【作者】 刘福水；

【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】 颈椎病(cervical spondylosis,CS)是全球范围内常见的公共健康问题,在职业性多发病和致残因素中占很大比例,给个人、家庭、社会、医疗系统带来严重的影响和经济负担。颈椎病的发病率逐年上升,呈现出普遍化、年轻化趋势。颈椎病临床表现复杂多样,可表现为疼痛、神经根和脊髓等受压引起的一系列症状和体征,常见颈肩背疼痛、头痛头晕、颈部板硬、上肢麻木、上肢肌力减弱和感觉减退缺失等。颈椎病的防治工作已引起各界广泛关注,对颈椎病的研究有重要的现实意义。针刀疗法作为一种中医新疗法自上世纪70年代末发明至今在临床上推广较快,针刀疗法已成为治疗颈椎病常用方法之一,颈椎病是针刀疗法的优势病种和适应症。针刀治疗颈椎病临床疗效肯定,但针刀疗法基础研究薄弱,针刀治疗颈椎病疗效机制的研究亟待开展。研究目的本研究通过模拟人类颈椎病好发因素长期低头位造成颈椎病兔模型,HE染色光镜观察颈后伸肌和颈椎间盘改变以及造模前、造模后、干预后3个时间节点拍颈椎X线片,探讨颈肌劳损在颈椎病发病中的作用。造模后针刀干预颈椎病兔模型并与电针对照,观察针刀干预对颈后伸肌TUNEL阳性细胞核计数和凋亡因子Bcl-2.Bax.Caspase-3的基因和蛋白表达水平,以期从影响颈后伸肌细胞凋亡的角度探揭示针刀治疗颈椎病可能的作用机制,为临床针刀治疗颈椎病和经筋病提供实验依据。研究方法选用6月龄健康成年新西兰兔(雌雄各半)随机分为空白组、模型组、针刀组、电针组,采用长期低头位方法制备颈椎病模型。造模后针刀组、电针组进行治疗干预,针刀组每周干预1次,电针组每周干预3次,针刀电针干预均为三周。各组动物于造模前、造模后、干预后3个时间节点拍颈椎X线片；HE染色光镜观察颈后伸肌、颈椎间盘的病理学表现；TUNEL法检测颈后伸肌TUNEL阳性细胞核数；Real-time PCR法检测Bcl-2 mRNA.Bax mRNA.Caspase-3 mRNA的表达水平,Western blot(?)去检测Bcl-2.Bax. Caspase-3蛋白的表达水平研究结果1.颈后伸肌组织和颈椎间盘HE染色光镜观察结果显示,模型组肌纤维着色不均,肌纤维变性,细胞核增多,纵横面均见核内移,结缔组织增生及瘢痕形成,见炎性细胞浸润,肌纤维呈波浪状、节段状扭曲改变,肌纤维溶解变性,肌纤维水肿,间质增生；针刀干预后颈肌组织肌纤维较完整,肌纤维较平直未见明显扭曲,针刀干预可有效促进颈肌组织的修复。模型组颈椎间盘髓核几乎全部纤维化,髓核组织中胶原纤维增生、满布软骨样细胞,大多数软骨样细胞的排列与胶原纤维的走行方向相一致；针刀和电针干预后椎间盘髓核纤维化程度较模型组稍轻,伸向髓核的增生纤维组织较模型组疏松。2.颈椎X线片显示造模后颈椎曲度改变,出现反张,椎间隙变窄,部分见椎间孔变小、椎体前后缘见骨质增生；针刀干预后见颈椎曲度改善较明显,曲度由反张变为较直,部分见椎间孔较治疗前变大、椎间隙有所改善,治疗前后骨质增生未见明显变化；电针干预后见部分兔颈椎反张有改善,治疗前后椎间隙、椎间孔、骨质增生未见明显变化。造模后,模型组、针刀组、电针组颈椎X线积分均明显升高,与空白组相比均有显著差异(P<0.01)；针刀干预后X线积分改善程度与模型组比较有显著差异(P<0.01)；电针干预后X线积分改善程度与模型组比较无统计差异(P>0.05)；针刀组干预后X线积分改善程度较电针明显(p<0.05)。3.TUNEL实验结果显示模型组颈后伸肌TUNEL阳性细胞核计数较空白组明显升高异(P<0.01)；针刀组较空白组略微升高,但二者无统计差异(P>0.05)；针刀组较模型组明显降低(P<0.01)；电针组较空白组明显升高(P<0.01)；电针组较模型组有所降低,但二者无统计差异(P>0.05)；针刀组较电针组明显降低(p<0.01)。4.Real-time PCR实验结果显示各组Bcl-2 mRNA表达水平无统计学差异(P>0.05)；模型组Bax mRNA.Caspase一3 mRNA表达水平较空白组明显升高(P<0.01,P<0.05),Bcl.2/Bax mRNA比值较空白组明显降低(P<0.01)；针刀组Bax mRNA.Caspase-3 mRNA表达水平较模型组明显降低(P<0.01,P<0.05),Bcl-2/Bax mRNA比值较模型组明显升高(P<0.01)；电针组Bax mRNA.Caspase-3 mRNA表达水平与模型组比较无统计差异(P>0.05),Bcl-2/Bax mRNA比值较模型组升高但二者无统计差异(P>0.05)。表明针刀干预可有效下调促凋亡因子Bax mRNA.Caspase-3 mRNA的表达水平和上调Bcl-2/Bax mRNA比值,针刀组效果优于电针组。5.Western blot实验结果显示各组Bcl-2蛋白表达水平无统计差异(P>0.05)；模型组Bax.Caspase-3蛋白表达水平较空白组明显升高(P<0.01,P<0.05),Bcl-2/Bax蛋白比值较空白组降低(P<0.05)；针刀组Bax.Caspase-3蛋白表达水平较模型组降低(P<0.05),Bcl-2/Bax蛋白比值较模型组升高(p<0.05)：电针组Bax.Caspase-3蛋白表达水平与模型组比较无统计差异(p>0.05),Bcl.2/Bax蛋白比值较模型组升高但二者无统计差异(P>0.05)。Western blot实验结果与Real-time PCR实验结果基本一致,表明针刀干预可有效降低促凋亡因子Bax.Caspase-3蛋白表达水平和上调Bcl-2/Bax蛋白比值，针刀组效果优于电针组。6.颈后伸肌TUNEL阳性细胞核计数与其他指标相关性分析结果：颈后伸肌TUNEL阳性细胞核计数与干预后颈推X线积分（r=0.832, P<0.01)、Bax mRNA ( r=0.669,P< 0.01)、Caspase-3 mRNA ( r=0.644,P< 0.01)、Bax蛋白（r=0.522,P< 0.01)、Caspase-3蛋白（r=0.667, P< 0.01 )均呈显著正相关；颈后伸肌TUNEL阳性细胞核计数与Bcl-2/Bax mRNA比值(r=-0.613 ,P< 0.01 )> Bcl-2/Bax蛋白比值（r=-0.700,P< 0.01 )均呈显著负相关；颈后伸肌TUNEL阳性细胞核计数与Bcl-2 mRNA表达水平（r=-0.145, P > 0.05 )、Bd-2蛋白表达水平（r=-0.247, P> 0.05 )均无明显相关性。研究结论1.针刀干预对颈椎病兔颈后伸肌细胞凋亡有良好的调节作用，可以降低颈后伸肌TUNEL阳性细胞核数。2.针刀干预可以下调颈推病兔颈后伸肌Bax、Caspase-3基因和蛋白表达水平，上调Bcl-2/Bax mRNA和蛋白比值。3.针刀干预对骨骼肌细胞凋亡的调节作用可能是其治疗颈推病和经筋病的重要机制之一。更多还原

