兔髓核组织中BNIP3表达与椎间盘退变的关系

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。

Preliminary research for correlation between BNIP3 expression and disc degeneration

Objective:To study the correlation between BNIP3 expression and disc degeneration.Method:Twenty-four one-month white New Zealand rabbits were randomly and equally assigned into four groups:post-surgery for 0 week(n=6),post-surgery for 2 weeks(n=6),post-surgery for 4 weeks(n=6),and post-surgery for 8 weeks(n=6).By puncturing the experimental disc annulus(L4/5,L5/6 and L6/7) with needle,we established a disc degeneration model in New Zealand white rabbit,the normal discs above the experimental discs(L1/2,L2/3 and L3/4) served as control.After the surgery for 0,2,4 and 8 weeks,the disc degeneration,BNIP3 expression and cell morphology were evaluated by using MRI,histochemistry staining,immunohistochemical staining and real-time-PCR,and the correlationship between the BNIP3 expression and the disc degeneration evaluated by MRI and histochemistry was also been analyzed.Result:The MRI showed that the normal discs and discs of post surgery for 0 week exhibited hyperintense in T2 weighting image with the average MRI grade of 1.0±0.0;the discs of post surgery for 2 weeks showed inhomogeneous hyperintense with the average MRI grade of 2.9±0.4;the discs of post surgery for 4 weeks showed intermediate to hypointense with the average MRI grade of 4.2±0.3;the discs of post surgery for 8 weeks showed a hypointense which the average MRI grade of 4.9±0.1.There were significant differences among normal discs and post surgery discs(P<0.05).The histochemistry staining also showed the normal discs and discs of post surgery for 0 week with intact structure and clear border between the annulus fibrosus and nucleus pulposus,the average histological grade was 4.0±0.0;the discs of post surgery for 2 weeks showed slight rupture in annulus fibrosus and minimally interrupted between annulus fibrosus and nucleus pulposus,the average histological grade was 7.5±0.2;the discs of post surgery for 4 weeks showed larger rupture in annulus fibrosus and moderate rupture between annulus fibrosus and nucleus pulposus,the average histological grade was 10.0±1.0;the discs of post surgery for 8 weeks showed the collapse of disc structure and fibrosis of nucleus pulposus,the average histological grade was 11.8±0.2.There were significant differences among normal discs and post surgery discs(P<0.05).The BNIP3 mRNA real-time-PCR of the nucleus pulposus region indicated increased expression of BNIP3 with 1.0±0.3-fold,2.0±0.1-fold,2.8±0.3-fold and 4.2±0.2-fold respectively at 0,2,4 and 8 weeks after surgery,and there was a high correlation between MRI grade and BNIP3 mRNA expression(r=0.92,P<0.01).The BNIP3 immunohistochemical staining of the nucleus pulposus region suggested increased positive reaction with 150.4±13.4,176.0±35.1 and 173.6±7.9 of gray intensity at 2,4 and 8 weeks after surgery,which had a high correlation with histological grade(r=0.92,P<0.01).Conclusion:The expression of BNIP3 mRNA has some relationship with the disc degeneration,and the increased expression of BNIP3 may play an important role in disc degeneration.