| Object:To explore the effect of knocking down NIPBL gene on chondrogenic differentiation of bone marrow mesenchymal stem cells induced by TGF-β1 and IGF-1 in vitro cell experiment,and to explore whether the effect of NIPBL gene deletion on chondrogenic differentiation of mesenchymal stem cells is related to TGF-βfactor.It was proved that the chondrogenic differentiation ability of bone marrow mesenchymal stem cells induced by TGF-β1 combined with IGF-1 was higher than that of TGF-β1 alone or IGF-1 alone.To explore the possible pathogenesis of NIPBL gene mutation CdLS,and to provide reference basis and ideas for the treatment and prognosis of skeletal developmental malformations in clinical CdLS.Methods:Mouse bone marrow mesenchymal stem cells with low NIPBL gene were obtained by lentivirus transfection,and the transfection efficiency was observed under fluorescence inversion microscope.NIPBL-sh RNA bone marrow mesenchymal stem cells were divided into TGF-β1 group,TGF-β1+IGF-1 group and NIPBL~-blank control group.Normal bone marrow mesenchymal stem cells without transfection were used to set up NIPBL~+control group.The above four groups of cells were induced to differentiate into cartilage.The morphological changes of cells at different stages were observed under inverted microscope,and the protein expression levels of Sox-9 and Collagen-Ⅱgenes were detected by Western blot on the 7th,14th and 21st day of induced differentiation.Results:(1)Morphological changes of bone marrow mesenchymal stem cells after lentivirus transfection:after lentivirus transfection,bone marrow mesenchymal stem cells grew normally under inverted microscope,and the morphology of bone marrow mesenchymal stem cells was fusiform,triangular or long fusiform,which was not significantly different from that of negative control group and blank control group.Under the fluorescence inverted microscope,a large number of green fluorescent protein could be seen in the transfection group and the negative control group,and the expression rate was more than 90%.There was no fluorescence in the blank control group.(2)Morphological changes of bone marrow mesenchymal stem cells after chondrogenic induction:after 3 days of chondrogenic induction,there was no significant difference in the morphology of bone marrow mesenchymal stem cells among the four groups under inverted microscope,and the morphology of long spindle cells gradually increased,and the cells gradually showed polygonal and triangular changes after 7 days of induction,and a large number of cells could be seen in NIPBL~+control group.After 14 days of induction,the cells showed aggregated distribution and formed colonies of different sizes.TGF-β1+IGF-1 group and NIPBL+control group had more extracellular matrix than the other two groups,and multiple processes could be seen after 21 days of induction.(3)Detection of Sox-9 and Collagen-Ⅱprotein expression by Western blot:the protein expression of Sox-9 in TGF-β1+IGF-1 group was higher than that in TGF-β1 group at 7,14 and 21 days,and the protein expression level of Collagen-Ⅱin TGF-β1 group was higher than that in TGF-β1 group at 7 and 14 days.The protein expression levels of Sox-9 and Collagen-Ⅱin NIPBL~+control group were higher than those in TGF-β1 group and NIPBL~-control group.The protein expression of Sox-9 in TGF-β1+IGF-1 group was higher than that in NIPBL~-control group at 7 and 21 days after induction.There was no significant difference in Sox-9 and Collagen-Ⅱprotein expression between TGF-β1 group and NIPBL control group,and there was no significant difference between TGF-β1+IGF-1 group and NIPBL~+control group.Conclusion:After knocking down NIPBL gene,the ability of bone marrow mesenchymal stem cells to differentiate into cartilage induced by TGF-β1 combined with IGF-1 decreased,suggesting that the abnormal skeletal development of limbs after NIPBL gene deletion in CdLS patients is related to TGF-βfactor. |
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