【作者】 张向涛；

【导师】 张哲敏；

【作者基本信息】 河北医科大学 ， 外科学， 2010， 硕士

【摘要】 目的:周围神经中含有感觉神经纤维和运动神经纤维两种成分,神经的功能是由这两种神经纤维的比例所决定的。在神经干中神经纤维并不是单独行走的,它们彼此组合,被神经束膜分隔成相对独立的神经束,对神经干内神经束分布规律及性质的判断,可以指导临床进行手术方法的选择。目前区别神经束的各种方法,因为各自的不足与缺陷难以应用于临床。自1970年以来,人们相继发现了多种神经元特异性蛋白,在免疫组织化学等方法的帮助下,人们对神经元的产生及生长过程有了更多的了解。免疫组织化学技术(immunohistochemistry),是应用免疫学基本原理—抗原抗体反应,即抗原与抗体特异性结合的原理,通过化学反应使标记抗体的显色剂(荧光素、酶、金属离子、同位素)显色来确定组织细胞内的抗原(多肽和蛋白质),对其进行定位、定性及定量的研究,又称为免疫细胞化学技术(immunocytochemistry)。免疫组织化学技术具有特异性强,快速等优点,但是应用此技术鉴别周围神经的关键是如何确定感觉神经与运动神经特有的标志物。膜联蛋白(Annexin V)是一种钙离子依赖的磷脂结合蛋白,分子量约为35KD,它具有抗凝、抗炎、钙离子通道等作用。据目前研究Annexin V主要分布在周围神经中的感觉神经轴突,在运动神经中尚未有发现的相关报道。本实验通过免疫组织化学技术证实膜联蛋白V在周围神经中特性,并应用这种特性鉴别人周围神经感觉束与运动束,希望达到证实临床应用快速免疫组化法鉴别人周围神经功能束具有可行性目的,从而构建科学实验与临床应用之间的桥梁。方法:实验1选正常健康雄性SD大鼠30只,体重250±10g,取大鼠脊神经前根、后根各5mm,暴露并取坐骨神经及其肌支、皮支各5mm,将上述组织立即做冰冻切片,片厚5um,行SABC免疫组化法,神经组织冰冻切片经抗原-抗体反应-抗体标记-DAB显色-苏木精复染,脱片、透明、封片处理后,显微镜下观察颜色。对照组采用替代试验,即用PBS代替一抗,其余步骤同实验组。实验2取新鲜废弃断肢前臂尺神经主干,尺神经腕背支(皮支)及正中神经大鱼际肌支各5mm,上述组织立即做冰冻切片,片厚5um,行S-P免疫组化法,神经组织切片经抗原-抗体反应-抗体标记-DAB显色-苏木精复染,脱片、透明、封片处理后,显微镜下观察颜色。对照组采用替代试验,即用PBS代替一抗,其余步骤同实验组。结果:实验1 SD大鼠脊神经根中,前根呈阴性反应,后根为阳性反应,肌支以未染的神经纤维为主,皮支以深染的神经纤维为主,其中坐骨神经内既有深染的神经纤维也有未染的神经纤维。对照组的神经组织均呈阴性反应。实验2正中神经大鱼际肌支以未染的神经纤维为主,尺神经腕背支以深染的神经纤维为主,尺神经主干内既有深染的神经纤维也有未染的神经纤维,对照组的神经组织均呈阴性反应。结论:在远端神经干内,神经的各分支在干内有明确的定位,即有特定的功能束存在,而在肢体近端,运动神经束和感觉神经束混合存在。在混合神经中,各类纤维所占的比例亦有差异。如果某一类纤维所占比例较大,在再生轴索长入远端神经内膜管的过程中,将有更多的机会长入相关的神经内膜管,因而周围神经断裂损伤后,在进行神经原位缝合或神经移位、移植修复术时,要求术者了解神经干内的精细结构,特别是神经干内神经功能束的定位知识,这样才有可能达到精确的选择性神经束支对位缝合,减少再生纤维迷走所致的无效再生,提高术后肢体功能的恢复率。因此临床需要一种快速、准确鉴别周围感觉神经束和运动神经束的方法,本实验通过膜联蛋白V在周围神经组织中的特性,并将此种方法应用于人体周围神经,验证了此法在人体周围神经鉴别方面的可行性,为周围神经束功能鉴别提供了新的手段,也为此种方法早日应用于临床搭建了桥梁。更多还原

