ABSTRACT
By co-culturing humm mesenchymal stem cells (hMSCs) and human umbilical rein endothelial cells (HUVECs) under hypoxia and creating a microenvironment similar to that of transplanted hMSCs for the treatment of avascular ni ANFH,the effect of hMSCs on survival,apoptosis,migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) under the hypoxic condition were investigated in vitro.hMSCs and HUVECs were cultured and identified in vitro.Three kinds of conditioned media,CdM-CdMNOR,CdM-CdMHYP and HUVEC-CdMHYP were prepared.HUVECs were cultured with these conditioned media under hypoxia.The survival rate,apoptosis rate,migration and angiogenesis of HUVECs were respectively detected by CCK-8,flow cytometry,Transwell and tube formation assay.The content of SDF-1α,VEGF and IL-6 in CdM was determined by ELISA.Our results showed that hMSCs and HUVECs were cultured and identified successfully.Compared with MSC-CdMNOR and HUVEC-CdMHYP groups,the survival rate,migration and angiogenesis of HUVECs in MSC-CdMHYP group were significantly increased while the apoptosis rate was declined (P＜0.05).Moreover,the expression of SDF-1a VEGF and IL-6 in MSC-CdMHYP group was up-regulated.Under hypoxia,the apoptosis of HUVECs was inhibited while survival,migration and angiogenesis were improved by co-culture of hMSCs and HUVECs.The underlying mechanism may be that hMSCs could secrete a number of cytokines and improve niche,which might be helpful in the treatment of femoral head necrosis.
ABSTRACT
The present study examined the role of Wnt/β-catenin signaling pathway in the degeneration of nucleus pulposus cells and the protective effect of DKK1 on nucleus pulposus cells.The model of nucleus pulposus cell degeneration was induced by intra-disc injection of TNF-α,and the expression of β-catenin protein was detected by Western blotting.The cultured rabbit nucleus pulposus cells were divided into 4 groups.In group A,the cells were cultured with normal medium and served as control group.In group B,the cells were cultured with TNF-α and acted as degeneration group.In group C,the cells were cultured with TNF-α and transfected with Adv-eGFP and was used as fluorescence control group.In group D,the cells were cultured with TNF-α and transfected with Adv-hDKK1-eGFP,serving as intervention group.The expression of type Ⅱ collagen,proteoglycan,β-catenin,and MMP-13 in each group was detected by immunocytochemistry and RT-PCR.The result showed that TNF-α increased the expression of β-catenin and MMP-13,and significantly inhibited the synthesis of type Ⅱ collagen and proteoglycan,which resulted in the degeneration of nucleus pulposus cells.This effect could be obviously reversed by DKK1.We are led to concluded that TNF-α could activate the Wnt/β-catenin signaling pathway,and increase the expression of MMP-13,thereby resulting in disc degeneration.Specifically blocking Wnt/β-catenin signaling pathway by DKK-1 could protect the normal metabolism of intervertebral disc tissue.The Wnt pathway plays an important role in the progression of the intervertebral disc degeneration.