Effect Of NIPBL Gene On Proliferation And Differentiation Of Mouse Bone Marrow Mesenchymal Stem Cells

Objective:Cornelia de Lange syndrome(Cd LS)is a hereditary disease,and NIPBL gene is the main pathogenic gene.NIPBL gene mutation may lead to skeletal dysplasia.Therefore,this study explored the effect of NIPBL gene on the proliferation and differentiation of mouse bone marrow mesenchymal stem cells through in vitro cytological experiment.To provide theoretical basis and experimental evidence for further exploration of the pathogenesis of Cd LS and the determination of new therapeutic regimens.Methods:The third generation of C57 Mouse Bone marrow mesenchymal stem cells were used in the experiment.Construction of 3 lentiviral vectors carrying NIPBL-si RNA,and then use them transfected into the cells.The cells were divided into experimental group,negative control group and blank control group.After lentiviral transfection,the results of lentiviral transfection were observed by inverted fluorescence microscope,and the expression of nipbl gene was detected by real-time PCR.CCK-8 method was used to detect the cell proliferation.The cells in each group were induced to differentiate into osteoblasts,cartilages and adipocytes.The m RNA and protein levels of OCN,BMP-2,Runx-2,Sox-9,COL2A1 and PPAR-γ were detected by real-time PCR and Western blot respectively.Alkaline phosphatase activity was detected and the results of alizarin red staining of calcium nodules,alcian blue staining chondrocytes and oil red O staining of lipid cells were observed.Results:(1)The results of three lentiviral vectors transfected mouse bone marrow mesenchymal stem cells: after lentiviral mediated nipbl gene transfected bone marrow mesenchymal stem cells,real-time PCR results showed that nipbl gene m RNA expression was significantly decreased(P < 0.05),and nipbl-sirna-3 silencing effect was the best,which was used for followup experimental study.(2)Comparison of cell proliferation ability after transfection: After inhibition of nipbl gene expression,CCK8 test results showed that the cell proliferation ability of experimental group was significantly lower than control group(P < 0.05),which proved that nipbl gene silencing reduced the proliferation ability of bone marrow mesenchymal stem cells.(3)Comparison of osteogenic induction culture and related gene expression: After osteogenic induction,m RNA and protein were extracted on the 7th,14 th and 21 st day.The results of real-time PCR and Western blot showed that the m RNA and protein expressions of OCN,BMP-2 and Runx-2 in experimental group were lower than control group(P < 0.05),in the experimental group the ALP activity was significantly decreased.The number of alizarin red stained calcium nodules in the experimental group was significantly less than control group(P < 0.05).(4)Comparison of chondrogenic induction culture and related gene expression: m RNA and protein were extracted from chondrocytes in seventh days,14 th days and 21 th days after induction.The results of real-time PCR and Western blot showed that the m RNA and protein expressions of Sox-9 and COL2A1 in experimental group were lower than control group(P <0.05),and alcian blue staining showed that the chondrocytes in the experimental group were significantly less than control group.(5)Comparison of adipogenic induction culture and related gene expression: On the 7th,14 th and 21 st day of adipogenic induction,m RNA and protein were extracted.The results of real-time PCR and Western blot showed that the m RNA and protein expressions of PPAR-γ in experimental group were lower than control group(P < 0.05).The results of oil red O staining showed that the lipid accumulation in the experimental group was less than control group(P <0.05).Conclusion:Lentivirus mediated silencing of nipbl gene reduced the proliferation and differentiation of mouse bone marrow mesenchymal stem cells,inhibited their osteogenic,chondrogenic and adipogenic differentiation,and reduced the proliferation and differentiation of bone marrow mesenchymal stem cells.