肝细胞生长因子对面神经运动终板损伤修复作用的实验研究
摘要
目的:近来国外研究发现肝细胞生长因子（HGF）具有神经营养样作用。而面神经损伤无论在平时还是战时均是多见的疾病，损伤后，尤其是离断后，成功的显微外科技术对修复虽然重要，但不能解决神经错向生长等问题，需行生物修复，且面神经的功能恢复最终要靠运动终板的修复，故本研究以新西兰家兔面神经硅胶生物再生室为模型，研究肝细胞生长因子（HGF）对面神经损伤后运动终板及肌纤维修复作用的影响,为临床寻找一种方便、有效的神经营养因子，创造一个良好的神经再生微环境提供初步的实验依据。
方法：实验设生理盐水对照组和肝细胞生长因子组，选用24只成年、健康新西兰家兔，暴露并分离双侧面神经颊支，将其横断后套入硅胶管内，用显微外科技术将硅胶管与神经外膜缝合以形成硅胶生物再生室，左侧再生室内注入HGF为实验侧，右侧注入生理盐水为对照
侧，于术后6周、12周分批取面神经颊支所支配的肌组织，行乙酰胆碱酯酶染色和HE染色以显示运动终板及肌纤维，进行组织形态学观察和形态定量分析。
结果：术后6周实验侧终板形态比对照侧规则，着色更深，具有显著性差异（P＜0.01），肌纤维横截面积无显著性差异（P＞0.05）。术后12周实验侧终板形态接近正常，对照侧终板结构不成熟，染色深浅具有显著性差异（P＜0.01），实验侧肌纤维比对照侧粗，其横截面积具有显著性差异（P＜0.01）。
结论：本研究初步表明在面神经硅胶生物再生室内外源性追加HGF对其运动终板及肌纤维的修复有促进作用，但具体的作用机制有待于进一步的研究。
Objective: Hepatocyte growth factor (HGF) exhibited properties on different types of neurons, including motor, sensory and parasympathetic neurons. Facial nerve (FN) injury was a common disease in practice medicine, successful microsurgery technic and microenviroment were equally essential for peripheral nerve regeneration .But there exited some problem and repairs of injured facial nerve needed biologic repair . Function repair of injured facial nerve finally depended on repair of motor end-plate. So our study was to establish the FN transected model, in which the buccal division of FN of adult New Zealand rabbit was transected and a nerve growth chamber created to elucidate the role of HGF in the regeneration of motor end-plate and myofiber of injured facial nerve.
Methods: The buccal branches of facial nerve were exposed and transected, a nerve growth chamber were created by suturing the epineurium and silicone chamber under microscope .The chambers of experimental sides(left sides of facial nerve) were injected with HGF/normal saline and that of the control sides with normal saline of
same amount (10μL ) .The acetylcholinesterase (AchE) in the motor end-plate and the myofiber of injured facial nerve were analysed by using histochemical staining and image analysis techique ofter 6 and 12 weeks of operation.
Results: Histochemical analysis of MEP AchE staining showed the color of MEP on HGF side was deeper than that on control side,and there was significan difference of the color of MEP between HGF and control sides after 6 and 12 weeks of operation (P<0.01).The structure of MEP on HGF side looked near normal ,while on control side it was still unmature after 12 weeks of operation.The area of the myofiber cross section on HGF side was bigger than that on control side, and there was significan difference of the area of myofiber cross section between HGF and control sides after 12 weeks of operation (P<0.01).But there was no significan difference of the area after 6 weeks of operation (P＞0.05).
Conclusion: HGF within a silicone chamber enhanced the recover of MEP and myofiber in New Zealand rabbits . But recovery of metabolism of HGF needed more experiment.
