本文關鍵詞：微創(chuàng)纖維環(huán)穿刺法誘導兔椎間盤退變模型　出處：《鄭州大學》2012年碩士論文　論文類型：學位論文

  更多相關文章： 微創(chuàng) 椎間盤退變 動物模型 兔

【摘要】：1研究背景 下腰背痛是骨科最常見的病癥,據(jù)統(tǒng)計,在中青年人群中多達4/5以上的人都會受到下腰背部疼痛的困擾,目前大多學者認為椎間盤退變是其最常見的原因。椎間盤退變是指椎間盤失去原有的正常結構,并伴有進行性的纖維化,其表現(xiàn)為纖維環(huán)層狀結構紊亂和進行性纖維化,凝膠樣髓核逐漸消失,纖維環(huán)與髓核原有界限消失。椎間盤退變發(fā)生的病因及機制尚無確切定論,大多數(shù)學者認為與生物力學、遺傳學、椎間盤營養(yǎng)、細胞衰老、自身免疫等多方面因素有關。目前針對椎間盤源性下腰痛(disc-genetic low back pain)主要采取理療及抗炎藥物應用等保守治療手術治療,這些治療方法只是治標而不治本。因此,我們需要針對IVDD發(fā)病機制進行研究,從發(fā)病原因入手,對因治療,從根本上治療、甚至是預防IVDD。 近年來新興的生物學再生治療方法以退變椎間盤為靶向治療目標,從病因出發(fā),以重建為理念,已經(jīng)備受關注,其包括有分子療法、基因療法、細胞療法及組織療法等。這些生物療法的優(yōu)勢在于能夠很好地維持正常椎間盤的生物力學穩(wěn)定性、并且不破壞周圍的解剖學結構、保持椎體間的相對運動等。 但要將這些方法從實驗研究應用到臨床,還需要我們更深入的研究IVDD的病理生理學發(fā)病機制。制作一種可靠、有效的IVDD動物模型有助于對IVDD的發(fā)病機制及預防治療的研究。 目前常用于制作退變模型的動物主要有小鼠、大鼠、兔、羊、狗、豬、靈長類動物等十余種。大小鼠由于體型較小,雖方便模型制作,但對于以治療為目的的模型卻難以進行椎間盤內注射,因此,對于以治療為目的的模型應選擇體型較大的動物,然而羊、狗、豬等動物價格較高,難以大量進行試驗,所以兔為最理想的模型動物,且其椎間盤內注射技術已有相關報道。兔作為腰椎間盤退變模型動物已有大量報道,但基本都是需要切開皮膚肌肉,肉眼直視椎間盤下行針刺或切開纖維環(huán)進行退變模型的制作。過程中容易損傷腹膜及周圍軟組織,給實驗動物帶來很大的創(chuàng)傷,且增加了感染的機會,術后動物死亡率高,造成實驗動物的浪費；手術過程中對腰椎周圍組織的破壞及術后瘢痕形成都對試驗結果造成影響,違背了自然退變過程的模仿。尚未有人用微創(chuàng)的方法進行兔退變椎間盤模型的制作。 2目的 建立新的微創(chuàng)兔腰椎間盤退變模型,為腰椎間盤退變機制與治療的進一步研究提供更理想的試驗對象。 3方法 本實驗在仔細研究家兔腰椎及周圍組織解剖結構的基礎上,經(jīng)過反復穿刺操作,找到了此安全微創(chuàng)的穿刺通道及方法。穿刺通路遠離腹膜及重要血管神經(jīng),且在C臂引導下進行,操作準確,一步到位,誤傷幾率小,有操作快速簡單,損傷小,感染機會小、動物存活率高、可重復性好等優(yōu)點。應用此方法對18只兔子的L3/4、L4/5、L5/6椎間盤進行造模損傷,術后將動物隨機分為A、B、C三個模型組,每組6只。分別于造模術后4、8、12周對三組進行MRI檢查,以Pfirrmann分級法作為評估標準進行分級,并前后對照。每組行MRI檢查后予以處死,取L3/4、L4/5、L5/6椎間盤髓核組織,分兩組,分別行RT-PCR檢測Ⅱ型膠原蛋白的表達及經(jīng)4%甲醛溶液在4℃下進行固定,然后脫水,石蠟包埋,組織切片,之后進行Ⅱ型膠原免疫組織化學染色及HE染色進行觀察,前后對照并且取L2/3及L6/7椎間盤髓核組織作為正常對照。 4結果 從術后4、8、12周及術前的兔腰椎MRI檢查結果可以看出,造模前后椎間盤髓核的信號強度有隨造模手術后的時間延長而逐漸降低的趨勢(L3/4、L4/5及L5/6)。由磁共振室兩名教授盲法采用Pfirrmann分級法將結果分級,采用有序分組資料的線性趨勢檢驗,計算得到P＜0.05。說明椎間盤的退變程度與造模后時間長短有顯著相關性,即造模后時間越長椎間盤退變越明顯。造模術后4周、8周、12周后取材采用RT-PCR技術檢測發(fā)現(xiàn)椎間盤Ⅱ型膠原蛋白均明顯表達,其中4周最強,8周、12周后逐漸次之。病理HE染色結果觀察可以看到,對照組正常的髓核組織中有大量的髓核細胞,隨著造模手術后時間的延長髓核細胞數(shù)量漸減少,到第12周時,髓核中只有極少量髓核細胞,卻有很多纖維軟骨組織。將對照組及術后4、8、12周組Ⅱ型膠原免疫組化染色的灰度值進行單因素方差分析結果：F=41.82(P＜0.05),組間比較采用LSD-t檢驗,結果顯示,除術后4周與8周組比較無統(tǒng)計學意義外,其余組間兩兩比較均有顯著差異(P＜0.05)。說明在造模以后,隨著時間的延長,髓核組織中Ⅱ型膠原的含量逐漸減少。 5結論 成功建立了新的微創(chuàng)兔腰椎間盤退變動物模型。
[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.

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R-332

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