1. Department of Orthopedics，The First Affiliated Hospital of Chongqing Medical University； 2. Department of Orthopedics，Jiangxi Provincial People’s Hospital

Abstract:

Objective：To investigate the expression of leukemia inhibit factor（LIF） in degenerative disc nucleus pulposus and its role in degenerative nucleus pulposus extracellular matrix synthesis. Methods：Forty adult New Zealand white rabbits were randomly divid－ed into 5 groups（1 group as the control group，the remaining 4 groups as the experimental group），8 rabbits in each group. Interverte－bral disc degeneration model was established by puncturing the L4/5 and L5/6 intervertebral discs in the experimental group. MRI scoring was performed on the discs at 0，2，4，8 weeks immediately after the operation，and the discs were obtained and stained with hematoxylin and eosin to get histological scores and to determine the degree of degeneration. Immuno-histochemical staining was used to semi-quantitatively detect the expression of LIF in nucleus pulposus. Rabbit nucleus pulposus cells were harvested for primary culture；the expressions of Aggrecan，COLLA2α1 in human in－tervertebral disc nucleus pulposus cells were detected by Western blot at 24，48，72 h after treated with gradient concentration hu－man recombinant LIF protein. In maximal concentration group and control group，Aggrecan expression was again verified by immunofluorescence and the expressions of tissue inhibitor of metalloproteinases-1（TIMP-1） and matrixmetalloproteinase-3（MMP-3） were detected by Western blot. Results：The imaging scores and histological scores showed that the degeneration of intervertebral disc increased with the prolongation of modeling time，and there was a significant difference（1.000±0.000，2.000±0.535，2.750±0.463，3.875±0.354，P=0.000；4.000±0.000，7.375±0.518，9.375±1.302，11.750±0.463，P=0.000）. The LIF expression in puncture 2-week group was significantly higher，as disc degen－eration aggravated，the expression decreased gradually，and the expression level of each experimental group was higher than that of the 0 week group，the difference was statistically significant（559.608±68.689，16 135.613±577.329，8 149.739±457.189，2 018.254±211.870，P=0.000）. Cytology experiments showed that after stimulation of different LIF concentrations，Aggrecan，COLLA2α1 protein expression and LIF stimulus concentration was positively correlated，and there was a significant difference between each concentration group and the control group（0.191±0.020，0.212±0.020，0.321±0.041，0.511±0.032，0.561±0.042，P=0.000），and Aggrecan immunofluorescence results were consistent. TIMP-1 expression in the treatment group was significantly higher than that of control group（0.454±0.022，0.211±0.012，P=0.000）；MMP-3 expression in the treatment group was significantly lower than that in the control group（0.243±0.013，0.362±0.021，P=0.000）. Conclusion：LIF expression in degenerative nucleus pulposus is increased，and LIF can promote the synthesis of extracellular matrix，and its underlying mechanism is related to up-regulating TIMP-1 and down-regulating MMP-3.