【Abstract】 Cervical spondylosis (CS) is becoming an increasingly common health problem throughout the world. It accounts for a large proportion in occupation-related diseases and disabilities. It causes a considerable impact and economic burden on individuals and their families, communities, health-care systems. The prevalence of CS has been increasing quickly, showing a trend of generalization and impacting the younger generation more. The clincal manifestations of CS are complicated and multilevel.It usually manifestates as pain on the neck and shoulder, headache, diziness, neck stiffness, numbness and weakness of upper limbs, reduction or loss of sensations. Prevention and treatment of CS has been a priority health problem, and study on CS has important significance in achieving this. As a new therapy of chinese medicine, acupotomy has been spreaded quickly since its advent in 1970s. Acupotomy has become a common therapy for CS. CS is a dominant disease indicating the value of necessitating acupotomy therapy. Acupotomy has achieved satisfactory efficacy, however its basic research on the efficacy mechanism is eagerly to be conducted.ObjectiveThis study established rabbit models of CS by the method of imitating human long-term flexed neck posture during work, a very common risk factor of human CS. Through observing the morphology of posterior cervical extensor muscles and intervertebral disc of rabbits with CS, changes of cervical X-rays taken before and after modeling, and after treatment, was used to assess the role of injury to the posterior cervical extensor muscles in the development of CS. Acupotomy therapy was applied after modeling, and compared to the group treated with electro-acupuncture. TUNEL-positive myonuclei count of posterior cervical extensor muscles, expression of apoptosis genes and proteins Bcl-2, Bax, Caspase-3 were tested. It is expected to explore the possible mechanism of acupotomy in the treatment of CS from the aspect of adjusting apoptosis of the posterior cervical extensor muscles and to provide evidences for acuptomy therapy.MethodsSix months old healthy adult New Zealand rabbits(with gender equality) were divided into four groups randomly:normal control group, model group, acupotomy group, and electro-acupuncture (EA) group. CS models were made by the method of flexed neck posture for long-term. After modeling, acupotomy and electro-acupuncture were applied to treat in acupotomy group and electro-acupuncture group. Both groups were treated for three weeks, once every week in acupotomy group, and three times in electro-acupuncture group. All the animals were subjected to cervical X-ray for three times:before and after modeling, and after treatment. Pathological changes of the muscles and intervertebral discs by hematoxylin-eosin staining were observed. TUNEL-positive myonuclei count of the muscles was conducted by TUNEL test. Expression of apoptosis genes Bcl-2 mRNA, Bax mRNA, Caspase-3 mRNA were conducted by Real-time PCR test, and expression of apoptosis proteins Bcl-2, Bax, Caspase-3 were conducted by Western blot test.Results1. Histomorphology of the muscle fibres in model group showed uneven staining, rhabdomylosis, increase