【作者】 李朝顶；

【导师】 杨惠林；

【作者基本信息】 苏州大学 ， 骨外科学， 2010， 博士

【摘要】 第一部分:兔腰椎间盘退变模型建立目的:纤维环损伤诱导椎间盘退变建立实验动物模型。方法:新西兰大白兔12只年龄2岁左右,体重2.0-3.5KG左右,雌雄不限。2只作为正常对照未行手术,其余10只进行手术制造椎间盘退变模型。手术采用10%水合氯醛3ml/kg耳缘静脉注射麻醉、备皮、碘伏消毒、铺巾。采用腰椎背侧纵切口,椎旁肌腹侧2cm,从胸廓下缘2cm至骨盆环,脊柱L1-L6左前外侧被暴露,钝性分离椎前软组织,骨盆环作为L5/6间隙的标志。用18G针头分别在L2/3、L4/5椎间盘进行穿刺,L3/4和L5/6作为自身对照。术后2、4、8、16、32周各随机选取2只兔,行脊柱MRI检查并行免疫组化及组织学观察。结果:术后第3～10周造模后的椎间盘MRI T2WI信号呈现持续减弱趋势,而内对照非手术节段髓核信号强度在观察期内无明显变化。各组椎间盘分级分值的变化显示,从术后2周开始,穿刺椎间隙与自身内对照椎间隙比较差异均有统计学意义(P < 0.05);穿刺髓核MRI T2相信号呈进行性下降,至术后16周呈快速下降。免疫组化及组织学观察发现髓核细胞的数量及Ⅱ型胶原含量较对照间盘进行性减少(P<0.01)。结论:纤维环穿刺诱导兔椎间盘退变的动物模型能再现IDD发展的客观规律而且模型制作简单、重复性好。第二部分:不同年龄组兔椎间盘细胞分布类型研究目的:分析脊索细胞和软骨样细胞细胞形态及随年龄增长时的分布情况方法:取3月龄组和3年龄组新西兰大白兔各5只,完整取出腰椎脊柱,兔腰椎椎间盘纤维环横行切开,完整取出髓核,放入petri培养皿,PPS洗液调制PH7.21%低熔点的琼脂糖覆盖,覆盖、固定组织,然后植入10%的中性福尔马林中固定,对于髓核和纤维环边缘不清的三度退变椎间盘,用6mm直径的穿刺针取出椎间盘中央部分,琼脂糖包埋的髓核。荧光抗淬灭封片剂封片,激光共聚焦显微镜下测量细胞大小以及CD44和vimentin在脊索细胞和软骨样髓核细胞内表达的平均荧光强度。结果:应用共聚焦显微镜,可以在新西兰大白兔椎间盘髓核标本中,发现两种细胞:脊索细胞和软骨细胞。两种细胞在大小上有明显的区别,在3月龄新西兰大白兔的椎间盘中,脊索细胞占主要部分。从细胞计数上分析,脊索细胞占全部的80.92%,与软骨样髓核细胞数量有着明显的差异;在3年组新西兰大白兔的椎间盘中,软骨样髓核细胞占主要部分,而脊索细胞数量较3月组明显减少,比例为32.8%,表明脊索细胞数量随年龄的增长而递减,软骨样髓核细胞的数量随年龄递增。Vimentin和CD44平均荧光强度在脊索细胞内明显高于软骨样髓核细胞,两者相比有显著统计学意义。结论:脊索细胞内这两种蛋白的表达强度显著高于软骨样髓核细胞(P<0.01)且与年龄呈负相关,年龄越小,表达越强。提示随着脊索细胞成熟和老化,与脊索细胞调控和发挥功能相关的蛋白表达量逐渐下降。其对周围环境发挥作用亦逐渐减少。第三部分:脊索细胞在兔椎间盘退变过程中的作用和转归目的:对脊索细胞在椎间盘退变过程中的作用的研究方法:新西兰大白兔28只,分3月龄和3年龄两组,每组共14只,3月龄组体重1.5±2.0kg,3年龄组体重3.0-4.5kg左右,雌雄不限。每组中有2只作为正常对照未行手术。每组其余12只进入手术制造椎间盘退变模型。术后2,4,8,16,24周行MRI影像学确认椎间盘退变后,经耳缘静脉注入空气10 mL处死实验动物,获取手术节段和正常自身对照的椎间盘髓核组织,手术节段髓核作为退变组,自身未作处理间隙作为对照组。取下的髓核组织分成两部分,一部分行免疫组织切片后进行HE染色与aggrecan,Ⅱ型胶原和PCNA染色,免疫组织化学染色观察。另一部分-70度保存,RT-PCR检测。结果:椎间盘退变模型显示脊索细胞逐渐消失,被软骨样髓核细胞和不规则的基质取代,3月组兔椎间盘退变模型,前16周脊索细胞PCNA阳性细胞表达率基本保持稳定,16周后显著减少,软骨样髓核细胞的PCNA阳性细胞表达率16周后逐渐增多。3年组脊索细胞PCNA阳性细胞表达率前8周逐渐增高,后逐渐下降;软骨样髓核细胞PCNA阳性表达细胞率前4周基本保持稳定,8周后逐渐增高。结论:实验两个年龄组在出现蛋白多糖和Ⅱ型胶原RT-PCR水平下降,MRI T2像信号出现明显降低后,术后相同时间点3月龄组组织学观测较3年组存在较多的脊索细胞,虽然该细胞在PCNA和aggrecan蛋白水平表达两组无显著差异,但髓核细胞PCNA和aggrecan蛋白水平3月组却明显高于3年组。一定程度显示髓核细胞增殖和蛋白多糖分泌的能力与脊索细胞的数目有关。更多还原

【Abstract】 Part I: rabbit lumbar intervertebral disc degeneration model buildingObjectives: To establish a rabbit model of intervertebral disc degeneration induced by puncturing the anulus fibrosus with needles of defined gaugesMethods: The L2/3 and L4/5 (L3 / 4 and L5 / 6 as self-control) intervetebral discs of New Zealand white rabbit in ordinary grade were stabbed by 16-gauge transfixion pin into a depth of 5 mm in the anterolateral annulus fibrosus magnetic resonance imaging scans of the stabbed discs and intact discs were performed preoperatively and at the2th,4th,8th.16th and 32th week af ter surgery. The histologic and immunohistochemical analyses were performed after the animals were killed and the materials were ext racted.Results: In the magnetic resonance imaging,the stabbed discs exhibited a progressive decrease of signal intensity in T2-weighted images which start at 2 week Statistically significant (P <0.05)after stabbing and last for 16 weeks.Immunohistochemical and histologic analyses