Experimential Study On The Canonical Wnt/β-catenin Pathway In Intervertebral Disc Degeneration

Partâ… Expression and Significance ofβ-catenin in thenucleus pulposus tissue of human degenerated lumberintervertebral discObjective To investigate the expression and significance ofβ-catenin in the nucleus pulposus tissue of human degenerated lumberintervertebral disc and clear whether there is relationship betweencanonical Wnt/β-catenin pathway and degeneration of lumberintervertebral disc.Methods Collect 28 cases of fresh nucleus pulposus of patients withlumbar disc herniation as experimental group (17 male and 11 female),mean age 45 years (26～63 years) ; and 12 cases of normal nucleuspulposus as control group(9 male and 3 female),mean age 35years(18～55 years). All thepatients were surgical operated in our hospital from May 2007 to March 2008. The expression ofβ-cateninmRNA and protein of nucleus pulposus were detected by real-timefluorescent quantitative PCR and western-blotting respectively.Resultsâ‘ The expression ofβ-catenin mRNA in experiment groupwas obviously higher than that in the control group(P＜0.05), thefluorescent quantitation ratio was 2.85/1 (P＜0.05) .â‘¡Expressionofβ-catenin protein has a similar difference contrast to mRNA,and a 37KD protein stripe was detected in every sample. Averagelight density ratio ofβ-catenin/GAPDH protein was 0.76±0.04 and0.13±0.02 respectively.Conclusion The expression ofβ-catenin , both mRNA and proteinlevel, was increased in the nucleus pulposus tissue of humandegenerated lumber intervertebral disc, which demonstrate thatthere are relationship between canonical Wnt/β-catenin pathwayand degeneration of lumber intervertebral disc.Partâ…¡Expression and Significance ofβ-catenin in thenucleus pulposus of rabbit lumber intervertebral discdegeneration model induced with TNF-α Objective To investigate the expression and significance ofβ-catenin in the nucleus pulposus of rabbit lumber intervertebraldisc degeneration model induced with TNF-αand to clear whetherthere is a relationship between canonical Wnt/β-catenin pathwayand TNF-αin the model.Methods 12 healthy white rabbits (2.5～3kg) were selected randomly,provided by Department of Experimental Animal of Tongji MedicalCollege of Huazhong Science and Technology University. Operate toexposure the L2～L5 lumber intervertebral discs of each rabbits.0.9% NaOl (as control) and various concentrations, of TNF-α(5ng/μl,10ng/μl and 20ng/μl) were injected to discs respectively.The models were randomly divided into 4 groups by concentration ofTNF-α. The nucleus pulposus were obtained after 8 weeks. Theexpression ofβ-catenin protein was detected by western-blottingand HE-Safranin-αStaining was done to clear the degree ofdegeneration.Resultsâ‘ The expression ofβ-catenin protein in TNF-αgroupwas obviously higher than that in the control group, average lightdensity ratio ofβ-catenin /β-actin protein was 0.142±0.036,0.351±0.041,0.472±0.052 and 0.710±0.063 respectively(P＜0.05), there was dose-effect relationship betweenβ-catenin protein andTNF-α.â‘¡HE-Safranin O Staining showed that amounts of nucleuspulposus cells reduced, cell framework was breakdown in TNF-αgroup. Degree of degeneration was according to the concentrationsof TNF-αConclusion The expression ofβ-catenin protein was increased inthe nucleus pulposus of rabbit lumber intervertebral discdegeneration model induced with TNF-α, and there was dose-effectrelationship between them, which demonstrate that canonical Wnt/βcatenin pathway could be dose-effect induced by TNF-αin rabbitlumber intervertebral disc degeneration model.Partâ…¢Construction of recombinant adenovirus vectorcontaining human Dickkopf-1 marked by eGFPObjective To construct,verificate and amplificate the adenovirusvector containing human Dickkopf-1 marked by eGFPMethods Primers containing matched sequence and digest site weresynthesized according to the Dkk-1 gene order. Dkk-1 cDNAobtained by RT-PCR from Dkk-1 plasmid was cloned into the linearizedpDC315-eGFP vector, and then the pDC315-eGFP -Dkk1 recombinedplasmid was transformed into E. coli host strain DH5, positive clonewas analyzed. The recombinant plasmid was co-transferred withadenoviral pack-vector-system into E. coli and homologouslyrecombinated in bacterial cells. After screening and amplification,the recombinant Ad plasmid was digested with Pac I and transfectedinto HEK293 cell. The replication-defective adenovirus AdEGFP-DKK-1was packed and amplified in the HEK293 cell. Fluorescenceexpression was observed after 24h, and EGFP protein was detectedby western-blotting. AdEGFP- Dkk1 titered in HEK293 cells bySpearman-Karber Method.Result PCR and gene sequencing confirmed that the recombinantcontained Dkk-1 cDNA. The viral particles was easily detected underelectron microscopy .Under fluorescence microscopy, thefluorescence sign was easily detected in HEK293 cells too. Theexpression of EGFP protein can be detected by western-blott Virustiter can reach as high as 2.5×10¹³ pfu/ml.Conclusion The construction of recombinant Adenovirus vector byhomologous recombination in bacterial cells can be quickly and easily performed. The AdEGFP- Dkk1 was had a high virus titer andcould expression Dkk-1 successfully.Partâ…£The Experimental Study on the inhibition of Wntpathway by DKK1 in the degenerated rabbit nucleus pulposuscells induced with TNF-αin vitro.Objective To study the biological property of Wnt pathway in thedegenerated rabbit nucleus pulposus cells induced with TNF-αinvitro, and to explore the effect of inhibition of Wnt pathway byDKK-1Methods The nucleus pulposus cells were isolated and cultured fromlumber intervertebral discs of young health rabbits. The nucleuspulposus cells degeneration model was induced with 20ng/ml TNF-αin the study. The cells were divided into 4 group; just normalmedium without TNF-αand treatment as control group, TNF-α20ng/ml as degenerated group, TNF-α20ng/ml and the cellstransfected by 200 MOI Adv-Egfp beforehand as fluorescence controlgroup, TNF-α20ng/ml and the cells transfected by 200 MOI Adv-hDKK-1 as experimental group. After 1 week culture, Theexpression ofβ-catenin,â…¡collagen,MMP-13 mRNA were examinedwith RT-PCR, theâ…¡collagen and proteoglycans protein weremeasured with immunocytochemistry and Safranin-o Stainingrespectively. Fluorescence expression was observed to know thetransfection efficeince.Results The expression ofβ-catenin mRNA was obviously increasedin the degenerated group and in experimental group there was noobvious difference with control group. The expression of MMP-13mRNA were justastheβ-catenin. The expression ofâ…¡collagen mRNAand protein were decreased in the degenerated group and inexperimental group there was no obvious change. The expression ofproteoglycans measured with afranin-o Staining was just as theâ…¡collagen protein.Conclusion The rabbit nucleus pulposus cells were obviouslydegenerated induced by 20ng/ml TNF-α, the ECM were decreasedcontrast with control group. The expression ofβ-catenin and MMP-13mRNA was obviously increased. In contrast, because of thetransfection of Adv-hDKK-1, the Wnt pathway was inhibited and thenucleus pulposus and the ECM of which were protected in the exper imental group. The Wnt pathway played an important role in theprogress of the intervertebral disc degeneration.