of myocyte nuclears, nuclears moved to the middle of myocytes, fibrosis and scar formation, inflammatory cells aggregated, wave-shaped muscle fibres, lysis of muscle fibres, swollen muscle fibres, overgrowth of intercellular substance. In acupotomy group, histomorphology showed strgight-shaped muslcle fibres. Acupotomy promoted the repair of injuried muscle tissues. Histomorphology of intervertebral disc in model group showed nucleus pulposus was almosted replaced by fibers; overgrowth of collagenous fiber with a lot of chondrocytes-like cells were seen, and the direction of chondrocytes-like cells was consistent to collagenous fibers. Fibrosis of nucleus pulposus in acupotomy and electro-acupuncture groups was a little less than the model group and overgrowth of collagenous fibers was a little less than the model group.2. After modeling, cervical X-rays showed that there were changes in cervical lordosis, even lordotic curve with posterior convexity, a decrease in discs height, narrowed intervertebrale foramen and overgrowth of osteophytes in anterior and posterior vertebral bodies in some rabbits. There was obvious improvement in X-rays after acupotomy treatment; cervical lordosis was recovered to straight, intervertebral foramen widened in some rabbits, and there was no obvious change of osteophytes before and after treatment. After modeling, the cervical X-ray scores of the model group, acupotomy group and electro-acupuncture group increased significantly in comparison with normal control group (P<0.01). The reduction of X-ray scores before and after treatment in acupotomy group had a significant difference in comparison with model group (P<0.01).The reduction of X-ray scores before and after treatment in electro-acupuncture group had no significant difference with model group (P >0.05). The reduction of X-ray scores before and after treatment in acupotomy group had a significant difference in correlation with the electro-acupuncture group (P<0.05).3. In comparison with normal control group after modeling, TUNEL-positive myonuclei count of posterior cervical extensor muscles increased significantly (P<0.01). In comparison with normal control group, TUNEL-positive myonuclei count in acupotomy group increased mildly but there was no significant difference between the two groups (P>0.05).In comparison with model group, TUNEL-positive myonuclei count in acupotomy group decreased significantly (P<0.01). TUNEL-positive myonuclei count in electro-acupuncture group was higher than normal control group (P<0.01). In comparison with model group, TUNEL-positive myonuclei count in electro-acupuncture group decreased mildly, but there was no significant difference between the two groups (P>0.05). TUNEL-positive myonuclei count in acupotomy group was significantly lower than electro-acupuncture group (P<0.01).4. The results of Real-time PCR tests showed that there was no significant difference of expression of Bcl-2 mRNA between all the groups (P>0.05). In comparison with normal control group after modeling, Bax mRNA and Caspase-3 