Study On Effect Of Hypoxia-Inducible Factor-1? Deficiency On Physiology Of Intervertebral Disc And MicroRNAs Expression In Mice Nucleus Pulposus

Objectives: 1.To investigate the expression of hypoxia-inducible factor(HIF)-1? in nucleus pulposus(NP)and the effectonfunction and degeneration of intervertebral disc in HIF-1? knocking out mice.2.To investigate differential expression of microRNAs in nucleus pulposus with HIF-1?-deficiencyandthe mechanism of HIF-1? involved in degeneration ofintervertebral disc.Methods: 1.Immunohistochemical staining studies were used to investigate the expression of HIF-1?in harvested intervertebral disc samples of human and mouse.2.In situ immunofluorescence staining studies were performed to identify the phenotype of NP cells compared with annulus fibrosus(AF)cells and endplate chondrocytes;Cre-lox P system was used to create the NP-HIF-1? deficiency mouse model.3.Imaging studies were used to evaluate NP dehydration and changes of disc height.Histology and molecular mechanisms studies were performed to evaluate the changes of matrix and cell number in disc.4.MicroRNAs microarray screening assay to investigate the changes of microRNAs expression,which may mediate expression of mRNAs in discs of HIF-1? deficiency miceResults: 1.The expression of HIF-1?was found in human and mice disc samples,which might play a role in degeneration of intervertebral disc.2.CD24 might be the specific cell marker for the healthy NP cells;HIF-1? in NP was successfully knocked out in nucleus pulposus of HIF-1?deficiency mice.3.NP cells apoptosis increased;the height of disc decreased and the signal of the NP area on MRI T2-weight image also decreased;the area and cell number of NP reduced,proteoglycan content including the protein level of collagen II and aggrecan decreased in NP-HIF-1?-/-micecompared with age matched control mice.4.Differential expression profiles of microRNAs(n=10)were obtained,which may degraded the NP and affected the expression of mRNAs in NP cells.Conclusions: 1.HIF-1? is highly expressed in NP of human and mice disc samples,which may be one of the phenotype of NP cells,CD24 specifically expressed in healthy NP cells.2.Protein level of collagen II and aggrecan was decreased,mass cells weredied in NP,microRNAswere aberrantly expressedanddegenerationof intervertebral disc occurredat early stage in NP-HIF-1? deficiency mice.3.HIF-1? is indispensable to the nucleus pulposus cell and may play a critical role in the protection ofnormalphysiology of intervertebral disc.