褪黑素受体在不同程度退变腰椎髓核组织中的表达

Expression of melatonin receptors in lumbar nucleus pulposus with degeneration of different grades

中文关键词:  褪黑素受体  核转录因子-κB  退行性变  腰椎  髓核组织

英文关键词:Melatonin receptors  Nuclear transcription factor -κB  Degeneration  Lumbar  Nucleus pulposus

中文摘要:

  【摘要】 目的：研究褪黑素受体褪黑素1型受体（MT1）、褪黑素2型受体（MT2）以及核转录因子-κB（NF-κB）在不同程度退变腰椎髓核组织中的表达。方法：选取2014年3月~2016年1月于我院住院患者31例（19例男性和12例女性，年龄47~62岁，平均56.9±4.9岁）。采用MRI对髓核退变程度进行Pfirrmann分级并经手术收集髓核标本，其中Ⅳ级标本11例纳入退变Ⅰ组；V级标本12例纳入退变Ⅱ组；8例分级小于Ⅲ级标本纳入对照组。实时定量PCR检测髓核组织中MT1、MT2、NF-κB mRNA的表达，免疫印迹和免疫组织化学技术检测MT1、MT2、p65 NF-κB和磷酸化p65（p-p65）NF-κB蛋白的表达，利用Spearman相关分析退变髓核组织中MT1、MT2、NF-κB蛋白表达的关系。结果：实时定量PCR结果显示对照组MT1和MT2 mRNA分别为1.00±0.04和1.00±0.04，退变Ⅰ组MT1和MT2 mRNA分别为0.68±0.06和0.77±0.04，退变Ⅱ组MT1和MT2 mRNA分别为0.52±0.03和0.68±0.06，各组间均存在统计学差异（P<0.05）。各组NF-κB mRNA分别为1.00±0.03、0.99±0.07、0.98±0.05，无统计学差异（P＞0.05）。免疫印迹结果显示对照组MT1和MT2蛋白表达分别为1.00±0.05和1.00±0.05，退变Ⅰ组为0.73±0.07和0.56±0.04，退变Ⅱ组别为0.62±0.05和0.55±0.04，各组间差异有统计学意义（P<0.05）。三组p65 NF-κB表达水平分别为1.00±0.05、1.00±0.06、0.99±0.04，无统计学差异（P=0.920）。对照组p-p65 NF-κB蛋白为1.00±0.07，退变Ⅰ组和退变Ⅱ组p-p65 NF-κB蛋白水平分别为1.43±0.08和1.49±0.08，各组间差异有统计学意义（P<0.05）。免疫组织化学技术检测结果显示退变髓核组织MT1和MT2蛋白与p-p65蛋白表达具有负相关关系（P<0.05），相关系数r分别为-0.6492和-0.6190。结论：髓核组织退变程度增加，褪黑素受体表达降低，而NF-κB活化增加，进而参与椎间盘组织退变过程。

英文摘要:

  【Abstract】 Objectives: To investigate the expression of melatonin receptor type 1(MT1), melatonin receptor type 2(MT2) and nuclear transcription factor-κB(NF-κB) in lumbar nucleus pulposus with degeneration of different grades. Methods: Thirty-one patients enrolled in our hospital from March 2014 to Janurary 2016 were selected(19 males and 12 females, age 47-62, mean age 56.9±4.9). Magnetic resonance imaging was used to identify the grade of intervertebral disc degeneration with Pfirrmann grading system. Then the nucleus pulposus samples were collected during surgery. 11 samples of grade Ⅳ were allocated into degeneration Ⅰ group; 12 samples of grade V into degeneration Ⅱ group; 8 samples lower than grade Ⅲ into control group. MRNA expressions of MT1, MT2, p65 NF-κB and phosphorylated p65(p-p65) NF-κB were examined by real-time quantitative PCR. Protein expressions of MT1, MT2, p65 NF-κB were also evaluated by immunoblot and immunohistochemistry. Correlation between MT1, MT2 and NF-κB was analyzed by using Spearman method. Results: The results of qRT-PCR showed that MT1 and MT2 mRNA expression in control group were 1.00±0.04 and 1.00±0.04, respectively, while the expressions of MT1 mRNA in degeneration I and degeneration Ⅱ group were 0.68±0.06 and 0.52±0.03. MT2 mRNA expressions were 0.77±0.04 and 0.68±0.06. The difference among these three groups was significant(P<0.05). NF-κB mRNA expressions in three groups were 1.00±0.03, 0.99±0.07 and 0.98±0.05, with insignificant difference(P＞0.05). Western blot results showed that MT1 and MT2 protein levels in control group were 1.00±0.05 and 1.00±0.05, while MT1 protein levels in degeneration I and degeneration Ⅱ group were 0.73±0.07 and 0.56±0.04. MT2 protein levels in degeneration I and degeneration Ⅱ group were 0.62±0.05 and 0.55±0.04. The difference among these three groups was significant(P<0.05). P65 protein levels in three groups were 1.00±0.05, 1.00±0.06 and 0.99±0.04, with insignificant difference(P=0.920). Phosphorylated p65 (p-p65) protein level in control group was 1.00±0.07, while those in degeneration I and degeneration Ⅱ group were 1.43±0.08 and 1.49±0.08, the difference among these three groups was also significant(P<0.05). The results of immunohistochemistry showed that MT1 and MT2 in degenerated nucleus pulposus were inversely correlated with the expression of p-p65 protein (P<0.05). The correlation coefficients were -0.6492 and -0.6190. Conclusions: With the increase of degeneration grade in human nucleus pulposus, the expression of melatonin receptors is suppressed and the activity of NF-κB is promoted.