mRNA expression increased significantly (P<0.01, P<0.05), and the ratio of Bcl-2/Bax mRNA decreased significantly (P<0.01). In comparison with model group after treatment of acupotomy, Bax mRNA and Caspase-3 mRNA expression decreased significantly (P<0.01, P<0.05) and the ratio of Bcl-2/Bax mRNA increased significantly(P<0.01). In comparison with model group after treatment of electro-acupuncture, there was no significant difference in Bax mRNA and Caspase-3 mRNA expression and the ratio of Bcl-2/Bax mRNA (P>0.05). The results of Real-time PCR tests showed that acupotomy therapy had significant effects in decreasing Bax mRNA and Caspase-3 mRNA expression and upregulating the ratio of Bcl-2/Bax mRNA. The effects of acupotomy group were superior to electro-acupuncture group.5. The results of western blot tests showed that there was no significant difference of Bcl-2 protein between all the groups (P>0.05). In comparison with normal control group after modeling, Bax and Caspase-3 proteins expression increased significantly (P<0.01, P<0.05),and the ratio of Bcl-2/Bax protein decreased significantly (P<0.05). In comparison with model group after treatment of acupotomy, Bax and Caspase-3 proteins expression decreased significantly(P< 0.05) and the ratio of Bcl-2/Bax protein increased significantly(P<0.05). In comparison with model group after treatment of electro-acupuncture, there was no difference in Bax and Caspase-3 protein expression and the ratio of Bcl-2/Bax protein (P> 0.05). The results of western blot tests were consistent to Real-time PCR tests, acupotomy therapy showing great significance in decreasing Bax and Caspase-3 protein expression, and upregulating the ratio of Bcl-2/Bax protein. The effects of acupotomy group were superior to electro-acupuncture group.6. Correlation analysis showed that TUNEL-positive myonuclei count of posterior cervical extensor muscles had a strong positive correlation with cervical X-ray scores after treatment(r=0.832, P<0.01), Bax mRNA expression(r=0.669, P<0.01), Caspase-3 mRNA expression(r=0.644, P<0.01), Bax protein expression(r=0.522, P<0.01), Caspase-3 protein expression (r=0.667, P<0.01). TUNEL-positive myonuclei count of posterior cervical extensor muscles had a strong negative correlation with the ratio of Bcl-2/Bax mRNA(r=-0.613, P< 0.01), ratio of Bcl-2/Bax protein(r=-0.700,P<0.01). There was no correlation between TUNEL-positive myonuclei count of posterior cervical extensor muscles with Bcl-2 mRNA expression(r=-0.145, P>0.05), Bcl-2 protein expression(r=-0.247, P>0.05).Conclusions1. Acupotomy therapy has good effects of regulating myocytes apoptosis in posterior cervical extensor muscles in rabbits with CS, reducing TUNEL-positive myonuclei count.2. Acupotomy therapy can down-regulate genes and proteins expression of Bax and caspase-3, up-regulate the ratio of Bcl-2/Bax mRNA and protein expression in posterior cervical extensor muscles in rabbits with CS. 3. Regulating myocytes apoptosis is the possible mechanism of acupotomy therapy to treat CS and diseases of meridian sinew.更多还原