方法：实验设生理盐水对照组和肝细胞生长因子组，选用24只成年、健康新西兰家兔，暴露并分离双侧面神经颊支，将其横断后套入硅胶管内，用显微外科技术将硅胶管与神经外膜缝合以形成硅胶生物再生室，左侧再生室内注入HGF为实验侧，右侧注入生理盐水为对照
侧，于术后6周、12周分批取面神经颊支所支配的肌组织，行乙酰胆碱酯酶染色和HE染色以显示运动终板及肌纤维，进行组织形态学观察和形态定量分析。
结果：术后6周实验侧终板形态比对照侧规则，着色更深，具有显著性差异（P＜0.01），肌纤维横截面积无显著性差异（P＞0.05）。术后12周实验侧终板形态接近正常，对照侧终板结构不成熟，染色深浅具有显著性差异（P＜0.01），实验侧肌纤维比对照侧粗，其横截面积具有显著性差异（P＜0.01）。
结论：本研究初步表明在面神经硅胶生物再生室内外源性追加HGF对其运动终板及肌纤维的修复有促进作用，但具体的作用机制有待于进一步的研究。
Objective: Hepatocyte growth factor (HGF) exhibited properties on different types of neurons, including motor, sensory and parasympathetic neurons. Facial nerve (FN) injury was a common disease in practice medicine, successful microsurgery technic and microenviroment were equally essential for peripheral nerve regeneration .But there exited some problem and repairs of injured facial nerve needed biologic repair . Function repair of injured facial nerve finally depended on repair of motor end-plate. So our study was to establish the FN transected model, in which the buccal division of FN of adult New Zealand rabbit was transected and a nerve growth chamber created to elucidate the role of HGF in the regeneration of motor end-plate and myofiber of injured facial nerve.
Methods: The buccal branches of facial nerve were exposed and transected, a nerve growth chamber were created by suturing the epineurium and silicone chamber under microscope .The chambers of experimental sides(left sides of facial nerve) were injected with HGF/normal saline and that of the control sides with normal saline of
same amount (10μL ) .The acetylcholinesterase (AchE) in the motor end-plate and the myofiber of injured facial nerve were analysed by using histochemical staining and image analysis techique ofter 6 and 12 weeks of operation.
Results: Histochemical analysis of MEP AchE staining showed the color of MEP on HGF side was deeper than that on control side,and there was significan difference of the color of MEP between HGF and control sides after 6 and 12 weeks of operation (P<0.01).The structure of MEP on HGF side looked near normal ,while on control side it was still unmature after 12 weeks of operation.The area of the myofiber cross section on HGF side was bigger than that on control side, and there was significan difference of the area of myofiber cross section between HGF and control sides after 12 weeks of operation (P<0.01).But there was no significan difference of the area after 6 weeks of operation (P＞0.05).
Conclusion: HGF within a silicone chamber enhanced the recover of MEP and myofiber in New Zealand rabbits . But recovery of metabolism of HGF needed more experiment.
引文
Edgerton MT,et al.Surgicalcorretion of facial pancent[J].Amer,J surg,1970;120:82.
骆文龙,林代诚等.面神经外膜与束膜缝合法的实验研究[J].1994;25(1):112-126.
骆文龙，林代诚，李永懋.面神经修复中再生室内髓鞘碱性蛋白的作用初探[J].中华耳鼻咽喉科杂志,2001,36(1):14-17.
Echim CL,Katyal S,Wiley CA et al.Expression of HGF and cMet in the developing and adult brain［J］. Brain-Res-Dev-Brain-Res, 1997, 102(2): 299～303.
Yang XM, Toma JG, Bamji SX et al. Autocrine hepatocyte growth factor provides a local mechanism for promoting axonal growth [J]. J Neurosci,1998, 18(20):8369-8381.
Maina F, Hilton MC, Andres R et al. Multiple roles for hepatocyte growth factor in sympathetic neuron development [J]. Neuron, 1998 , 20(5):835-846.
Ma JZ,Luo YX,Wu XM,Ding H.The study on NGF effect on degenerated and reinnervated motor end-plate histochemical and ultrastructural changes [J].Chin J Microsury,1998,21(5):118-120.
