微创纤维环穿刺法诱导兔椎间盘退变模型

发布时间：2018-01-16 05:14

本文关键词：微创纤维环穿刺法诱导兔椎间盘退变模型　出处：《郑州大学》2012年硕士论文　论文类型：学位论文

【摘要】：1研究背景下腰背痛是骨科最常见的病症,据统计,在中青年人群中多达4/5以上的人都会受到下腰背部疼痛的困扰,目前大多学者认为椎间盘退变是其最常见的原因。椎间盘退变是指椎间盘失去原有的正常结构,并伴有进行性的纤维化,其表现为纤维环层状结构紊乱和进行性纤维化,凝胶样髓核逐渐消失,纤维环与髓核原有界限消失。椎间盘退变发生的病因及机制尚无确切定论,大多数学者认为与生物力学、遗传学、椎间盘营养、细胞衰老、自身免疫等多方面因素有关。目前针对椎间盘源性下腰痛(disc-genetic low back pain)主要采取理疗及抗炎药物应用等保守治疗手术治疗,这些治疗方法只是治标而不治本。因此,我们需要针对IVDD发病机制进行研究,从发病原因入手,对因治疗,从根本上治疗、甚至是预防IVDD。近年来新兴的生物学再生治疗方法以退变椎间盘为靶向治疗目标,从病因出发,以重建为理念,已经备受关注,其包括有分子疗法、基因疗法、细胞疗法及组织疗法等。这些生物疗法的优势在于能够很好地维持正常椎间盘的生物力学稳定性、并且不破坏周围的解剖学结构、保持椎体间的相对运动等。但要将这些方法从实验研究应用到临床,还需要我们更深入的研究IVDD的病理生理学发病机制。制作一种可靠、有效的IVDD动物模型有助于对IVDD的发病机制及预防治疗的研究。目前常用于制作退变模型的动物主要有小鼠、大鼠、兔、羊、狗、猪、灵长类动物等十余种。大小鼠由于体型较小,虽方便模型制作,但对于以治疗为目的的模型却难以进行椎间盘内注射,因此,对于以治疗为目的的模型应选择体型较大的动物,然而羊、狗、猪等动物价格较高,难以大量进行试验,所以兔为最理想的模型动物,且其椎间盘内注射技术已有相关报道。兔作为腰椎间盘退变模型动物已有大量报道,但基本都是需要切开皮肤肌肉,肉眼直视椎间盘下行针刺或切开纤维环进行退变模型的制作。过程中容易损伤腹膜及周围软组织,给实验动物带来很大的创伤,且增加了感染的机会,术后动物死亡率高,造成实验动物的浪费；手术过程中对腰椎周围组织的破坏及术后瘢痕形成都对试验结果造成影响,违背了自然退变过程的模仿。尚未有人用微创的方法进行兔退变椎间盘模型的制作。 2目的建立新的微创兔腰椎间盘退变模型,为腰椎间盘退变机制与治疗的进一步研究提供更理想的试验对象。 3方法本实验在仔细研究家兔腰椎及周围组织解剖结构的基础上,经过反复穿刺操作,找到了此安全微创的穿刺通道及方法。穿刺通路远离腹膜及重要血管神经,且在C臂引导下进行,操作准确,一步到位,误伤几率小,有操作快速简单,损伤小,感染机会小、动物存活率高、可重复性好等优点。应用此方法对18只兔子的L3/4、L4/5、L5/6椎间盘进行造模损伤,术后将动物随机分为A、B、C三个模型组,每组6只。分别于造模术后4、8、12周对三组进行MRI检查,以Pfirrmann分级法作为评估标准进行分级,并前后对照。每组行MRI检查后予以处死,取L3/4、L4/5、L5/6椎间盘髓核组织,分两组,分别行RT-PCR检测Ⅱ型胶原蛋白的表达及经4%甲醛溶液在4℃下进行固定,然后脱水,石蜡包埋,组织切片,之后进行Ⅱ型胶原免疫组织化学染色及HE染色进行观察,前后对照并且取L2/3及L6/7椎间盘髓核组织作为正常对照。 4结果从术后4、8、12周及术前的兔腰椎MRI检查结果可以看出,造模前后椎间盘髓核的信号强度有随造模手术后的时间延长而逐渐降低的趋势(L3/4、L4/5及L5/6)。由磁共振室两名教授盲法采用Pfirrmann分级法将结果分级,采用有序分组资料的线性趋势检验,计算得到P＜0.05。说明椎间盘的退变程度与造模后时间长短有显著相关性,即造模后时间越长椎间盘退变越明显。造模术后4周、8周、12周后取材采用RT-PCR技术检测发现椎间盘Ⅱ型胶原蛋白均明显表达,其中4周最强,8周、12周后逐渐次之。病理HE染色结果观察可以看到,对照组正常的髓核组织中有大量的髓核细胞,随着造模手术后时间的延长髓核细胞数量渐减少,到第12周时,髓核中只有极少量髓核细胞,却有很多纤维软骨组织。将对照组及术后4、8、12周组Ⅱ型胶原免疫组化染色的灰度值进行单因素方差分析结果：F=41.82(P＜0.05),组间比较采用LSD-t检验,结果显示,除术后4周与8周组比较无统计学意义外,其余组间两两比较均有显著差异(P＜0.05)。说明在造模以后,随着时间的延长,髓核组织中Ⅱ型胶原的含量逐渐减少。 5结论成功建立了新的微创兔腰椎间盘退变动物模型。
[Abstract]:1 background of research
Low back pain is the most common disease in Department of orthopedics, according to statistics, up to more than 4/5 people are influenced by the low back pain in young people in distress, most scholars believe that the degeneration of intervertebral disc is the most common cause. Intervertebral disc degeneration refers to the intervertebral disc lost the normal structure of the original, and associated with progressive fibrosis the performance of fiber ring, layered structure disorder and progressive fibrosis, gelatinous nucleus pulposus gradually disappear, annulus and nucleus original boundaries disappear. The etiology and mechanism of intervertebral disc degeneration. There is no definite conclusion, most scholars believe that the intervertebral disc with biomechanics, genetics, nutrition, cell senescence, autoimmune factors etc. now for discogenic low back pain (disc-genetic low back pain) mainly adopt physiotherapy and anti-inflammatory drugs application of conservative treatment such as surgical treatment, these treatments are only temporary and not Therefore, we need to study the pathogenesis of IVDD, from the cause of the disease, the treatment, the treatment, and even the prevention of IVDD. from the cause of the disease.
In recent years new biological regeneration method for treatment of degenerative intervertebral disc for targeted therapy, from the cause of the reconstruction for the concept, have attracted much attention, including molecular therapy, gene therapy, cell therapy and tissue therapy. These biological therapy has the advantage of being able to maintain good biomechanical stability of normal intervertebral disc, and does not destroy the anatomical structure around, keep the relative movement between the vertebrae.
But if we want to apply these methods from experimental research to clinical practice, we need to further study the pathophysiology mechanism of IVDD. Making a reliable and effective IVDD animal model is helpful for the study of IVDD pathogenesis, prevention and treatment.
