快速免疫组化法鉴别人体周围神经感觉纤维的实验研究
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。
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.
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。
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.
引文
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7韩震,胥少汀等.桡、尺、正中神经的自然分述在临床上的应用.中华外科杂志,1986,24:23
8 Watchmaker GP,Gnmucio GA,Grandall RE,Vannier MA,Weeks PM.Fascicular to photography of the median nerve:A Comprter based studyto identify branding patterns.J Hand Surg,1991,16A:53-59
9 Hakstian RW.Funicular orientation by direct stimulation,an aid toperipheral nerve repair[J].J Bone Joint Surg,1986,50(Am):1178-1186
10 Grube H. Identification of motor and sensory funiculi in cut nerves and their selective reunion.Br J Plast Surg,1976,29:70
11何蕴韶,钟世镇.乙酰胆碱脂酶组化法在手术中鉴别神经束可行性的探讨.临床解剖学杂志,1987,5:13
12 Girsch W,Deutinger M,Bayer GS,et al.sensorimotor differentiated reconstruction of peripheral nerves-a methods of intraoperative identification of motor fascicles.Handchir Mikrochir Plast Chir,1996,28(4):181-186
13 Carson KA, Tezis JK. Carbonic anhyrace histochemistry A potential disgnostic method for peripheral nerve repair. Clin Plast Surg, 1985, 12:227-232
14周长满.鉴别神经功能束的碳酸酐酶显示法.临床应用解剖学杂志,1986,3(3):148
15 Gandl A,Farine I,et al. Intraoperative nerve fascicle identification using choline acetyltransferase:a. preliminary report. Clin Orthop, 1982,165:228
16 Urakami H and Chiu AY. A monoclonal antibody that recognizes somaticmotoneurons in themature rat nervous system.J N eurosci, 1990,10:620
17 Erkman L, M attenberger L, Kato AC. A monclonal anti-body distinguishes anterior horncells of human embryonic spinal cord during a transcient period of development. Dev Brain Res, 1992, 66: 109
19张沛云,顾晓松等.识别脊神经感觉神经元单克隆抗体的制备.神经解剖学杂志,1991,7(1):130
20顾晓松,陈昌富,严志强等.免疫组化法鉴别感觉与运动神经纤维.中国临床解剖学杂志,1992,10:121
21严志强,王友华,张沛云等.快速免疫组化法区分人周围神经束性质.中华显微外科杂志,1999,19(1),3
22刘璠,王友华,顾晓松等.分子免疫染色法鉴别人体周围神经感觉纤维的实验研究及临床应用.中华手外科杂志,1996,12:5
23蒲小平,汪家政,李长龄.Western Blot测定脊神经感觉神经元一35KD蛋白的初步研究.中国实验临床免疫学杂志,1999,11(2):2
24 Ping XP, Wang JZ. Purification of a sensory-specific protein of 35kDa(SSP-35). Biol Pharm Bull, 2000, 23: 542-544
25解建勋,蒲小平,包金风.大鼠脊神经节感觉神经特异蛋白35kD( SSP-35)的纯化及鉴定.Chinese Journal of Biochemistry and MolecularBiology,2001,17 (3):306-310
26 Quan Li, Hongzhi Guan, Xiaoping Pu.Identication of a 35 kDa protein in rat spinal ganglia and sensory fibers. Developmental Neuroscience, 2004, 15: 2083