马宏敏,黄维国,王锦玲等. 豚鼠面神经损伤后运动终板及面神经核乙酰胆碱脂酶含量的变化[J]. 第四军医科大学学报,1996,17(4): 248-250.
Misulis KE,Deltham WD.Is fast fiber innervation responsible for increased acetylcholinesterase activity in reinnervating soleus muscles [J]?Exp Neurol,1985,89:204-215.
徐建广,顾玉东,李继峰.大鼠失神经支配骨骼肌退变的实验研究[J]. 上海医科大学学报,1999,26(4):265-267.
Davey F, Hilton M, Davies AM. Cooperation between HGF and CNTF in promoting the survival and growth of sensory and parasympathetic neurons [J].
Mol Cell Neurosci , 2000,15(1):79-88.
Yang XM, Toma JG, Bamji SX et al. Autocrine hepatocyte growth factor provides a local mechanism for promoting axonal growth [J]. J Neurosci,1998, 18(20):8369-8381.
Michalopoulos GK, Zarnegar R.Hepatocyte growth factor.[J]. Hepatology, 1992, 15: 149-155.
Hashimoto N, Yamanaka H, Fukuoka T et al. Expression of HGF and cMet in the peripheral nervous system of adult rats following sciatic nerve injury[J].Neuroreport ,2001 May 25,12(7):1403-1407
Okura Y, Arimoto H, Tanuma N et al.Miyazawa hypoglossal nerve axotomy model[J]. Eur J Neurosci, 1999 Nov,11(11):4139-4144.
Spector JG, Lee P, Derby A et al.Early stages of facial nerve regeneration though silicone chambers in the rabbit[J].Laryngoscope, 1991; 101(10):1109-23.
魏泓主编.实验动物学.四川科技出版社.1998,3.
张哲，陈辉主编.实用病理组织染色技术.辽宁科学技术出版社.1988,6.
骆文龙,林代诚等.家兔面神经钳压伤病理组织学研究[J].华西医学;1992;23(12):371-373.
Seckel BR. Enhancement of peripheral nerve regeneration[J]. Muscle Nerve ,1990,13:785.
Perry VH, et al. Role of macrophage in peripheral nerve degeneration and repair.Bioessays[J].1992,14:401-406
骆文龙,林代诚等.面神经外膜与束膜缝合法的实验研究[J].1994;25(1):112-126.
Millesi H. Progress in peripheral nerve reconstruction[J].World J Surg, 1990, 14:733
Lundborg G, Zhao Q, Kanje M,et al.Can sensory and motor collateral sprouting be induced from intact peripheral nerve by end to side anatomosis[J]? J Hand Surg,1994,19B:277
Barde YA, Trophic factors and neuronal survival[J]. Neuron, 1989;2:1525-34.
Lundborg G. Nerve regeneration and repair[J].Acta Orthop Scand,1987,58:145
Doi K.New reconstructive procedure for brachial plexus injury[J].Clin Plast Surg,1997,24:75-85
Spector JG, Lee P, Derby A et al. Rabbit facial nerve regeneration in NGFcontaining silastic tubes[J]. Laryngoscope,1993;103(6):548-558.
Chen YS, Wang-Bennet LT,Coker NJ.Facial nerve regeneration in the silicone chamber:the influence of nerve growth factor[J].Exp Neurol,1989,103(1):52-60.
Krull CE, Koblar SA. Motor axon pathfinding in the peripheral nervous system[J].Brain Res Bull , 2000, 15,53(5):479-487.
Funakoshi H,Nakamura T.Identification of HGF-like protein as a novel neurotrophic factor for avian dorsal root ganglion sensory neurons[J].Biochem Biophys Res Commun 2001;283(3):606-12.
Hashimoto N, Yamanaka H, Fukuoka T et al. Expression of HGF and cMet in the peripheral nervous system of adult rats following sciatic nerve injury[J].Neuroreport ,2001 May 25,12(7):1403-1407.
