| Background:Degenerative disc disease, represented by cervical spondylosis and lumbar disc herniation, is a serious hazard to human health.Its pathogenes is mul tifactorial and complex. How to delay disc degeneration has become the focus of international research, and also been an important part of "degenerative and aging diseases research" of the "population and health project" of our country. With the arrival of aging society, disc degeneration has been a threat to people’s heal th, and ser ious ly affected the qual ity of life of patients. With the advent of the information age and the widespread use of the computer, the number of patients with degenerative disc disease is rising, and the patients tend to be younger. Therefor, degenerative disc disease has become a hot research topic today.Currently, there are mainly two types of treatment of degenerative disc disease:conservative treatment and surgical treatment. Conservative treatment primarily consist bed rest, analgesics, anti-inflammatory, physical therapy. Generally, after conservative treatment, the patient’s pain and discomfort could be mitigated. However, conservative treatment could only relieve symptoms. If the effect is not obvious, surgical treatment must be carried out. In consideration of its high cost and risk, uncertain long-term effect, surgical treatment is not the ideal treatment either.lntervertebral disc is a complete structural unit, consist of three parts: annulus f ibrosus, nucleus pulposus and cartilage endplate. Due to physiological and pathological factors, intervertebral disc is prone to degenerat. For a long time, intervertebral disc degeneration caused by annulus fibrosus and nucleus pulposus degeneration has been studid a lot, while less research have been done to investigate cartilage endplate degeneration. However, there is some evidence indicate that, intervertebral disc degeneration is caused by cartilage endplate degeneration, and the treatment of endplate cartilage would help the repairment and regeneration of degenerated intervertebral disc.Recent studies have confirmed that the reduced nutrient supply caused by cartilage endplate calcification may be the key factor initiating intervertebral disc degeneration. When disc aging or nutrient supply obstacles happen, disc cells would synthesis some cytokines, which affect cell activity and the information exchange of cells, leading to apoptosis. With the environmental changed, the intradiscal degrading enzymes is activated from the latent state, which accelerate the decomposition of the intervertebral disc matrix, resulting in a push of intervertebral disc degeneration.The ideal solution of spine degenerative disorders is to delay and prevent the degenerat ion of the intervertebral disc t issue, and promote its regenerat ion. This is a hot point in intervertebral disc degeneration research field. Deeply understanding the changes of appreciation rate of cells in intervertebral disc tissue through degeneration have important implications for the prevention, diagnosis and treatment of disc degenerative disease.Objective:The objective of this experiment is to investigate the effect of1igustrazine on endplate cartilage cells cul tured in the three-dimens ional cell culture microcarriers in vitro, via observeing cell morphology, detecting cell count, measuring MTT (OD value), and drawing cell growth curve, supply the basis for the subsequent animal experiments (reveal the effect of ligustrazine on the generation of matrix and changes in gene expression in disc degeneration process under the hydrostatic pressure intervention), so as to provide a new model to further prevent and delay disc degeneration in Western medicine treatment.Method:Accroding to different ligustrazine intervention, the cells are divided into medium control group, ligustrazine groups of0.01mg/L,0.02mg/L,0.04mg/L,0.08mg/L,0.16mg/L. The effect of1igustrazine on endplate chondrocytes is observed in3-D cell cultrue carrier. Endplate chondrocytes morphology is observed by inverted microscope after24h and48h,cell survival rate is detected by trypan blue staining, cell proliferation effect is observed by MTT. All data are expressed with x±s, using SPSS13.0software for analysis of variance.Results:①Endplate cartilage cell morphology in3D cytoskeleton are Observed by inverted Microscope.Recovery of endplate cartilage cell adhered to the wall normal, the cells are triangular, polygonal, spindle, etc after3-5days, which are colony-like growth, contacted inhibit ion, nuclear round, located in the side of the cell body.0. Olmg/ml,0.02mg/ml,0.04mg/ml,0.08mg/ml,0.16mg/ml of ligustrazine groups can promote different levels of endplate cartilage cell proliferation, differentiation, compared with the control group, the number of endplate cartilage cell is largest in0.02mg/ml and0.04mg/ml dose group, cells were colony-like growth, extended pseudopodia increased cell multi-spindle. As the time of ligustrazine is extended, the promotion of cell proliferation is decreased. The effect of0.08mg/ml and0.16mg/ml of ligustrazine groups on cell proliferation is not obvious.②The rate of endplate cartilage cell survival≥90%③0.04mg/L dose endplate cartilage cell group after cultured24h, comparing with the OD value of blank control group and the groups is significantly different, statistically significant (P<0.01); OD value is measured to compare each dose group after48h cultured and blank control group is no significant difference.Conclusion:Ligustrazine can promote proliferation of endplate cartilage cells in the3D cytoskeleton in time and dose-dependent. It is suggests that it may have a direct promotion of endplate cartilage cells proliferation. The impact of ligustrazine on endplate cartilage cells in vitro three-dimensional cell culture suggest that3D cytoskeleton have good biocompatibility, and provides a theoretical basis for composite applications of the follow-up animal experiments research and provide a new model to further prevent and delay disc degeneration in Western medicine treatment. |
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