Research On Adiponectin: The Expression In Nucleus Pulposus Cells And The Anti-inflammatory Effect On Intervertebral Disc Degeneration

Low back pain(LBP)is a common symptom in 70%-85% of people who have a history of LBP in their lifetime.Below 45 years of age,LBP is the leading cause of disability in human.Billions of dollars are spent every year treating this disease,which bring the tremendous in mind and economy for families and society.Intervertebral disc(IVD)degeneration is considered to be the major cause of LBP.The function of nucleus pulposus(NP)cells would be changed under the IVD,such as loss of cellular structure,the accumulation of cellular metabolites,the degeneration of extracellular matrix,increased expression of inflammatory molecules.The pathogenesis of intervertebral disc degeneration is often accompanied by the inflammatory responses.There are many inflammatory factors in local expression,such as TNF-??IL-6?IL-8?IL-9?IL-16?PGE2 and MMPs.Previous studies on the mechanism of the role of inflammatory response in intervertebral disc degeneration showed thatinflammation may promote the pathogenesis of disc degeneration and cause LBP and sciatica.However,the therapeutic methods to relieve the LBP by controlling inflammation have less efficacy at present.Adipocytokines are considered to be widely involved in human energy metabolism,which may play an important role in the pathophysiology of many diseases.In a recent study,adiponectin,the major member of the adipocytokines,was reported to play an important regulatory effect in the process of inflammatory disease.Adiponectin can play an anti-inflammatory role in vascular endothelial cells and protect endothelial cells from inflammatory lesions.Moreover,adiponectin and its receptors have been found to be widely involved in pathophysiological processes in the bone and cartilage disease.Meanwhile,the study also found that a mutual inhibition mechanism between adiponectin and TNF-?.Althogh the IVD tissue as a special organ play an important role in the body,the correlation between IVD and adiponectin has not been reported.Therefore,the expression of adiponectin and its receptors in IVD,the role of adiponectin played in maintaining the normal function of IVD,the relationship between intervertebral disc degeneration grades and adiponectin,and whether adiponectin would play an anti-inflammatory role in disc degeneration were studied in this project.The development of these experiments will provide a theoretical basis for searching for a new therapeutic target of disc degeneration.The study were divided into three parts as below.Part ?: In vitro culture of human NP cells and phenotype identification of NP cells.Backgrounds.The human NP cells is derived from notochord cells.The previous use of chondrocyte phenotype for the identification of NP cells is considered to be not scientific enough.It is more accurate to analyze the NP cell phenotype based on the notochord cells.The conventional method to obtain the NP tissues from IVD surgery has many shortcomings,such as inefficient,sample composition complex,complicated process and easy tocontaminate.Therefore,the better methods for collection of the IVD tissue need to be found.Objective.To culture the NP cells in vitro from the the tissue obtained from the operation and to identify the phenotype of NP cells.Moreover,to verify the feasibility of using surgical microscope to obtain the NP cells.Methods.Surgical microscope was used to isolate the nucleus pulposus.In vitro cell culture was performed by conventional methods.The expression of KRT19 and CDH2 were analyzed by immunohistochemical staining techniques in tissue slicing and cells cultured in vitro.Results.The immunohistochemical staining showed that the expression of KRT19 and CDH2 were all positive in tissue section and cells cultured in vitro.Conclusion.The surgical microscope can be used to obtain tissue samples from intervertebral disc surgery.The NP tissue sample is pure and meets the experimental requirements.Part ?: The expression of adiponectin and its receptors in NP tissue and NP cells and the mechanisms involved in inflammatory response.Backgrounds.Adiponectin and its receptors are widely expressed in multiple tissue organs.Whether it is expressed in human NP tissue and NP cells and whether there is difference in the expression of adiponectin between normal and degenerative NP tissue and NP cells have not yet been reported.Objective.To find out the expression level of adiponectin and its receptor in NP tissue and NP cells.Methods.Immunohistochemical staining,RT-PCR and Western-Blotting analysis were carried out to detect the expression of adiponectin and itsreceptor in NP tissue and NP cells.Meanwhile,the same analysis is performed for TNF-?.Results.Adiponectin expression levels were down-regulated,while adiponectin receptor R1 and R2 expression levels were up-regulated in degenerated IVD tissues and degenerated NP cells compared with those in healthy IVD tissues and healthy NP cells.There was no significant difference in T-cadherin levels between the two groups.TNF-? expression levels were up-regulated in degenerated IVD tissues and degenerated NP cells compared with those in healthy IVD tissues and healthy NP cells.Adiponectin expression levels are negatively correlated with TNF-? expression levels.Conclusion.Adiponectin may protect NP cells from degeneration by binding with its receptors.The increases in adiponectin receptor expression on NP cells from degenerated IVD specimens may represent a compensatory response to the decreases in adiponectin expression intended to increase tissue sensitivity to adiponectin.Part ? : Study on the relationship between adiponectin and IVD degeneration grades and the anti-inflammatory mechanism of adiponectin in degenerative NP cells.Backgrounds.The correlation between adiponectin and IVD degeneration grades is still unknown.Whether adiponectin affects the expression of TNF-? in the NP cells and whether adiponectin could play an anti-inflammatory role in degenerative IVD are still unclear.Objective.To determine the correlation between adiponectin and IVD degeneration grades and to determine the effect of adiponectin on the expression of TNF-? in cultured degenerative NP cells in vitro.Methods.The concentrations of adiponectin in the tissue lysate supernatants from the 30 degenerated IVDs and 20 healthy IVDs were detected with ELISA for the quantitative of adiponectin.The effects of adiponectin on TNF-? production in degenerated NP cells were also detected by ELISA.Results.The results of the comparison showed that adiponectin levels decreased with increasing IVD degeneration grades.The level of TNF-? secreted by degenerated NP cells decreased in a dose-dependent manner after adiponectin treatment.TNF-? secretion tended to decrease in a time-dependent manner when NP cells were incubated with the same concentration of adiponectin for 1 to 5 days.Conclusion.Adiponectin can significantly inhibit the secretion of inflammatory cytokines TNF-? in the degenerated NP cells and play an anti-inflammatory role in IVD degeneration.