Okura Y, Arimoto H, Tanuma N et al.Miyazawa hypoglossal nerve axotomy model[J]. Eur J Neurosci, 1999 Nov,11(11):4139-4144.
周翠英,骆文龙.肝细胞生长因子对面神经损伤修复作用的实验初探[J].重庆医学,2003,32(4):456-459.
石阳,任重.豚鼠面神经失神经支配后口轮匝肌琥珀酸脱氢酶、乙酰胆碱酯酶酶活性观察[J].辽宁医学杂志,2001,15(1):15-16.
Okura Y, Arimoto H, Tanuma N et al.Miyazawa hypoglossal nerve axotomy model[J]. Eur J Neurosci, 1999 Nov,11(11):4139-4144.
Yan Q, Elliott J, Snider WD.BDNF rescues spinal motor neurons from axotomy-induced cell death[J]. Nature,1993, 360:753.
Chen YS, Yao CH, Chen TH.Effect of acupuncture stimulation on peripheral
nerve regeneration using silicone rubber[J]. Am J Chin Med.2001; 29(3): 377-385.
马金忠,罗永湘,吴晓明等.神经生长因子对运动终板变性与再生的研究[J].中华显微外科杂志,1998,21(5):118-120.
Yoshimoto Y, Lin Q, Collier TJ, et al. Astrocytes retrovirally transduced with BDNF elicts behavioral improvement in a rat model of Parkinsons` disease[J]. Brain Res, 1995 ,691 :25.
骆文龙,林代诚等.面神经外膜与束膜缝合法的实验研究[J].1994;25(1):112-126.
骆文龙，林代诚，李永懋.面神经修复中再生室内髓鞘碱性蛋白的作用初探[J].中华耳鼻咽喉科杂志,2001,36(1):14-17.
Echim CL,Katyal S,Wiley CA et al.Expression of HGF and cMet in the developing and adult brain［J］. Brain-Res-Dev-Brain-Res, 1997, 102(2): 299～303.
Yang XM, Toma JG, Bamji SX et al. Autocrine hepatocyte growth factor provides a local mechanism for promoting axonal growth [J]. J Neurosci,1998, 18(20):8369-8381.
Maina F, Hilton MC, Andres R et al. Multiple roles for hepatocyte growth factor in sympathetic neuron development [J]. Neuron, 1998 , 20(5):835-846.
Ma JZ,Luo YX,Wu XM,Ding H.The study on NGF effect on degenerated and reinnervated motor end-plate histochemical and ultrastructural changes [J].Chin J Microsury,1998,21(5):118-120.
马宏敏,黄维国,王锦玲等. 豚鼠面神经损伤后运动终板及面神经核乙酰胆碱脂酶含量的变化[J]. 第四军医科大学学报,1996,17(4): 248-250.
Misulis KE,Deltham WD.Is fast fiber innervation responsible for increased acetylcholinesterase activity in reinnervating soleus muscles [J]?Exp Neurol,1985,89:204-215.
徐建广,顾玉东,李继峰.大鼠失神经支配骨骼肌退变的实验研究[J]. 上海医科大学学报,1999,26(4):265-267.
Davey F, Hilton M, Davies AM. Cooperation between HGF and CNTF in promoting the survival and growth of sensory and parasympathetic neurons [J].
Mol Cell Neurosci , 2000,15(1):79-88.
Yang XM, Toma JG, Bamji SX et al. Autocrine hepatocyte growth factor provides a local mechanism for promoting axonal growth [J]. J Neurosci,1998, 18(20):8369-8381.
Michalopoulos GK, Zarnegar R.Hepatocyte growth factor.[J]. Hepatology, 1992, 15: 149-155.
Hashimoto N, Yamanaka H, Fukuoka T et al. Expression of HGF and cMet in the peripheral nervous system of adult rats following sciatic nerve injury[J].Neuroreport ,2001 May 25,12(7):1403-1407
Okura Y, Arimoto H, Tanuma N et al.Miyazawa hypoglossal nerve axotomy model[J]. Eur J Neurosci, 1999 Nov,11(11):4139-4144.
