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目的:探讨兔椎间盘髓核组织中BNIP3表达量与椎间盘退变的相关性。方法:健康1月龄新西兰白兔24只,随机分为4组,每组6只。在每只动物的L4/5、L5/6和L6/7椎间盘进行纤维环穿刺术,建立椎间盘退变模型,作为实验椎间盘;穿刺椎间盘近端3个椎间盘(L1/2、L2/3、L3/4)作为正常对照椎间盘。分别于术后即刻、2、4、8周对椎间盘进行MRI及组织学评分,应用Real-time PCR定量检测髓核组织BNIP3 mRNA表达,免疫组织化学染色半定量髓核组织BNIP3表达,同时分析同一椎间盘BNIP3表达与MRI评分、组织学评分之间的相关性,并与正常椎间盘进行对照。结果:正常及手术后即刻实验椎间盘在MRI T2加权像上呈高密度,评分为1.0±0.0;手术后2周实验椎间盘信号呈不均一的高密度,评分为2.9±0.4;手术后4周实验椎间盘信号呈不均一中低密度,评分4.2±0.3;手术后8周实验椎间盘信号呈低密度,评分4.9±0.1,各时间点MRI评分比较有显著性差异(P<0.05)。组织学染色显示正常及手术后即刻椎间盘结构整齐,髓核与纤维环交界清晰,组织学评分4.0±0.0;手术后2周实验椎间盘纤维环出现小裂隙,髓核与纤维环交界轻度不清,组织学评分7.5±0.2;手术后4周实验椎间盘纤维环出现较大裂隙,髓核与纤维环交界中度不清,组织学评分10.0±1.0;手术后8周实验椎间盘正常结构丧失,髓核组织纤维化,组织学评分11.8±0.2,各时间点间组织学评分比较有显著性差异(P<0.05)。手术后即刻、2周、4周及8周实验椎间盘BNIP3 mRNA表达是正常组的1.0±0.3倍、2.0±0.1倍、2.8±0.3倍和4.2±0.2倍,与椎间盘MRI退变评分呈显著性正相关(r=0.92,P<0.01)。正常及手术后即刻实验椎间盘髓核组织BNIP3灰度值分别为55.3±6.2和60.7±4.4;而手术后2周、4周及8周实验椎间盘分别为150.4±13.4、176.0±35.1和173.6±7.9,其灰度值与椎间盘组织学评分呈显著性正相关(r=0.92,P<0.01)。结论:兔椎间盘髓核组织中BNIP3 mRNA表达量与椎间盘退变相关,BNIP3表达上调可能在椎间盘退变过程中发挥了重要作用。
Objective: To investigate the correlation between the expression of BNIP3 and the degeneration of intervertebral disc in rabbit nucleus pulposus. Methods: Twenty-four healthy 1-month-old New Zealand white rabbits were randomly divided into 4 groups with 6 in each. The lumbar intervertebral disc of L4 / 5, L5 / 6 and L6 / 7 was perfused with pericardial puncture in each animal to establish a model of disc degeneration as an experimental disc. Three discs (L1 / 2, L2 / 3, L3 / 4) as a normal control disc. The MRI and histological scores of the intervertebral discs were evaluated immediately after the operation. The expression of BNIP3 mRNA in the nucleus pulposus was detected by Real-time PCR. The expression of BNIP3 in the semi-quantitative nucleus pulposus was detected by immunohistochemical staining. Intervertebral disc BNIP3 expression was correlated with MRI score and histological score, and was compared with normal disc. Results: The normal and postoperative immediately after operation showed high density on the MRI T2 weighted image, with a score of 1.0 ± 0.0. The two weeks after operation, the experimental disc showed a nonuniform high density with a score of 2.9 ± 0.4. After 4 weeks of operation The intervertebral disc signal was inhomogeneous medium and low density with a score of 4.2 ± 0.3. At 8 weeks after operation, the intervertebral disc signal showed a low density with a score of 4.9 ± 0.1. MRI scores at each time point showed significant difference (P <0.05). Histological staining showed that the structure of the intervertebral disc was normal and the junction of the nucleus pulposus and annulus fibrosus was clear. The histological score was 4.0 ± 0.0. Small annulus fibrosus of experimental intervertebral disc appeared 2 weeks after operation, and the junction of nucleus pulposus and annulus fibrosus was slightly unclear , Histological score 7.5 ± 0.2; 4 weeks after operation, there was a large fissure in the annulus fibrosus of the experimental intervertebral disc, the junction between nucleus pulposus and annulus fibrosus was unclear, histological score was 10.0 ± 1.0; 8 weeks after operation, The histological score of nuclear tissue was 11.8 ± 0.2. There was significant difference in histological score between different time points (P <0.05). The expression of BNIP3 mRNA in the experimental disc immediately after operation was 1.0 ± 0.3 times, 2.0 ± 0.1 times, 2.8 ± 0.3 times and 4.2 ± 0.2 times higher than those in the normal group, which were significantly different from those of the MRI disc degeneration Positive correlation (r = 0.92, P <0.01). The gray values of BNIP3 in the nucleus pulposus were 55.3 ± 6.2 and 60.7 ± 4.4, respectively, in the normal and postoperative immediately after operation. The experimental discs were 150.4 ± 13.4, 176.0 ± 35.1 and 173.6 ± 7.9 respectively at 2, 4 and 8 weeks after operation There was a significant positive correlation between the gray value and the histological score of intervertebral disc (r = 0.92, P <0.01). CONCLUSION: The expression of BNIP3 mRNA in the nucleus pulposus of rabbit is related to the degeneration of intervertebral disc. The up-regulation of BNIP3 may play an important role in the process of degeneration of intervertebral disc.