The role of apoptosis in the pathogenesis of intervertebral disc degeneration (IDD) remains enigmatic. Accumulating evidence has shown that the apoptotic machinery is regulated by miRNAs. We hypothesized that miRNAs might contribute to apoptosis in IDD. We have found that 29 miRNAs were differentially expressed and miR-155 was down-regulated in degenerative nucleus pulposus (NP). The deregulation of miR-155 was further verified using real-time PCR (0.56 fold, p < 0.05). Bioinformatics target prediction identified FADD and caspase-3 as putative targets of miR-155. Furthermore, miR-155 inhibited FADD and caspase-3 expression by directly targeting their 3'-UTRs, which was abolished by mutation of the miR-155 binding sites. In vitro up-regulation of miR-155 in human NP cells by transfection with lentiviral pre-miR-155 resulted in repression of FADD and caspase-3; whereas knockdown of miR-155 with lentiviral antigomiR-155 led to over-expression of FADD and caspase-3. Also, Fas-mediated apoptosis was increased when antagonizing miR-155 and decreased when using pre-miR-155 in human NP cells. In addition, we presented direct evidence of NP cells undergoing apoptosis in IDD tissues using transmission electron microscopy analysis. Moreover, a combination of in situ hybridization (ISH) and immunohistochemistry (IHC) revealed that miR-155 expressed in the cytoplasm of human NP cells with reverse correlation with FADD and caspase-3. In summary, this is the first study addressing the underlying mechanisms of IDD in terms of apoptosis and miRNAs. Furthermore, caspase-3 is identified as a novel target of miR-155. Our results suggest that deregulated miR-155 promotes Fas-mediated apoptosis in human IDD by targeting FADD and caspase-3, implicating an aetiological and therapeutic role of miR-155 in IDD.

The mechanisms of immune privilege in human nucleus pulposus (NP) remain unclear. Accumulating evidence indicates that Fas ligand (FasL) might play an important role in the immune privilege of the disc. We aimed for addressing the role of FasL expression in human intervertebral disc degeneration (IDD) and immune privilege in terms of the interaction between NP cells and immunocytes via the FasL-Fas machinery. We collected NP specimens from 20 patients with IDD as degenerative group and 8 normal cadaveric donors as control. FasL expression was detected by qRT-PCR, western blotting and flow cytometry (FCM). We also collected macrophages and CD8+ T cells from the peripheral blood of patients with IDD for co-cultures with NP cells. And macrophages and CD8+ T cells were harvested for apoptosis analysis by FCM after 2 days of co-cultures. We found that FasL expression in mRNA, protein and cellular resolutions demonstrated a significant decrease in degenerative group compared with normal control (p<0.05). FCM analysis found that human NP cells with increased FasL expression resulted in significantly increased apoptosis ratio of macrophages and CD8+ T cells. Our study demonstrated that FasL expression tends to decrease in degenerated discs and FasL plays an important role in human disc immune privilege, which might provide a novel target for the treatment strategies for IDD.

Biologic and cellular treatment strategies aiming for curing intervertebral disc degeneration (IDD) have been proposed recently. Given the convenient availability and expansion potential, adipose-derived stromal cells (ADSCs) might be an ideal cell candidate. However, the interaction between ADSCs and nucleus pulposus (NP) cells still remains ambiguous, especially in direct cocultures of the two types of cells. Nevertheless, NP markers in ADSCs after co-cultures were unidentified. Here, we addressed the interaction of human ADSCs and NP cells in a direct co-culture system for the first time. As a result, ADSCs could differentiate to the NP cell phenotype with a significant up-regulated expression of multiple genes and proteins in extracellular matrix (ECM) (SOX9, COL2A1, ACAN, and COL6A2), relative NP markers (FOXF1, PAX1, CA12, and HBB) and pertinent growth factors (CDMP-1, TGF-b1, IGF-1, and CTGF). Moreover, the gene expression of COL2A1, ACAN, and COL6A2 of degenerate NP cells was also up-regulated. Collectively, these results suggest that direct co-cultures of ADSCs and NP cells may exert a reciprocal impact, that is, both stimulating ADSCs differentiation to the NP cell phenotype and inducing NP cells to regain functional phenotype. Accordingly, ADSCs might be a potential candidate in the development of cellular treatment strategies for IDD. Keywords: direct co-culture; intervertebral disc degeneration; adipose-derived stem cells; nucleus pulposusIntervertebral disc degeneration (IDD) is tightly linked with low back pain, which results in worldwide socioeconomic implications, wage losses, and consequently morbidity.1 Current treatment options including conservative and surgical therapies aim for relieving symptoms rather than modifying the underlying pathological processes of IDD. Therefore, biologic and cellular treatment strategies aiming for curing disc degeneration have been attracting more and more attention of investigators these years.Macroscopically, normal intervertebral disc consists of three sub-parts, the central nucleus pulposus (NP), the outer anulus fibrosus (AF) and the cartilage endplates connecting adjacent vertebras. In normal human disc, the central NP is made of abundant extracellular matrix (ECM) interspersed by a small number of NP cells that make up about 1% of the total volume.2 As the disc degenerates, proteoglycan together with water content in NP decreases. NP becomes less gelatinous and more fibrous with the formation of cracks and fissures in AF, which eventually leads to IDD. The etiology of IDD is ascribed to numerous aspects, amongst which cell death is thought to play a crucial role.3 NP cells are the vital machinery for the synthesis of functional ECM, the production of cytokines and the maintenance of relevant enzymes' activities. Cytology analysis of the disc has identified a variety of cellular alterations and provides gross information in molecular level. However, the therapeutic modalities are still in its infancy in terms of NP cell function restoration or ...

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