| Objective: To establish a simple and effective, repeatable animal model of Intervertebral disc degeneration, and to have an Intervention study on the animal model of the intervertebral disc degeneration using receptor tyrosine enzyme inhibitors(Genistein) to study the therapeutic value of Genistein in the treatment of degenerative intervertebral disc disease.Intervention: The first part: 24 three-months-old clean level male SD rats, randomly divided into 4 groups in digital table method: a blank group and 3 experimental groups with 6 rats in each group. Positioning Co7/8, Co8/9 and Co9/10 intervertebral space and puncture with 16 G, 18 G and 26 G hypodermic needle respectively to establish intervertebral disc degeneration model through annulus puncture. In the experimental group, 6 rats were randomly selected at 1 week, 2 week and 4 week respectively after operation to perform magnetic resonance imaging of T2 images, calculated Thompson score according to the intervertebral disc signal. Rats were killed after the inspection and Co7/8, Co8/9 and Co9/10 intervertebral discs were taken for HE staining and immunohistochemical staining for type II collagen. Calculate the histological scores according to HE staining and observe nucleus pulposus expression of collagen type Ⅱ trend. The second part: 30 three-month-old clean level male SD rats, randomly divided into 5 groups in digital table method: a Blank group, a normal group and 3 experimental groups with 6 rats in each group. Blank group for Modeling, and not Genistein injection; after modeling the experimental group was divided into 3 groups according to the dose of Genistein(5mg/ml, 10mg/ml and 20mg/ml). MRI scanning, HE staining and immunohistochemical staining of type II collagen were the same as the first part.Results: The first part: Co7/8 disc signal declined in the week after the puncture, and Co8/9, Co9/10 signal presented as high signal. Two weeks later, the Co7/8 intervertebral disc signal further reduced, showing a "disc Dark", and the intervertebral space becomes smaller, Co8/9 intervertebral disc signal began to decrease, showing the degeneration performance. During the observation period Co9/10 always showed high signal, compared the puncture a week after the Co7/8 with the normal intervertebral disc, the difference of the Thompson scores was statistically significant(P < 0.05); two weeks after the puncture, 18 G puncture group Thompson scores decrease, and the difference was statistically significant, the signal of Co9/10 intervertebral disc after four weeks of puncture slightly decreased, but compared with the normal group, there was no statistical difference(P > 0.05); after one week of puncture, the nucleus pulposus of Co7/8 intervertebral disc was reduced, and even disappeared, Annulus structural disorder, some were crushed shape and no clear boundary between annulus fibrosus and nucleus pulposus, volume of nucleus pulposus with narrow puncture time. At four weeks, the nucleus pulposus was replaced by fibrous tissue, which showed the expression of fibrosis, when Co8/9 intervertebral disc in the puncture for two weeks, the nucleus began to shrink, annulus twisted, showing degeneration performance; at four weeks, the nucleus pulposus cells were further reduced, and the annulus was disordered, during the modeling observation period of Co9/10 intervertebral disc, there was a small amount of nucleus pulposus cells decreased and the annulus was slightly damaged during the four week, according to the histological grading standard, the difference of the histological score of Co7/8 intervertebral disc a week after the model compared with the normal intervertebral disc was statistically significant(P < 0.05), and the score increased gradually with time; there was a statistical difference in the score of Co8/9 intervertebral disc in the model two weeks; there was no statistical difference in histological grading of Co9/10 intervertebral disc(P > 0.05); after one week of puncture, the Co7/8 intervertebral disc was stained lighter with the color of type II collagen, and the positive staining decreased gradually with time increasing, The positive staining of type II collagen in the Co8/9 intervertebral disc began to fade in the two week time, and the positive staining area became smaller, There was no significant difference in the immunohistochemistry of type II collagen in Co9/10 intervertebral disc compared with the normal intervertebral disc, yellow or yellow brown staining was widely seen in the nucleus pulposus. The second part: one week after modeling the intervention group compared with the blank group, the disc signals have different degrees of improvement,The signal intensity of the intervertebral disc after drug intervention increased with the increase of the drug dose, but with the increase of time, the intervertebral disc signal gradually decreased, at a week of drug intervention,compared the Thompson score of the 5mg / ml dose group of intervertebral disc with the blank group, the difference was not statistically significant(P > 0.05), the difference of 10 mg / ml dose injection group and 20 mg / ml dose injection group has statistical significance(P < 0.05), 20 mg / ml dose injection group compared with the blank group, the difference was still statistically significant(P <0.05) in the four week drug intervention; compared with the blank group, there was no statistical significance(P > 0.05) at each time point compared with the blank group in the 5mg/ml dose group, the histological scores of 10 mg / ml dose injection group and 20 mg / ml dose injection group were statistically significant(P <0.05) in the intervention of one week, however, when drug intervention four weeks, There was no significant difference between the 10mg/ml dose group and the blank group, 20 mg / ml dose injection group compared with the blank group, the difference was still statistically significant(P <0.05); after one week intervention of 5mg/ml dose injection group, the collagen type II collagen was stained with light yellow, with the time increasing, the color gradually became pale, there was no positive staining in the nucleus of the nucleus in intervention of four weeks; after the intervention of one week, nucleus pulposus of 10 mg / ml dose injection group and 20 mg / ml dose injection group increase in positive staining region, and exhibit yellow or brown yellow. At two weeks of intervention, the positive staining of 10 mg / ml dose injection group began to fade. In the 20mg/ml dose group, only the positive staining area was slightly smaller when at four weeks in the event of intervention.Conclusions: 1. Acupuncture Annulus modeling method is convenient and feasible method of Intervertebral disc degeneration model, and 18 G needle is best suited for modeling, 26 G needle is suitable for injecting drug intervertebral. 2.Genistein can retard Intervertebral disc degeneration in rats, but can not prevent the occurrence of degeneration.3.The efficacy of Genistein was related to the drug concentration and the intervention time, the greater the drug concentration is, the more obvious curative effect will be. 4. There is a certain application value of Genistein in the prevention of intervertebral disc degeneration. |
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