针刀松解法对颈椎病兔颈后肌纤维化影响的研究

【作者】 钟鼎文；

【导师】 郭长青；

【作者基本信息】 北京中医药大学 ， 针灸推拿学， 2012， 博士

【摘要】 研究目的通过兔颈椎病动物模型,进行针刀干预治疗以及电针对照治疗。本实验采用低头位方法造颈椎病模型,首先采用HE染色法观察兔颈肌形态学观察,以及采用电镜观察肌外膜形态学,X线观察颈椎和椎间隙之间的改变情况。用RT-PCR法检测Ⅰ型胶原和转化生长因子β1(TGFβ1的基因水平表达以及Western-blot法检测Ⅰ型胶原和转化生长因子β1(TGFβ1)的蛋白水平表达,观察颈椎病兔颈肌肉结缔组织中Ⅰ型胶原和转化生子因子β1 (TGFβ1)相互影响的局势。探讨颈肌肌间组织纤维化改变与颈椎病的关系,丰富对颈椎病的发病机制的认识,并且从针刀治疗颈椎病的作用机制研究以及疗效判定提供客观依据。研究方法选用由北京市海淀区兴隆实验动物养殖场提供的健康成年新西兰家兔30只,6月龄,雌雄各半,每只体重2.5±0.5kg。按体重编号,分为空白组、模型组、针刀组和电针组,每组6只动物,其余6只动物同期造模,以补充模型组动物在造模过程中的缺失。采用低头位固定的方法制作兔颈椎病动物模型,使颈椎处于低头屈曲45度位。每天固定一次时间定为5小时,松解后笼中自由饲养。针刀组、电针组于造模第90天开始进行治疗干预,针刀组每周干预1次,电针组每周干预3次(隔日一次),共三周。采用HE染色法观察兔颈后肌形态学观察,X线观察颈椎和椎间隙之间的改变情况、电镜观察颈肌外膜形态学观察。然而采用Western-blot法检测Ⅰ型胶原和转化生长因子β1(TGFβ1)的蛋白水平表达和RT-PCR法检测Ⅰ型胶原和转化生长因子β1(TGFβ1)的基因水平表达。结果1.本实验分别对颈椎病兔放入特制的低头位兔架方式制造了颈椎病模型,本模型模拟了颈椎病初步病情状态的病理特征,从X线的变化过程以及组织形态学的变化确定模型的可靠性。结果显示针刀治疗可有效改善局部炎症反应,改善局部组织瘢痕的增生以及促进损伤组织修复,且针刀的效果优于电针。2.通过HE染色观察颈后肌肉以及电镜观察颈后肌外膜的形态学中模型组明显有肌肉组织结构破坏,肌纤维肿胀、变形如肌纤维吞噬被Ⅰ型胶原纤维代替等现象。经过针刀松解明显出现整体上修复过程状态。可以推断针刀通过恢复颈椎的生物力学平衡,改善局部代谢,提高致痛物质和炎症物质的吸收。3.颈椎病兔颈后肌组织中Ⅰ型胶原基因水平表达量的研究结果显示,造模后模型组、针刀组、电针组的模型兔颈肌中Ⅰ型胶原基因水平表达量较空白组明显升高,模型组与空白组之间有极显著性差异(P<0.01)；针刀组与空白组之间均无显著性差异(P>0.05)；电针组与空白组之间有极显著性差异(P<0.01)；针刀组与电针组之间有显著性差异(P<0.05)。4.造模后,模型兔颈后肌中Ⅰ型胶原蛋白水平表达量显示造模后,模型组、针刀组、电针组的模型兔颈肌中Ⅰ型胶原蛋白水平表达量较空白组明显升高,模型组与空白组间有极显著性差异(P<0.01)；模型组与针刀组之间有显著性差异(P<0.05)；电针组与空白组之间有极显著性差异(P<0.01)；针刀组与电针组之间有显著性差异(P<0.05)。5.