Function And Mechanism Study Of Ossification Related TWIST In Intervertebral Disc Degeneration

Background and objectiveSince the publication of SPINE,the authoritative magazine of spine surgery in 1996,on low back pain as a century problem for human beings,low back pain has attracted more and more attention.Chronic back pain and neck and shoulder pain caused by disc degeneration is a serious problem that threatens human health.There is currently no effective treatment.Chronic low back pain is a common cause of labor loss.For patients with severe degenerative intervertebral disc disease,surgical treatment is currently the main method of treatment: lumbar interbody fusion,artificial intervertebral disc replacement,minimally invasive nucleotomy,etc.Surgical treatment can relieve the patient’s symptoms to a certain extent,but the surgery incorporates many complications,such as adjacent segment disease,intervertebral space infection,etc.,which often require further surgery.Although surgery can reduce clinical symptoms,it neither prevents the progression of degeneration nor restores the natural functional status of IVD.Based on the above conditions and limitations of surgical interference,therefore,research on the mechanism of degenerative disc degeneration and the search for non-invasive treatments have not stopped.Intervertebral disc degeneration is a complicated multi-factorial process.Although a lot of long-term research work has been done,the exact mechanism is not fully understood.The current study focuses on inflammatory pathways.A large number of studies have shown that degenerated intervertebral disc cells can produce inflammatory mediators,including TNF-α,IL-1α/β,IL-6,IL-10,IFN-γ and so on.These factors cause the inflammatory cascade to promote matrix degradation,chemokine production,and changes in cell phenotypes.This resulted in a serious imbalance between the decomposition and synthesis of the matrix components that eventually led to disc degeneration.Inflammatory factors on the role of intervertebral disc does not exist disease-specific,with limitations,such as poor conversion efficiency.The intervertebral disc is the largest avascular tissue in the human body.The intervertebral disc consists of two parts: the inner nucleus pulposus which is rich in water and collagen,and the outer fiber ring which is rich in fiber.Degeneration of the intervertebral discs is characterized by a significantly lower content of type II collagen and proteoglycans in the extracellular matrix,which directly leads to decreased water-binding capacity,reduced water content in the intervertebral disc,reduced number of nucleus pulposus cells,and nucleus pulposus cells are replaced by the fibroblast-like cells.Disc as a special cartilage tissue,the degeneration process may be associated with ossification-related genes are closely linked.The research group has found in previous studies reported that ossification-related gene RUNX2 can cause disc degeneration.TWIST as an important member of ossification-related genes involved in the early inhibition of RUNX2 regulation of osteoblastic.Based on the above,we speculate that the TWIST gene plays a crucial role in the process of disc degeneration.Part Ⅰ: TWIST gene expression in nucleus pulposus Objective: To study the expression of TWIST gene in human nucleus pulposus tissue.Methods:(1)The ossification related genes in disc degeneration were screened by GEO database and bioinformatics.(2)Collecting intervertebral disc specimens of patients with lumbar disc herniation from March 2016 to June 2017 in our hospital.According to the magnetic resonance Pfirrmann grading system,grade I-II was mild degenerative control group,Group III-V grade severe degeneration degeneration group.Totally 32 patients were collected,including 12 cases in control group and 20 cases in degenerative group.The collected disc tissue was identified.(3)Immunohistochemistry of the ossification-related genes of intervertebral disc tissue cells against screening.(4)RNA extraction and verification of intervertebral disc tissue,through real-time quantitative PCR detection method to determine the expression of ossification gene in disc degeneration.Results:(1)The ossification-related IBSP,ENPP,OPN and so on exist in the disc degeneration network,suggesting that ossification-related genes may play an important role in the degeneration of intervertebral disc.(2)The histological identification,disc degeneration and Pfirrmann grade consistent,grouping reasonable.