Expression Of ADAMTS-4、ADAMTS-5、TIMP-3in Human Degenerate Nucleus Pulposus Cells Induced By TGF-β1or IL-1β In Vitro

[Objective] The homeostasis of intervertebral disc (IVD) tissues is accomplished through a complex and precise coordination of a variety of substances, including cytokines, growth factors, enzymes and enzyme inhibitors.Recent biological therapeutic strategies for disc degeneration have included attempts to up-regulate the production of key matrix proteins or to down-regulate the catabolic events induced by pro-inflammatory cytokines. The rate of synthesis of proteoglycans (PGs) by intervetebral disc(IVD) cells increases several-fold following the addition of transforming growth factor-b (TGF-β). IL-1β regulates catabolic metabolism. Our study include all of the below:the results of cells proliferation with TGF-β and IL-10treatment in vitro, the balance between anabolic and catabolic metabolism induced by ADAMTS-4、-5and TIMP-3,and the expression of them in human degenerate nucleus pulposus cells.[Methods]1Tissues from nucleus in IVD of Christian MRI degree type3were obtained in patients(<40-years-old). Cells from NP tissues were expanded in monolayer Cell culture. Cell Proliferation and cell Morphology were observed. Qualification of the nucleus pulposus cells proceeded.,and cell viability was tested using CCK8method.2The proliferation of human nucleus pulposus cells was tested using CCK8method after2days exposure of TGF-β or IL-1β (1ng/ml、10ng/ml).3Cells were induced by TGF-β1or IL-1β in vitro with different dose and time. Analysis of expression of ADAMTS-4、ADAMTS-5、TIMP3mRNA was performed using quantitative real-time polymerase chain reaction (PCR).4Twenty-three formalin-fixed embedded intervertebral disc samples of Christian MRI grade3or4of degeneration were used for immunohistochemical analyses. 5Both tissues from cervical or lumbar disc were test by Western blot method to prove if ADAMTS-4、-5、TIMP-3、TGFβ-1、IL-1β were contained.[Results]1TGF-β lOng/ml stimulates the cell proliferation of human nucleus pulposus cells after2days exposure. IL-1β10ng/ml inhibits the proliferation of human nucleus pulposus cells after2days exposure.2①Expression of TIMP-3mRNA was significantly upregulated.with increasing the dose of TGFβ-1.②TGFβ-1(10ng/ml) induced the expression of ADAMTS-4mRNA.③IL-1β (1ng/ml、10ng/ml) up-regulate the expression of ADAMTS-4mRNA of human nucleus pulposus cells after2days exposure.④IL-1β (0.1ng/ml、1ng/ml、10ng/ml) downregulate the expression of ADAMTS-5mRNA of human nucleus pulposus cells after16h exposure.⑤Expression of TIMP-3mRNA was not upregulated by IL-13.3Immunohistochemical staining showed that ADAMTS-4or ADAMTS-5was mainly localized in extracellular matrix, TIMP-3was mainly localized in nucleolus and cytoplasm, and TGFβ-1was mainly localized in cell membrane.4Western blot method suggested these aggrecanases(ADAMTS-4、-5)、TIMP-3、TGF β-1、IL-1β were also detected in cervical or lumbar disc tissue.[Conclusion] TGF-β10ng/ml stimulates the cell proliferation of human nucleus pulposus cells after2days exposure. IL-1β10ng/ml inhibits the proliferation of human nucleus pulposus cells after2days exposure.We have shown that TGF β-1or IL-1β is produced in the degenerate IVD.,. They are synthesized by native disc cells,and treatment of human disc cells with TGFβ-1or IL-1β induces an imbalance between ADAMTS-4.-5and TIMP-3, responses that represent the changes seen during disc degeneration.