【Abstract】 Objective: It is well known that the peripheral nerves is composed of motor and sensory fibers, neural function is by these two kinds of nerve bundles of proportion of the decision. Nerve fibers and not exist alone. They each combination and nerve bundle membrane separates nerve fibers composition bundle of nerves. Neural stem function beam distribution and nature of the judge to guide the choice of surgical methods of clinical. The current distinction between nerve bundles, because there are a lot of their own deficiencies and shortcomings in the clinical practice it is difficult. Since 1970, people found many neurons specific protein in immunohistochemical method under the help of the people, the development process of neurons have further understanding. Immunohistochemical techniques (immunohistochemistry) is the basic principle, application of immunology antigen-antibody reaction antigen and antibody specificity, namely, through the theory of chemical reaction to mark the antibody colour-display reagent (fluorescent element, enzyme, metal ions, isotopes) color to determine the organization intracellular antigens (peptide and protein), carries on the orientation, qualitative and quantitative research and immune cells called chemical technology (immunocytochemistry). Although immunohistochemical technology has advantages, but the key is how to determine the sensory nerve and nerve characteristic marker, through the literature review, this experiment target in the membrane protein V, hoped through al immunohistochemical techniques to reveal membrane proteins in peripheral nerve V al the characteristics, so as to achieve the objective identification nerve bundle nature. Membrane protein (al Annexin V) is a kind of phospholipids on calcium ions, molecular binding proteins for 35KD, it has many functions such as anticoagulation, anti-inflammatory, calcium ion channels, in peripheral nerve in Annexin V mainly distributed in sensory nerve of axons, not found in the motor. This experiment by means of fast yow-chyi method to confirm the specificity of membrane protein V al, and using the characteristics of this application from the fault of peripheral nerve, hope to achieve rapid identify clinical guidelines of peripheral nerve function, thus establishing the scientific experiment and clinical application of Bridges.Methods: Experiment 1.Choose normal healthy male SD rats, weight only 250±10g, Cut out rat spinal nerve root 5mm, before after spinal nerve root 5mm, exposure and siatic nerve and muscle and skin, 5mm, lumbav immediately above freezing crosscut slice organization, thick slices 5um ,Starting SABC immunohistochemical experiment, Nerve biopsy via the antigen-antibody reaction - DAB color - hematoxylin-eosin stain, pure complex, transparent, wafer processing, microscope color, By using the substitution test group, an antioxidant, the substitute PBS experimental procedure. Experiment 2 Taking fresh abandoned landmine forearm ulnar nerve trunk, ulnar wrist back teams (skin) and the median nerve big teams each 5mm. Yu muscle,the organization immediately frozen section, thick slices 5um, Starting S-P immunohistochemical experiment, nerve biopsy via the antigen-antibody reaction - DAB color - hematoxylin-eosin stain, pure complex, transparent, wafer processing, microscope color, By using the substitution test group, an antioxidant, the substitute PBS experimental procedure.Results: Experiment 1 SD rat spinal nerve root, with a negative response, before the heel for positive reaction to dye, muscle teams of nerve fibers, skin with hyperchromatic teams, including positive nerve fibers sciatic nerve fibers in both positive and negative has the nerve fiber dyed, at high magnification observation, the nerve fiber immune dyeing of axons, myelin not colouring. The glial tissue is a negative response.Experiment 2 The median nerve, with thenar of nerve fibers, its feet nerve with hyperchromatic wrist back a nerve fiber, the positive one nerve fibers in both positive and negative has the nerve fiber dyed, ulnar nerve trunk, The glial tissue is a negative response . Conclusion: We know that in the distal nerve dry, dry branches in the nerve in positioning, which has specific functions in the body, and girded proximal, each bundle of nerves, no single function of mixed bundle. In the mix of fiber bundle of nerves, the proportion of differences. If a large proportion of fiber in axonal regeneration, into the distal nerve endometrial tube process, will have more opportunities of endometriosis neural tube into the related peripheral nerve injury, thus breaking in situ, nerve or shift, the nerve repair when transplanted neural stem in understanding performer fine structure, especially within the neural function of the nerve stem of positioning knowledge, so that it was possible to precise selective nerve bundle branch, reducing regeneration fibers showed the vagus caused by void regeneration, improve the recovery rate of postoperative limb function. Therefore the clinical needs a rapid, accurate identification of sensory nerve bundle around and nerve bundle method, this experiment through membrane proteins in peripheral nerve V al organizational characteristics, broke the immune method identified no exact molecular properties bundle of nerves, and the bottleneck of the method is applied to human peripheral nerve, validated in human peripheral nerve identification method of peripheral nerve bundle for feasibility, provides a new means to identify function, this kind of method applies early clinical built bridge.更多还原