(3)Overexpression of osteogenesis-related genes showed up-regulation by histochemical staining,whereas TWIST1 and TWIST2 in ostasis-related genes decreased gradually with degeneration.(4)The results of RNA detection through the disc nucleus showed that TWIST1 and TWIST2 did appear to be significantly down-regulated in the degeneration as shown by the histochemistry.Conclusion: We use GEO database,immunohistochemistry of intervertebral disc specimens,RNA extraction and verification of intervertebral disc tissue to detect the ossification gene in degenerative intervertebral disc,to clarify the abnormal expression of ossification related gene in degenerative intervertebral disc,TWIST1 and TWIST2 in degenerative intervertebral disc In significantly reduced.Part Ⅱ: The role of TWIST in the degeneration of human nucleus pulposus cells Objective: To study the expression of TWIST gene in human nucleus pulposus cells.Methods:(1)Primary human nucleus pulposus cell culture was collected from May 2016 to July 2017 for Pfirrmann class I-II disc tissue in patients with spinal cord injury or scoliosis.(2)The TWIST expression CDS region was purified by TWIST1 and 2 full-length PCR,cloned into the expression vector,and TWIST1 and 2 genes were over-expressed in nucleus pulposus cells using TWIST1 and TWIST2 to construct TWIST overexpression and interference vector.(3)Inflammatory stimulus of in vitro nucleus pulposus cells with inflammatory cytokines IL-1b and TNF-α was used to establish a degenerative model of in vitro nucleus pulposus cells.(4)Through the transfection of TWIST1/TWIST2 overexpression and interference vector in nucleus pulposus cells,after 24 h of culture,the expression of MMP and ADAMTS in nucleus pulposus cells were detected by Real-time PCR.(5)The expression of ECM-related enzymes such as CHSY,MMP,ADAMTS in nucleus pulposus cells detected by Western Blot after being treated as above.Results:(1)Through the primary culture of nucleus pulposus,6 in vitro nucleus pulposus cell lines were successfully established.(2)The detection of TWIST1 and 2 expression in cells did indeed appear up-regulated,suggesting that over-expression and interference vectors were successfully constructed.(3)The expression of matrix related genes such as CHSY,ACAN and COL2A1 in nucleus pulposus cells decreased after stimulation,and the expression of MMP,ADAMTS related to matrix degradation was significantly increased.(4)Overexpression of TWIST1 and 2 genes,nucleus pulposus cells CHSY,ACAN,COL2A1 and other matrix-related gene expression was significantly increased compared with the control group,matrix degradation associated MMP,ADAMTS expression was significantly reduced.Conclusions: We constructed TWIST overexpression vector,constructed in vitro degenerate model and transfected overexpression vector through primary culture of nucleus pulposus cells.It was clarified that TWIST gene could indeed inhibit the degeneration of intervertebral disc and protect the intervertebral disc in vitro.Part Ⅲ: The mechanism of TWIST gene in protection intervertebral disc degeneration Objective: To study the mechanism of TWIST gene protection in disc degeneration.Methods:(1)Construction of RUNX2 overexpression and interference vector.(2)Overexpression of RUNX2 gene in human nucleus pulposus cells and detection of ECM-related enzymes such as CHSY,MMP,ADAMTS.(3)Co-transfection of TWIST and RUNX2 co-expression in the degenerative nucleus,the detection of ECM-related CHSY,MMP,ADAMTS and other enzymes.(4)Construction of RUNX2 promoter double fluorescent reporter vector to detect RUNX2 promoter activity after TWIST over-expression.Results:(1)RUNX2 overexpression and interference vector were successfully constructed.(2)Genes such as ACAN and COL2A1 related to nucleus pulposus synthesis after RUNX2 overexpression showed significant downregulation,suggesting that they have the role of promoting degeneration.(3)Co-transfection of TWIST and RUNX2 in degenerating nucleus pulposus resulted in a certain degree of alleviation of the pro-degeneration effect induced by RUNX2 gene.(4)Using dual luciferase reporter assay to detect the effect of TWIST on RUNX2 downstream function,it was found that TWIST can significantly inhibit RUNX2 activity.CONCLUSION: The purpose of this study is to further clarify the degeneration of intervertebral disc caused by TWIST by constructing RUNX2 overexpression and interference vectors,co-transfection of TWIST and RUNX2,dual luciferase reporter gene detection and so on.The protection of intervertebral disc is mainly achieved by inhibiting the activity of RUNX2.