revealed progressive decrease of chondrocyte-like cells and type II collagen (P<0.01).Conclusions: The stabbing approach results in a slowly progressive intervertebral disc degeneration in rabbit model.This model is available for studying the status intervertebral disc degeneration.Although the rabbits of the spine in bio-mechanics and anatomy of human existence with some differences, but the disc tissue structure similar to humans, it can be used as animal models of IDD research, and may for this kind of research a large sample of experimental data. Part II: different age groups of rabbit intervertebral disc cells in the distribution of types of researchObjectives: To evaluate notochordal cells and chondrocyte cell morphology and distribution with each stage of ages.Methods: The experimental group to take 10 New Zealand white rabbits, weighing 2±0.5kg, 2 years old, complete, remove the lumbar spine, rampant rabbit lumbar intervertebral disc annulus incision, complete remove the nucleus, into the petri dish, PPS lotion modulation PH7. 21% of low melting point agarose coverage, coverage, fixed tissue, and then implanted in 10% neutral formalin fixed, for the edge of nucleus pulposus and annulus fibrosus unclear degenerative disc three times, with 6mm diameter needle out disc central part of the agarose-embedded nucleus. Fluorescence quenching of anti-sealing tablet Fengpian laser confocal microscope observation, semi-quantitative.Results: Confocal microscopy can be in New Zealand white rabbit nucleus pulposus samples, we found two kinds of cells, notochord cells and chondrocytes in cell size between the two although there are obvious differences, but the 3-month-old when the intervertebral discs account for large-cell the main part, from the cell counts on the analysis of large cells accounted for all of the 80.92%, and the small cells have very different; in three years, when the sharp increase in the number of small cells, while the large number of cells is decreased, the proportion of 32.8% that large cell from 3 months to 3 years is to reduce the number, while a small number of cells increased faster. Vimentin immunofluorescence intensity of the average optical density of semi-quantitative analysis, and average optical density of CD44 immunofluorescence intensity of semi-quantitative analysis (P <0.01, P <0.05), there are statistically significant.Conclusions: Select notochord cells in this experiment the characteristic marker CD44 and Vimentin fluorescent mark was measured in notochord cells and cartilage-like nucleus pulposus cells, the average fluorescence intensity, results showed that: notochord cells expression intensity of these two proteins was significantly higher than medullary nucleated cells (P<0.01), and negatively correlated with age, younger, stronger expression. Tip cell maturation and aging as the notochord, and notochord cells, regulation and function-related protein expression gradually decreased. To play a role in