颈椎病兔颈后肌组织中TGF-β1基因水平表达量的研究结果显示,造模后,模型组、针刀组、电针组的模型兔颈肌中TGF-β1的PCR表达量较空白组明显升高,模型组和空白组两者之间有显著性差异(P<0.05)；针刀组与模型组之间有显著性差异(P<0.05)；电针组与空白组之间有极显著性差异(P<0.01)；针刀组与电针组之间有显著性差异(P<0.05)。6.模型兔颈后肌中TGF-β1蛋白水平表达量的研究结果显示,造模后,模型组、针刀组、电针组的模型兔颈肌中TGF-β1蛋白水平表达量较正常组明显升高模型组和空白组两者之间有极显著性差异(P<0.01)；针刀组与模型组之间有显著性差异(P<0.05)；电针组与正常组之间有极显著性差异(P<0.01)；针刀组与电针组之间有显著性差异(P<0.05)。结论1.本实验通过X线观察不仅可以显示病变的部位、范围和程度,从平片中C3-C6之间显示生理弯曲变形、椎体骨质增生、椎间隙变化等影像学改变等提供了造模成功的可靠依据。同时考虑造模时间为3个月的因素本实验中的颈椎病定为颈型颈椎病,为颈椎病的初步阶段。提供的有利的依据。2.从局部的组织的HE染色以及电镜结果显示,针刀治疗可以有效缩小病变区的胶原疲痕组织形成,针刀松解法可以改善颈后肌纤维化。针刀治疗后,肌纤维排列恢复整齐,说明针刀治疗不仅可以有效阻止病理性瘫痕的形成,而且可以恢复局部静态及动态平衡状态,使组织恢复后不影响肌肉功能活动。这与针刀直接对瘫痕等局部组织进行铲拨、切割有关。3.实验中观察针刀和电针对兔颈后肌Ⅰ型胶原的影响,在模型兔颈后肌肉中RT-PCR检测显示Ⅰ型胶原基因水平表达结果显示,造模后模型组与空白组之间Ⅰ型胶原基因水平表达量明显增加又有极显著性差异(P<0.01),针刀组跟模型组之间Ⅰ型胶原基因水平表达量有明显降低又有显著性差异(P<0.05),且与空白组之间无显著差异(P>0.05),针刀组检测接近于空白组,说明针刀治疗颈椎病兔模型中的Ⅰ型胶原基因水平表达量有良好的调节作用。4.在模型兔颈后肌肉中Western-Blot检测显示Ⅰ型胶原蛋白水平表达量明显增加,模型组与空白组相比有极显著性(P<0.01),说明了颈椎病兔模型通过纤维化主要因素的Ⅰ型胶原中引起肌肉纤维化的现象。针刀组跟模型组之间Ⅰ型胶原蛋白水平表达量有明显降低又有显著性差异(P<0.05),针刀组检测接近于空白组,说明针刀治疗颈椎病兔模型中的Ⅰ型胶原蛋白水平表达量有良好的调节作用。5.在模型兔颈后肌肉中RT-PCR检测显示造模后颈椎病兔模型的TGF-β1的基因水平表达量明显增加,模型组与空白组相比有极显著性(P<0.01),说明了颈椎病兔模型通过TGF-β1基因释放纤维化物质的信号。针刀治疗后TGF-β1的基因水平表达量较模型明显降低,针刀组与模型组之间出现显著性变化(P<0.05),较接近空白组,表现出良好的调节作用。针刀组与电针组之间有显著性差异(P<0.05),显示针刀优于电针等差异。6.从模型兔颈后肌肉中Western-Blot检测显示TGF-β1的蛋白水平表达量明显增加,模型组与空白组相比有极显著性差异(P<0.01)说明颈椎病兔模型引起局部长期劳损使机体为了能够适应应变而增加TGF-β1的蛋白表达量,TGF-β1是胶原合成最迅速的刺激因子从而而促进肌肉纤维化的过程。针刀组与模型组之间出现显著性变化(P<0.05)和针刀组与电针组之间有显著性差异(P<0.05),说明针刀治疗颈椎病兔模型中的TGF-β1有良好的调节作用以及接近正常水平以及针刀治疗优势与电针治疗。更多还原

