Study About The Property Of Nucleus Pulposus: Secreting Inflammatory Related Cytokines

PART 1 The Expression and Characterization of Inflammatory Cytokines in Lumbar Disc HerniationObjective: To observe the histopathological changes of intervertebral disc(IVD) from different types of lumbar disc herniation(LDH), and to discuss the sources of inflammatory cytokines and shed a light on the role of nucleus pulposus in the pathogenesis of LDH.Method: The enrolled patients can be divided into two groups, according to the integrity of the annulus pulposus, namely(1)ruptrued group(Rgroup)(2)non-ruptrued group(NR group). The pathological changes of disc tissues from different groups were observed by HE staining, and the expression of inflammatory cytokines, such as IL-23,IL-17, NLRP3 and Caspase-1, were measured quantitatively and qualitively by immune-histological staining and western-blot.Result: The pathological changes of disc tissues from R group were more complex than NR group: more cracks and fissures were observed in the disc tissues, and the collagen arrangement of the annulus fibrosus was more disordered, loose and even disrupted; cellulose like necrosis, mucus degeneration, synovial metaplasia, calcium crystal deposition, the ingrowth of neovascularization, infiltration of inflammatory cells and the formation of cartilage cell clusters can be seen in the margins of herniated disc tissues. Higher expression of IL-17?IL-23?NLRP3 and caspase-1 can be found in the ruptured group, especially in the nucleus pulposus and cell clusters around the cracks and in the inflammatory area.Conclusion: Inflammatory cytokines may be derived from both disc cells and infiltrated inflammatory cells, which may together play an important role in the local inflammation and radiculopathy resulted by LDH.PART 2 The Ability and Characterization of Nucleus Pulposus to Secrete Inflammatory CytokinesObjective: Inflammatory cytokines have been found in disc tissues, which may play an important role in radiculopathy. However, whether and how they can be secreted by nucleus pulposus cells is still much unknown. Thus in this part, we aimed to make a preliminary study on this question.Methods: Nucleus pulposus from the non-ruptured group were used for cell culture after carefully separated from the annulus fibrosus and endplate and the second passage were used for the following experiment. Nucleus pulposus cells were identified by Safranin O, toluidine blue, Switzerland Giemsa staining and Type II collagen immunohistochemical staining. NP cells were then cultured without FBS or exposed to LPS for 4h,16 h,24h and 48 h, and the gene expression of inflammatory cytokines, such as IL-1??IL-12?IL-17?IL-23 and TNF-? were then measured by RT-PCR, and the protein level of IL-1??IL-17 and TNF-? in the medium was then detected by ELISA.Result: The cultured cells were identified as nucleus pulposus. In the basal conditions, gene expression of several inflammatory cytokines, such as IL-1??IL-12?IL-17?IL-23 and TNF-?, and anti-inflammatory cytokines(IL-35, IL-37) were detected. When the cells were cultured in serum-free medium or in complete medium supplemented with LPS, more gene expression of inflammatory related cytokines were detected by RT-PCR. There were a similar trend for the expression of IL-1??IL-17 and TNF-? in protein level detected by ELISA.Conclusion: Several inflammatory related cytokines can be secreted by nucleus pulposus, which may play an important role in the disc degeneration and in the pathogenesis of radiculopathy. Works on this characterization of nucleus pulposus may enlarge our knowledge about disc and lay a foundation for treatment of disc diseases by tissue engineering.