Expression And Significance Of Pleiotrophin And Vascular Endothelial Growth Factor In Intervertebral Disc

ObjectiveDegeneration of intervertebral disc is a term to describe the loss of normal structure of intervertebral disc accompanied with the progressive fibrosis.The manifestations are gelatinous disappearance of pulpiform nucleus,rough lamellar bone of annulus fibrosis and progressive fibrosis.Finally,fissure and senile chromatosis ensure.The degeneration of intervertebral disc is associated with the release of some cytokines and the changes of enzyme activity in the intervertebral disc,pleiotrophin and vascular endothelial growth factor are two important cytokines in angiopoiesis.It is not clear how the cytokines affect the degeneration of the intervertebral disc.In this study,we investigate the morphological changes of intervertebral disc and the expression of pleiotrophin and vascular endothelial growth factor in rats at different months,so as to probe into the association of pleiotrophin and vascular endothelial growth factor with degeneration of intervertebral disc.Materials and Methods1.Grouping and Specimen Selection50 male Wistar rats were selected and divided into five groups(10 each group of 1,3,6,12,18 months old) by different months.Killed the rats by over dose pentobarbital and got the intervertebral discs of certain segments.Fixed one half specimens in 4% paraformaldehyde and then decalcificatiom in 10%ethylene diamine tetraacetic acid (EDTA).After that,tissue samples embeded in paraffin were cut into 4μm-thick sections.Other specimens were conserved in fridge at -70℃.2,Methods(1)Hematoxylin-Eosin StainingSections were deparaffinized with xylene,and rehydrated with a series of graded alcohol.Then the slides were stained with hematoxylin and eosine.After that the sections were dehydrated and cleared and mounted with resin.(2)Strept Actividin-Biotin Complex(SABC)Immunohistochemical StainingSections were deparaffinized with xylene,and rehydrated with a series of graded alcohol.The slides were immersed in 3%H₂O₂ to block endogenous peroxidase activity.After heat antigen retrieve processing,the sections were treated with bovine serum albumin(BSA) to block nonspecific staining.Sections were then incubated with anti-PTN and anti-VEGF primary antibodies respectively.This was followed by incubation with a biotinylated secondary antibody and thereafter incubation with Strept Actividin-Biotin Complex(SABC).Immunostaining was visualized using, 3V-diaminobenzidine tetrahydrochloide substrate.Slides were counterstained with hematoxylin,then the sections were dehydrated and cleared and mounted with resin. PBS replacing the primary antibodies was used as negative controls.(3) Western-BlotDiced tissues into very small pieces,further disrupted and homogenized tissues with a sonicator.Then transfered tissues to microcentrifuge tubes,centrifuged at 10,000xg for 10 minutes at 4℃.Removed supernatant and centrifuged again.The supernatant fluid was the total cell lysate.Certain proteins of different groups boiled for 5 minutes in sample buffer and were separated in 12%and 8%SDS-PAGE and transfered onto nitrocellulose membranes.Blocked non-specific binding by incubating membrane in bovine serum albumin(BSA).Membranes were then incubated with anti-PTN and anti-VEGF primary antibodies respectively at 4℃overnight in a bag,this was followed by incubation with a biotinylated secondary antibody at room temperature for 2 hours.Washed membranes and incubated membranes in visualization reagent.β-actin was used as internal controls.PBS replacing the primary antibodies was used as negative controls.(4)Results JudgmentThe interspongioplastic substance stain was considered as PTN and VEGF positive.The stain results of PTN and VEGF were measured by the percentage of positive cells number in all cells.Gene Tools from Syngene gel image analysis system was used for the systematic result analysis.(5)Statistical Analysis The data was represented by(?)±s,and SPSS13.0 statistical software was used for ANOVA,and the difference was statistically significant(P＜0.05).ResultsAll the 50 rats were included in the result analysis.1.Hematoxylin-Eosin StainingHistological structure of intervertebral discs in the rats changed along with the rat aging.The matrix of nucleus pulposus degenerated and collagen fibers were thick,hyperplastic and disorganized.2.Expression of PTN and VEGFThe expression of PTN and VEGF in intervertebral discs decreased as the rat aging(1 to 12month),but it increased when it comes to 18-month.And that in the 6-month and 12-month groups were significantly lower than that in the 1-month group(P＜0.01),18-month group was significantly higher than the 12-month group(P＜0.01).In the same month group,the expression of PTN and VEGF was higher in cartilage end plate than in annulus fibrosus and nucleus pulposus(P＜0.05),and there was no statistical difference of the expression of PTN and VEGF in nnulus fibrosus and nucleus pulposus(P＞0.05).Conclusion1.The intervertebral discs of rats degenerate along with their age of months.2.PTN and VEGF may induce new vascularization in intervertebral disc tissues and delay the degeneration of intervertebral disc.