its surrounding environment is also reduced gradually.Part III: notochord cells in the rabbit intervertebral disc degeneration in the process of the role and fateObjectives: To evaluate the roles of notochord cells in the process of disc degeneration.Methods: To take New Zealand white rabbits divided into 3-month-old and 3 years two groups of a total of 14, in March body weight 1.5±2.0kg, 3 year-old group of about 3.0-4.5kg body weight of 28. Each has two-line operation is not as normal controls. The remaining 12 rats in each group into the manufacture of disc degeneration model of operation. Lines at each time point imaging MRI confirmed disc degeneration, via ear vein injection of 10 mL of air experimental animals were sacrificed, access to surgery and the normal self-control segment of the intervertebral disc nucleus pulposus, surgical segmental degeneration of nucleus pulposus as a group, self - the absence of treatment gap for a control group. Remove the nucleus pulposus is divided into two parts, part of the line-free tissue sections stained with HE after aggrecan,Ⅱ-type collagen and PCNA staining, immunohistochemical staining. Another part of -70 degrees to save, RT-PCR detection.Results: Intervertebral disc degeneration models show that notochordal cells gradually disappear, being irregular cartilage-like matrix of nucleus pulposus cells and replaced in March of rabbit disc degeneration model, before 16 weeks of notochord cells showed PCNA expression, 16 weeks after the spinal cord cells were significantly reduced, did not express PCNA, group cartilage-like cells PCNA16 weeks after an increase in cartilage-like nucleus pulposus cells, showing positive staining; March group notochord cells PCNA-positive cells gradually increased the rate of eight weeks before 16 weeks, decreased gradually. Cartilage-like nucleus pulposus cells, PCNA-positive cells 4 weeks before the rate remained stable, increased gradually after 8 weeks. Nucleus pulposus cells, PCNA and aggrecan protein levels in March group was significantly higher than 3-year group, notochord cells in the first 2 weeks of basic stability, the latter dropped significantly. Cartilage-like nucleus pulposus cells in the first 8 weeks of basic stability, the latter slightly increased.Conclusions: The experimental two age groups in the event of proteoglycan and collagen typeⅡRT-PCR reduction in the level, MRI T2 signal and decreased significantly as the following time points after the same group in March compared with the histological observation of the 3-year group there are more notochord cells, Although the cellular protein level of PCNA and expression of aggrecan was no significant difference between the two groups, but the nucleus pulposus cells, PCNA and aggrecan protein levels in March group was significantly higher than 3-year group. Nucleus pulposus cells show a certain degree of proliferation and proteoglycan secretion capacity and the number of cells in the spinal cord.更多还原