27 Cameron L. Hoover,Lutz G.W.The COOH-terminal domain of agrin signals via a synaptic receptor in central nervous system neurons.The Journal of Cell Biology, 2003,161: 923-932
28 Ma J, Lugo B, Shah S. Synaptic localization and axonal targeting of agrin secreted by ventral spinal cord neurons in culture. J Neurobiol, 2000, 15,43(4): 338-351
29王虎,路来金等.微区喇曼光谱法区分周围神经束性质.中国临床康复,2006,10:104-106
30刘淑红,吕艺,吴海涛等.大鼠中枢和外周神经损伤后胞体分布区神经组织的红外光谱分析.军事医学科学院院刊,2004,28:441-444
31岳喜军,曹晓建等.近红外光谱和聚类分析法快速鉴别神经束性质的初步研究.南京医科大学学报(自然科学版),2006,5:313-317
2徐建光,胡韶楠.颈7神经根的组化研究及其临床意义[J].中国临床解剖学杂志,1996,14(4):243-244
3 Ping XP and Wang JZ.Purification of a sensory-specific protein of 35 kDa(SSP-35).Biol Pharm Bull,2000,23:542-544
4解建勋,蒲小平,包金风.大鼠脊神经节感觉神经特异蛋白35 kD(SSP-35)的纯化及鉴定. Chinese Journal of Biochemistry and MolecularBiology,2001,17(3):306-310
5蒲小平,汪家政,李长龄.Western Blot测定脊神经感觉神经元一种
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6 Quan Li,Hongzhi Guan and Xiaoping Pu.Identication of a 35 kDa protein in rat spinal ganglia and sensory fibers.Developmental Neuroscience,2004,15:2083
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23周长满,钟世镇.坐骨神经及其分支自然分支的分离解剖.中华骨科杂志,1987,7(1):51-55
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27常万绅,褚寅.神经束移位重建屈肘功能.中华显微外科杂志,2001,24(3):180-182
28董震,成效敏.肱二头肌和三角肌肌支在臂丛的定位及临床意义.中国临床解剖学杂志,1999,17:223-224
29王岩,朱盛修.骨间前神经旋前方肌支移位修复鱼际肌支、尺神经深支的解剖研究及临床应用.中国修复重建外科杂志,1997,11:335-337
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31 Hakstian RW.Funicular orientation by direct stimulation, an aid to peripheral nerve repair. Bone Joint surg, 1986, 50(Am): 1178-1186
32张沛云,顾晓松等.识别脊神经感觉神经元单克隆抗体的制备.神经顾晓松,陈昌富,严志强等.免疫组化法鉴别感觉与运动神经纤维.中国临床解剖学杂志,1992,10:121
34王友华等.识别人脊神经感觉特异蛋白单克隆抗体的制备及临床应用.南通医学院学报,1996,16(3):321
35严志强,王友华,张沛云等.快速免疫组化法区分人周围神经束性质.中华显微外科杂志,1999,19(1),3
36刘璠,王友华,顾晓松等.分子免疫染色法鉴别人体周围神经感觉纤维的实验研究及临床应用.中华手外科杂志,1996,12:5
37于昌玉,张咸中,胡王弘等.酶联免疫吸附试验技术用于周围神经束性质染色的实验研究。中华手外科杂志,1944,10:176
38蒲小平,汪家政,李长龄.Western Blot测定脊神经感觉神经元一35KD蛋白的初步研究.中国实验临床免疫学杂志,1999,11(2):2
1钟世镇.选择周围神经缝和方式的解剖学依据[J].临床解剖学杂志1986,4(1):3
2徐建光,胡韶楠.颈7神经根的组化研究及其临床意义.中国临床解剖学杂志,1996,14(4):243-244
3 Sunderland S. The intraneural topography of peripheral of the radial, median and rlnar nerves.Brain, 1945, 68:243
4 Sunderland S. Nerves and Nerve injuries 2ed, Churchill living stone, lodon, 1987
5 Jabaley, et al. Internal Topography of Major Nerves of the Forearm and Hand: A current View. J Hand Surg, 1980, 5A:1-18
6周长满,钟世镇等.坐骨神经及其分支自然分述的分离解剖.中华骨科杂志,1987,7:51-55
7韩震,胥少汀等.桡、尺、正中神经的自然分述在临床上的应用.中华外科杂志,1986,24:23
8 Watchmaker GP,Gnmucio GA,Grandall RE,Vannier MA,Weeks PM.Fascicular to photography of the median nerve:A Comprter based studyto identify branding patterns.J Hand Surg,1991,16A:53-59