【Abstract】 ObjectiveThis study established rabbit models of cervical spondylosis (CS) by the method of imitating human long-term flexed neck posture,applied acupotomy intervention after modeling, and compared to the group intervented by electro-acupuncture. Morphology of rabbit’s cervical muscles by hematoxylin-eosin staining,and epimysium of rabbit’s cervical muscles by electronic speculum were observed. All the animals were cervical X-rayed for the observation of changes of the cervical vertebrae and intervertebrale.Expression of apoptosis genes of type I collagen and TGFβ1 by RT-PCR test,and expression of apoptosis protein of type I collagen and TGFβ1 by Western blot test were conducted.Thus to observe the interaction relationship between type I collagen and TGFβ1, and it is expected to explore the relationship between fibrosis and cervical spondylosis in the neck muscle, enrich understanding of the pathogenesis of cervical spondylosis,and provide evidences for the mechanism research and efficacy of acupuncture therapy.Methods30 Six-month-old healthy adult New Zealand rabbits, equal between genders, provided by Xinglong experimental animals farms in Haidian District, Beijing,from which each weight 2.5±0.5kg.. number by weight and divided into four groups randomly:normal control group,model group, acupotomy group,electro-acupuncture(EA) group.Each group had 6 rabbits,and the rest 6 rabbits are raised to be the back-ups of model group. CS models were made by the method of imitating human long-term flexed neck posture of 45 degrees for 5 hours a day, After modeling, acupotomy and electro-acupuncture were applied to treat in acupotomy group and electro-acupuncture group. Once a week in acupotomy group, three times a week in electro-acupuncture group, both groups were treated for three weeks.Morphology of rabbit’s posterior cervical muscles by hematoxylin-eosin staining,and epimysium by electronic speculum were observed.All the animals were cervical X-rayed for the observation of changes of the cervical vertebrae and intervertebrale, Expression of apoptosis genes of type I collagen and TGFβ1 by RT-PCR test,and expression of apoptosis protein of type I collagen and TGFβ1 by Western blot test were conducted.Results1.In this study, CS models were made by the method of fixing the rabbits’neck posture in specialized boxes thus simulated the pathological features of the initial status of cervical spondylosis. With the observation of morphology of cervical muscles and manifestations of cervical X-ray, the results verified model of CS was established successfully and the modeling method is reliable.And acupotomy therapy has good effects in regulating the local inflammatory response, and promoting damaged tissue repairation,and the effect of the acupotomy therapy is better than EA.2.The results of Real-time PCR tests showed that,in comparison with normal control group after modeling,expression of apoptosis genes of type I collagen increased significantly in other 3 groups, there was a highly significant difference between the model group and normal control group (P<0.01);also with the EA group and normal control group (P<0.01); and significant difference between the model group and acupotomy group (P<0.05); also significant difference between the acupotomy group and EA group (P<0.05).3. The results of Western blot tests showed that, in comparison with normal control group after modeling, expression of apoptosis protein of type I collagen increased significantly in other 3 groups, and there was a highly significant difference between the model group and normal control group (P<0.01);also with the EA group and normal control group (P<0.01);and significant difference between the model group and acupotomy group (P<0.05);also significant difference between the acupotomy group and EA group (P<0.05).4. The results of Real-time PCR tests showed that, in comparison with normal control group after modeling, expression of apoptosis genes of TGF-β1 increased significantly in other 3 groups, there was a significant difference between the model group and normal control group (P<0.05);also with the acupotomy group and normal control group (P<0.05);the acupotomy group and EA group (P<0.05).;and highly significant difference between the EA group and normal control group (P<0.01).5. The results of Western blot tests showed that, in comparison with normal control group after modeling, expression of apoptosis protein of TGF-β1 increased significantly in other 3 groups,and there was a highly significant difference between the model group and normal control group (P<0.01);also with the EA group and normal control group (P<0.01);and significant difference between the model group and acupotomy group (P<0.05);also significant difference between the acupotomy group and EA group (P<0.05).ConclusionIn this experiment, the X-ray observations can not only display lesion location, extent and degree of physiological bending, vertebral hyperostosis,intervertebral space changes the image changes from a flat film, C3-C6 between the making model was successful and reliable basis. Taking into account the modeling time of the cervical syndrome, cervical spondylosis in the 3 months of the factors in this experiment as a preliminary stage of cervical spondylosis. Provided a favorable basis.From the local organization of HE staining and electron microscopy results showed that acupuncture therapy can effectively reduce the collagen weakness of the lesion scar tissue formation, needle knife method can improve the back of the neck muscle fibrosis. Acupuncture therapy, the muscle fibers arranged in recovery neat, indicating that acupuncture therapy can not only effectively prevent pathological scar formation, and the local static and dynamic equilibrium can be restored, so that the organization does not affect muscle function after recovery. Directly with the needle knife scar local tissue shovel aside, cutting related.Three experimental observation of the needle knife and electric rabbit back of the neck muscle of type I collagen in the back of the neck muscles in the rabbit model RT-PCR analysis showed that the level of type I collagen gene expression results showed that after modeling, model group and blank group between the level of type I collagen gene expression increased significantly there are very significant differences (P<0.01)between the knife group with the model group, the level of type I collagen gene expression levels have decreased significantly and there are significant differences (P<0.05) with a blank between groups was no significant difference (P> 0.05), the knife group detected close to the blank group, indicating that acupuncture therapy in cervical spondylosis rabbit model of type I collagen gene level expression of good regulation.Western-Blot shows in the back of the neck muscles in the rabbit model of type I collagen protein levels increased significantly, compared to the model group and blank group had significantly (P<0.01), indicating a rabbit model of cervical disease through fibrosis The main factors of type I collagen cause muscle fibrosis. Type I collagen levels between the knife group with the model group expression levels have significantly reduced there are significant differences (P<0.05),the knife group detected close to the blank group, indicating that acupuncture therapy in cervical spondylosis rabbit model of type I the level of collagen expression of good regulation.In the back of the neck muscles in the rabbit model RT-PCR analysis showed that the level of cervical spondylosis rabbit model of TGF-β1 gene expression was significantly increased after modeling, model group and blank group was highly significant (P<0.01). illustrates the rabbit model of cervical disease and fibrosis of the signal of the substance released by TGF-β1 gene. The expression levels of TGF-β1 gene level compared with the model in acupuncture therapy significantly reduced, significant changes (P<0.05)between the knife group and model group, closer to the blank control group, showed good regulation. Significant difference (P<0.05) between the knife group and EA group. Show acupotomology better than the EA differences.From the Western-Blot back of the neck muscles in the rabbit model of TGF-β1 protein level was significantly increased compared to the model group and blank group there was a significant difference(P<0.01) a rabbit model of cervical spondylosis caused by the local long-term strain so that the body in order to be able to adapt the strain increase of TGF-β1 protein expression of TGF-β1 is the most rapid collagen synthesis stimulating factor to promote the process of muscle fibrosis.Changes between the knife group and model group were significantly (P<0.05) and the knife group and EA group, significant differences (P<0.05), indicating the knife treatment of cervical spondylosis rabbit model of TGF-β1 good regulation, as well as close to normal levels, as well as acupuncture therapy strengths and electro-acupuncture treatment.更多还原