髓核中p16^(INK4a)的表达及其对椎间盘退变的影响

EXPRESSION OF p16^(INK4a) IN NUCLEUS PULPOSUS AND ITS EFFECT ON DEGENERATED INTERVERTEBRAL DISCS

目的探讨同一年龄段、不同程度腰椎间盘退变患者髓核中p16INK4a的表达,明确其与椎间盘退变的关系,为退变椎间盘的生物学修复提供依据。方法收集腰椎间盘退变患者退变髓核标本17例,年龄40~50岁;退变按Pfi rrmann分级为Ⅲ级10例、Ⅳ级7例。检测衰老相关-β-半乳糖苷酶(senescence-associatedβ-galactosidase,SA-β-gal)活性,评价细胞衰老情况;免疫组织化学染色和Western blot检测聚集蛋白聚糖(Aggrecan)、含血小板凝血酶敏感蛋白的解聚素与金属蛋白酶5(A disintegrin and metalloproteinase with thrombospondin motifs 5,ADAMTS 5)、Sry相关的HMG盒转录因子9(Sry-related HMG box transcription factor 9,Sox-9)、p16INK4a蛋白表达。采用Bivariate过程分析ADAMTS 5和p16INK4a蛋白表达的相关性。结果Ⅲ、Ⅳ级髓核组织内均可见到绿染的SA-β-gal阳性髓核细胞(nucleus pulposus cells,NPCs),多呈簇状分布,其中Ⅲ级中也可见少量单个分布的绿染NPCs,呈圆形,其外周由多层细胞外基质包裹,而Ⅳ级中难以见到单个分布的绿染NPCs;Ⅲ、Ⅳ级髓核组织阳性细胞百分比分别为22.7%±5.4%和37.1%±7.6%,差异有统计学意义(t=-9.666,P=0.000)。免疫组织化学染色和Western blot检测示,Ⅳ级髓核组织中p16INK4a、ADAMTS5染色阳性细胞百分比和蛋白相对表达量明显多于Ⅲ级,而Aggrecan、Sox-9明显少于Ⅲ级,差异均有统计学意义(P<0.05)。退变髓核组织中ADAMTS 5与p16INK4a的蛋白表达成线性相关(r=0.908,P=0.000)。结论过早衰老NPCs的累积与椎间盘退变程度密切相关,p16INK4a参与的NPCs过早衰老对椎间盘退变进程起着主导作用。

Objective To investigate the expression of p16^INK4a in nucleus pulposus（NP） and to clarify its relationship with intervertebral disc degeneration so as to provide evidence for biological repair of intervertebral disc.Methods The NP specimens were obtained from 17 patients with intervertebral disc degeneration undergoing discectomy,who aged 40-50 years（mean,45.4 years）.Based on the preoperative MRI,there were 10 cases of grade Ⅲ degeneration,and 7 cases of grade Ⅳ degeneration.Cell senescence was evaluated by detecting senescence-associated β-galactosidase（SA-β-gal） activity.Senescence marker（p16^INK4a） and disc degeneration markers[A disintegrin and metalloproteinase with thrombospondin motifs 5（ADAMTS 5）,Aggrecan,and Sryrelated HMG box transcription factor 9（Sox-9）]were determined in the NP specimens with immunohistochemistry and Western blot.The correlation between ADAMTS 5 and p16^INK4a was analyzed.Results Clustered distribution of green SA-p-gal-positive cells was seen in the NP with grade Ⅲ and Ⅳ degeneration.A few single round SA-β-gal-positive NP cells（NPCs） wrapped by the layered extracellular matrix were also seen in the NP with grade Ⅲ degeneration.It was difficult to see single distribution of NPCs in the NP with grade Ⅳ degeneration.The percentage of SA-β-gal-positive cells was 22.7%± 5.4% and 37.1%± 7.6%in the NP with grade Ⅲ and Ⅳ degeneration respectively,showing significant difference（t=9.666,P=0.000）.The percentages of p16^INK4a-positive and ADAMTS 5-positive NPCs in the NP with grade Ⅳ degeneration were significantly higher than those with grade Ⅲdegeneration（P〈0.05）.The percentages of Aggrecan-positive and Sox-9-positive NPCs in the NP with grade Ⅳ degeneration were significantly lower than those in the NP with grade Ⅲ degeneration（P〈0.05）.The protein expressions of Aggrecan and Sox-9 in the NP with grade Ⅳ degeneration were significantly lower than those in the NP with grade Ⅲ degeneration（P〈0.05）.The NP with grade Ⅳ degeneration showed significantly higher protein expressions of p16^INK4a and ADAMTS 5（P〈0.05）.Importantly,there was a good correlation between p16^INK4a and ADAMTS 5 protein expressions（r=0.908,P=0.000）.Conclusion Premature senescent NPCs increase in the NP with the advancing disc degeneration.The expression of p16^INK4a and its association with degeneration grades suggest that the p16^INK4a may play a significant role in the pathogenesis of intervertebral disc degeneration.