At present commonly used in the production of the degeneration of animal mainly in mice, rats, rabbits, sheep, dog, pig and primate animal. More than ten kinds of rats due to the smaller size, although convenient for model making, but for the purpose of medical treatment is difficult to model of intradiscal injection for treatment, therefore, for the purpose of the model should choose the larger animal, however, sheep, dogs, pigs and other animal prices higher, so it is difficult to test, so is the ideal animal model of rabbit, and the intradiscal injection technology has been reported. As a rabbit lumbar disc degeneration has been reported in many animal models, but the basic is the need to cut the skin muscle, making direct eye disc or cut fiber ring down acupuncture. Peritoneal degeneration model and soft tissue injury prone process, bring great trauma to the animal, and increase the chance of infection, postoperative dynamic From the mortality rate is high, resulting in experimental animal waste; influence of operation process of damage to the surrounding tissue of lumbar and postoperative scar formation in Chengdu caused by the test results, contrary to the natural degeneration process of imitation. Yet making rabbit model of degenerative intervertebral disc by minimally invasive method.
2 purposes
A new minimally invasive rabbit lumbar disc degeneration model is established to provide more ideal experimental objects for the further study of the mechanism and treatment of the lumbar intervertebral disc degeneration.
3 method
In this experiment, based on careful study of rabbit lumbar and surrounding tissue anatomical structure, after repeated puncture operation, find the puncture channel and method. The safety of minimally invasive puncture pathway from the peritoneum and important blood vessels and nerves, and, in the C arm under the guidance of accurate operation, step in place, there are accidental injury probability is small, fast operation simple, less damage, less infection, animal survival rate is high, the repeatability is good. The application of this method in 18 rabbits of L3/4, L4/5, L5/6 disc modeling after injury, the animal were randomly divided into A, B, C three model group, 6 rats in each group respectively. Model 4,8,12 weeks after operation of three groups were examined by MRI, using Pfirrmann classification method as evaluation criteria for classification, and the control group. Before and after MRI were to be killed, L3/4, L4/5, L5/6 of nucleus pulposus, divided into two groups, respectively, for detection of RT-PCR expression of type II collagen and 4% Formaldehyde Solution was fixed at 4 degrees, then dehydrated, paraffin embedded and tissue sectioning. Then type II collagen immunohistochemical staining and HE staining were used for observation. Before and after comparison, L2/3 and L6/7 intervertebral disc nucleus pulposus tissue was used as normal control.
4 Results
From the rabbit lumbar MRI examination results 4,8,12 weeks after operation and before operation can be seen before and after modeling intervertebral disc nucleus signal intensity is gradually decreased with the molding time after operation (L3/4, L4/5 and L5/6). The MRI room two professors by blind method Pfirrmann classification results classification, linear trend test using ordered packet data, the calculated P < 0.05. degeneration of intervertebral disc was significantly correlated with the length of time after modeling, namely the model for the length of time after intervertebral disc degeneration is more obvious. 4 weeks after model establishment, 8 weeks, 12 weeks using RT-PCR detection technology that disc type II collagen were significantly expressed in the 4 week, the strongest, 8 weeks, 12 weeks after the second. Gradually observations can be seen by HE staining, the control group has a large number of nucleus pulposus cells of normal nucleus pulposus tissues, with the time of the operation of the model after the extension of the nucleus pulposus cells Gradually reduce the number to twelfth weeks, only a very small amount of nucleus pulposus cells, but there are a lot of fibrous cartilage. The controls were the result of the analysis of variance and gray value of group 4,8,12 weeks after operation group of type II collagen immunohistochemical staining: F=41.82 (P < 0.05), compared with LSD-t test results showed that in 4 weeks after operation and 8 week group was not statistically significant, significant differences between the other groups were 22 (P < 0.05). In the model, with the extension of time, the content of type II collagen in nucleus pulposus decreased gradually.
5 Conclusion
A new minimally invasive rabbit model of lumbar intervertebral disc degeneration was established.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332

【参考文献】