Spector JG, Lee P, Derby A et al.Early stages of facial nerve regeneration though silicone chambers in the rabbit[J].Laryngoscope, 1991; 101(10):1109-23.
魏泓主编.实验动物学.四川科技出版社.1998,3.
张哲，陈辉主编.实用病理组织染色技术.辽宁科学技术出版社.1988,6.
骆文龙,林代诚等.家兔面神经钳压伤病理组织学研究[J].华西医学;1992;23(12):371-373.
Seckel BR. Enhancement of peripheral nerve regeneration[J]. Muscle Nerve ,1990,13:785.
Perry VH, et al. Role of macrophage in peripheral nerve degeneration and repair.Bioessays[J].1992,14:401-406
骆文龙,林代诚等.面神经外膜与束膜缝合法的实验研究[J].1994;25(1):112-126.
Millesi H. Progress in peripheral nerve reconstruction[J].World J Surg, 1990, 14:733
Lundborg G, Zhao Q, Kanje M,et al.Can sensory and motor collateral sprouting be induced from intact peripheral nerve by end to side anatomosis[J]? J Hand Surg,1994,19B:277
Barde YA, Trophic factors and neuronal survival[J]. Neuron, 1989;2:1525-34.
Lundborg G. Nerve regeneration and repair[J].Acta Orthop Scand,1987,58:145
Doi K.New reconstructive procedure for brachial plexus injury[J].Clin Plast Surg,1997,24:75-85
Spector JG, Lee P, Derby A et al. Rabbit facial nerve regeneration in NGFcontaining silastic tubes[J]. Laryngoscope,1993;103(6):548-558.
Chen YS, Wang-Bennet LT,Coker NJ.Facial nerve regeneration in the silicone chamber:the influence of nerve growth factor[J].Exp Neurol,1989,103(1):52-60.
Krull CE, Koblar SA. Motor axon pathfinding in the peripheral nervous system[J].Brain Res Bull , 2000, 15,53(5):479-487.
Funakoshi H,Nakamura T.Identification of HGF-like protein as a novel neurotrophic factor for avian dorsal root ganglion sensory neurons[J].Biochem Biophys Res Commun 2001;283(3):606-12.
Hashimoto N, Yamanaka H, Fukuoka T et al. Expression of HGF and cMet in the peripheral nervous system of adult rats following sciatic nerve injury[J].Neuroreport ,2001 May 25,12(7):1403-1407.
Okura Y, Arimoto H, Tanuma N et al.Miyazawa hypoglossal nerve axotomy model[J]. Eur J Neurosci, 1999 Nov,11(11):4139-4144.
周翠英,骆文龙.肝细胞生长因子对面神经损伤修复作用的实验初探[J].重庆医学,2003,32(4):456-459.
石阳,任重.豚鼠面神经失神经支配后口轮匝肌琥珀酸脱氢酶、乙酰胆碱酯酶酶活性观察[J].辽宁医学杂志,2001,15(1):15-16.
Okura Y, Arimoto H, Tanuma N et al.Miyazawa hypoglossal nerve axotomy model[J]. Eur J Neurosci, 1999 Nov,11(11):4139-4144.
Yan Q, Elliott J, Snider WD.BDNF rescues spinal motor neurons from axotomy-induced cell death[J]. Nature,1993, 360:753.
Chen YS, Yao CH, Chen TH.Effect of acupuncture stimulation on peripheral
nerve regeneration using silicone rubber[J]. Am J Chin Med.2001; 29(3): 377-385.
马金忠,罗永湘,吴晓明等.神经生长因子对运动终板变性与再生的研究[J].中华显微外科杂志,1998,21(5):118-120.
Yoshimoto Y, Lin Q, Collier TJ, et al. Astrocytes retrovirally transduced with BDNF elicts behavioral improvement in a rat model of Parkinsons` disease[J]. Brain Res, 1995 ,691 :25.