9 Hakstian RW.Funicular orientation by direct stimulation,an aid toperipheral nerve repair[J].J Bone Joint Surg,1986,50(Am):1178-1186
10 Grube H. Identification of motor and sensory funiculi in cut nerves and their selective reunion.Br J Plast Surg,1976,29:70
11何蕴韶,钟世镇.乙酰胆碱脂酶组化法在手术中鉴别神经束可行性的探讨.临床解剖学杂志,1987,5:13
12 Girsch W,Deutinger M,Bayer GS,et al.sensorimotor differentiated reconstruction of peripheral nerves-a methods of intraoperative identification of motor fascicles.Handchir Mikrochir Plast Chir,1996,28(4):181-186
13 Carson KA, Tezis JK. Carbonic anhyrace histochemistry A potential disgnostic method for peripheral nerve repair. Clin Plast Surg, 1985, 12:227-232
14周长满.鉴别神经功能束的碳酸酐酶显示法.临床应用解剖学杂志,1986,3(3):148
15 Gandl A,Farine I,et al. Intraoperative nerve fascicle identification using choline acetyltransferase:a. preliminary report. Clin Orthop, 1982,165:228
16 Urakami H and Chiu AY. A monoclonal antibody that recognizes somaticmotoneurons in themature rat nervous system.J N eurosci, 1990,10:620
17 Erkman L, M attenberger L, Kato AC. A monclonal anti-body distinguishes anterior horncells of human embryonic spinal cord during a transcient period of development. Dev Brain Res, 1992, 66: 109
19张沛云,顾晓松等.识别脊神经感觉神经元单克隆抗体的制备.神经解剖学杂志,1991,7(1):130
20顾晓松,陈昌富,严志强等.免疫组化法鉴别感觉与运动神经纤维.中国临床解剖学杂志,1992,10:121
21严志强,王友华,张沛云等.快速免疫组化法区分人周围神经束性质.中华显微外科杂志,1999,19(1),3
22刘璠,王友华,顾晓松等.分子免疫染色法鉴别人体周围神经感觉纤维的实验研究及临床应用.中华手外科杂志,1996,12:5
23蒲小平,汪家政,李长龄.Western Blot测定脊神经感觉神经元一35KD蛋白的初步研究.中国实验临床免疫学杂志,1999,11(2):2
24 Ping XP, Wang JZ. Purification of a sensory-specific protein of 35kDa(SSP-35). Biol Pharm Bull, 2000, 23: 542-544
25解建勋,蒲小平,包金风.大鼠脊神经节感觉神经特异蛋白35kD( SSP-35)的纯化及鉴定.Chinese Journal of Biochemistry and MolecularBiology,2001,17 (3):306-310
26 Quan Li, Hongzhi Guan, Xiaoping Pu.Identication of a 35 kDa protein in rat spinal ganglia and sensory fibers. Developmental Neuroscience, 2004, 15: 2083
27 Cameron L. Hoover,Lutz G.W.The COOH-terminal domain of agrin signals via a synaptic receptor in central nervous system neurons.The Journal of Cell Biology, 2003,161: 923-932
28 Ma J, Lugo B, Shah S. Synaptic localization and axonal targeting of agrin secreted by ventral spinal cord neurons in culture. J Neurobiol, 2000, 15,43(4): 338-351
29王虎,路来金等.微区喇曼光谱法区分周围神经束性质.中国临床康复,2006,10:104-106
30刘淑红,吕艺,吴海涛等.大鼠中枢和外周神经损伤后胞体分布区神经组织的红外光谱分析.军事医学科学院院刊,2004,28:441-444
31岳喜军,曹晓建等.近红外光谱和聚类分析法快速鉴别神经束性质的初步研究.南京医科大学学报(自然科学版),2006,5:313-317