue intrachondral acidic mucopolysaccharides were observed in the sh-NC group than that in the sh-NIPBL group after alician blue staining under an inverted microscope. (2) After induction into chondrogenic differentiation, the expression levels of Collagen II and Sox-9 in bone marrow mesenchymal stem cells of the sh-NIPBL group were lower than those in the sh-NC and blank control groups (P < 0.05). (3) In the process of chondrogenic induction, the expression levels of TGF-β1, Smad2, and Smad4 mRNA of the sh-NIPBL group were lower than those in the sh-NC and blank control groups (P < 0.05). (4) These findings suggest that lentiviral knockdown of NIPBL gene expression reduced the chondrogenic differentiation of bone marrow mesenchymal stem cells, and this process was mediated by the TGF-β1/Smad signaling pathway.
Key words: NIPBL gene, skeletal developmental defect, lentiviral transfection, chondrogenic differentiation, TGF-β1/Smad signaling pathway, Cornelia de Lange syndrome