针刀干预对颈椎间盘退变兔整合素α5、β1表达的影响

目的观察针刀干预对颈椎间盘退变兔颈部肌肉形态学以及整合素α5、β1含量的影响,探讨针刀延缓颈椎间盘退变的机制。方法将24只新西兰家兔按随机数字表法分为空白组、模型组、针刀组,每组8只,采用"低头位加风寒湿刺激"法建立异常应力诱导下的兔颈椎间盘退变模型,通过颈椎核磁共振(MRI)影像学验证造模是否成功。针刀组在造模结束1周后进行干预,干预在无菌状态下以针刀治疗四步曲为参考,每周1次,共治疗4周。空白组、模型组与针刀组采用相同固定方式固定。结束后采用HE染色法观察各组实验兔颈部肌肉组织形态学变化;Western Blot法和Real-time PCR法检测椎间盘髓核中整合素α5、β1含量的变化。结果颈椎MRI结果显示,空白组矢状面可见髓核呈高亮信号状态、椎间盘高度正常,横断面可见髓核内信号均匀且与纤维环边界清晰;与空白组相比,模型组椎间盘的高度出现轻度降低,矢状面可见部分椎间盘髓核颜色较暗,呈灰色,信号强度较空白组明显减低,与空白组椎间盘横断面所见的髓核均匀分布相比,模型组髓核内部结构明显分布不均,且纤维环和髓核之间界限模糊,难以分清。HE染色显示,空白组可见肌纤维结构清晰,呈长条状平行排列,肌核少量散在纤维附近,肌细胞无变形水肿;与空白组相比,模型组肌纤维出现粗细不皱缩、变形,肌核聚集在附近,肌节不清晰,炎性浸润片状散在出现;与模型组相比,针刀组可见肌纤维挛缩改善,肌核规律分布,炎性细胞浸润明显减少。Western Blot法结果显示:与空白组相比,模型组椎间盘髓核中整合素α5、β1蛋白表达量显著降低(P<0.01);与模型组相比,针刀组整合素α5、β1蛋白含量明显增加(P<0.01)。Real-time PCR法结果显示,与空白组相比,模型组椎间盘髓核中整合素α5、β1 mRNA相对表达量均显著降低(P<0.01);与模型组相比,针刀组整合素α5 mRNA显著升高(P<0.01),β1mRNA相对表达量差异无统计学意义(P>0.05)。结论本实验所选取的"低头位加风寒湿刺激"模型具有可行性;针刀可以修复肌肉的紊乱排列,促进组织修复;针刀延缓颈椎间盘退变的可能机制是通过力学干预调整整合素α5、β1的表达含量,达到延缓颈椎间盘退变的目的。

Effect of Acupotomy Intervention on Expressions of Integrin α5 and β1 in Rabbits with Cervical Disc Degeneration

Objective To observe the effect of acupotomy intervention on the morphology of cervical intervertebral disc degeneration rabbit neck muscles and the contents of integrin α5 and β1 and explore the mechanism of acupotomy delaying cervical disc degeneration. Methods According to the random number table method, 24 New Zealand rabbits were divided into blank group, model group and acupotomy group, with 8 rabbits in each group. The rabbit cervical intervertebral disc degeneration model induced by abnormal stress was established by "low head position and wind-cold-dampness stimulation", and the success of the model was verified by cervical magnetic resonance(MRI) imaging. The acupotomy group was intervened one week after the modeling. Under the sterile state, the intervention took the four steps of acupotomy treatment as the reference, once a week for a total of 4 weeks. The blank group, model group and acupotomy group were fixed in the same way. At the end of the experiment, HE staining was used to observe the morphological changes of neck muscles of rabbits in each group. The contents of integrin α5 and β1 in nucleus pulposus of intervertebral disc were detected by Western Blot and Real time PCR. Results The MRI results of cervical spine showed that in the blank group, the nucleus pulposus showed a bright signal state in the sagittal plane, the height of intervertebral disc was normal, and the signal in the nucleus pulposus was uniform and the boundary with the fibrous ring was clear in the cross section. Compared with that of the blank group, the height of the intervertebral disc in the model group was decreased slightly, and the color of some intervertebral disc nucleus pulposus in the sagittal plane was dark and gray, and the signal intensity was significantly lower than that in the blank group. Compared with the uniform distribution of nucleus pulposus in the cross section of the intervertebral disc in the blank group, the internal structure of the nucleus pulposus in the model group was obviously uneven, and the boundary between the fibrous ring and nucleus pulposus was blurred, which was difficult to distinguish. HE staining showed that in the blank group, the structure of muscle fibers was clear, arranged in long strips in parallel, a small amount of muscle nuclei were scattered near the fibers, and the muscle cells had no deformation and edema. Compared with those of the blank group, the muscle fibers in the model group did not shrink and deform, the muscle nuclei gathered nearby, the sarcomeres were not clear, and inflammatory infiltration was scattered. Compared with that of the model group, the muscle fiber contracture was improved, muscle nucleus was regularly distributed, and inflammatory cell infiltration was significantly reduced in the acupotomy group. The results of Western Blot showed that compared with those of the blank group, the expressions of integrin α5 and β1 protein in the intervertebral disc nucleus pulposus of the model group were decreased significantly(P<0.01). Compared with those of the model group, the contents of integrin α5 and β1 protein in the acupotomy group were increased significantly(P<0.01). The results of Real time PCR showed that compared with those of the blank group, the relative expression of integrin α 5 and β1 mRNA in the nucleus pulposus of the intervertebral disc in the model group were decreased significantly(P<0.01). Compared with that of the model group, the integrin α5 in the acupotomy group was increased significantly(P<0.01). There was no significant difference in the relative expression of β1 mRNA(P>0.05). Conclusion The "low head position and wind-cold-dampness stimulation" model selected in this experiment is feasible. Acupotomy can repair the disordered arrangement of muscles and promote tissue repair. The possible mechanism of acupotomy delaying cervical disc degeneration is to adjust integrin α5 and β1 through mechanical intervention to delay the degeneration of cervical intervertebral disc.