| Background : Cornelia de Lange syndrome(Cd LS)is genetic disorder with multiple phylogenetic defects.Its main clinical features include: mental retardation,deformity in upper limbs and spinal,and hirsutism among numerous other signs and symptoms.Mutations in seven genes(NIPBL,SMC1 A,RAD21,BRD4,HDAC8,SMC3,ANKRD11)have been found to be involve in the occurrence of CDLS,and about 70% patients are caused by NIPBL mutations.Objective : to investigate the effect of NIPBL gene on proliferation and osteogenic differentiation of bene marrow mesenchymal stem cells.Methods: The NIPBL+/-gene defective heterozygous mouse constructed by NIPBL-Loxp mice and Cre mice was used as the experimental group,and the wild-type NIPBL+/+ mice as the control group.Their bone marrow mesenchymal stem cells were isolated and cultured on basal medium by using the whole bone marrow adherence method.The cell morphology was observed under inverted microscope on day 3.In order to identify cell types,flow cytometry was used to detect cell surface antigen expression when the cells were cultured to the third generation.CCK8 method were used to detect cell proliferation ability,and then basal medium was changed by osteogenesis induction medium for further culture.The expression levels of Runx2 and OCN genes and their protein were detected by RT-PCR and Western Blot.Alkaline phosphatase activity was detected on day 3,7 and 10 after induction.Morphological changes of the two group of cells were observed after induction on day 14.Calcium nodule production of the two kinds of cells was compared by alizarin red staining on day 21.Results:(1)NIPBL gene knockout(NIPBL+/-)mouse made by a hybrid of NIPBL-loxp and Cre mice were born with small size and slow growth and development,and NIPBL gene deletion was identified by RT-PCR.(2)Identification of bone marrow mesenchymal stem cells in two group of mice: NIPBL+/-mouse bone marrow mesenchymal stem cells in the experimental group showed negative expression of hematopoietic specific marker molecules: CD34(0.25%),CD11b(0.64%)and CD45(0.78%),and positive expression of mesenchymal stem cell specific marker molecule:Sca-1(95.19%),CD44(78.23%)and CD106(39.26%).NIPBL+/+ mouse bone marrow mesenchymal stem cells in the control group showed negative expression of hematopoietic specific marker molecules: CD34(0.30%),CD11b(0.23%)and CD45(0.47%),and positive expression of mesenchymal stem cell specific marker molecule: sca-1(94.46%),CD44(77.49%)and CD106(36.35).The results suggested that the primary cultured cells were derived from mouse bone marrow mesenchymal.(3)Growth and proliferation of bone marrow mesenchymal stem cells in the two groups:compared with the control group,the proliferation ability of cells in the experimental group was not significantly different in the first 5 days;after the 6th day,the proliferation ability of cells in the control group was significantly improved,while that in the experimental group was relatively slow.(4)Comparison in osteogenesis ability between the two groups of mice bone marrow mesenchymal stem cells: observation from the morphological found that in the control group between bone marrow mesenchymal stem cells induced osteogenesis arrange more closely after day7,cell morphological changes gradually,from the long spindle to irregular,appear in the cell nodules,structure layer increases,the experimental group the bone marrow mesenchymal stem cell morphology change between similar with control group,but nodule significantly less;After osteogenesis,the cells in the control group showed a "paving stone"-like change on day 14,and the cell morphology was still full,while the cell particle quantity in the experimental group was low.The activity of alkaline phosphatase in both groups reached the peak on the 7th day,and the activity of alkaline phosphatase in the experimental group was significantly lower than that in the control group(P < 0.05).The expression levels of OCN and Runx2 genes in the experimental group were significantly lower than those in the control group(P < 0.05),and the levels of Runx2 and OCN genes increased with the increase of osteogenic induction time.On day 7,14,21,28 after osteogenic induction,Runx2 protein expression in the experimental group was significantly lower than that in the control group(P7,21d< 0.05,P14,28 d < 0.01).On day 21,28 of osteogenic induction,OCN protein expression in the experimental group was significantly lower than that in the control group(P21d < 0.05,P28d< 0.001),Runx2 and OCN protein levels increased with the increase of osteogenic induction time before day 21,and then decreased.On the 21 st day of osteogenic induction,the number of calcium nodules in the control group was significantly higher than that in the experimental group after alizarin red staining.It suggested that NIPBL knockout down-regulated the osteogenic differentiation of mouse bone marrow mesenchymal stem cells.Conclusion : Knockout of NIPBL gene reduced the proliferation and osteogenic differentiation of mouse bone marrow mesenchymal stem cells. |
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