胶质源性神经营养因子对大鼠不完全脊髓损伤的保护作用及其机理的研究
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摘要
近二十年来，大量的研究工作证实损伤的中枢神经具有可塑性，也能再生；而
且在神经营养因子（NTFs）作用下再生速度及质量都有显著增强，故神经营养因子
对神经元细胞的支持及挽救损伤神经元存活作用已成为神经科学研究的“热点”之
一。GDNF是近年(1993）发现并已克隆其基因的一种神经营养因子，属于转化生
长因子β超家族成员，是目前特异性最强的多巴胺能神经元营养因子，对离休、在
体交感、感觉神经元的存活起支持作用。它在中枢神经、周围神经及其它运动系统
组织广泛表达，尤以胚胎期及损伤后周围神经较高表达，提示对组织发育和神经损
伤修复起营养作用。近来发现GDNF对离体运动神经元亦有挽救其死亡作用，但对
损伤脊髓神经元的作用尚未见报道。
SCI后NTFs及其受体表达增加，表现自我保护作用。虽然SCI后外周靶组织
和损伤神经元能产生有限和微量NTFs，但由于轴突受损，神经元失去与靶组织的
联系，NTFs就不能再经轴突转运到神经元胞体；加之大量神经元相互竞争NTFs，
这就使不能得到足够NTFs的神经元发生程序性死亡，为外源性GDNF治疗SCI提
供了理论依据。
本研究主要方法：
1．建立了大鼠AJlen脊髓不完全损伤模型
2．经蛛网膜下腔分别给予GDNF、SAL,在不同时间作（1）脊髓功能评价：Rivlin
斜板观察大鼠爬坡角度：（2）脊髓电生理检查：包括 MEP和 SEP；（3）脊髓形
态学检查：CHE、ACP组织化学染色，以阳性颗粒的平均灰度（MG）及等效
圆直径(ED）作为指标观察神经元存活情况。
3．分别于损伤后不同时间取伤段脊髓，用半定量RT-PCR方法测定GDNFmRNA
在伤后不同时期的表达。
4．观察脊髓损伤后脊髓含水量、细胞内游离钙含量变化及局部应用GDNF对该变
化趋势的影响。
本研究主要结果如下：
l
第二 学学业殷文
l一成赠立了大鼠脊腿伤模型。
2．首次证明大鼠脊髓损伤后脊髓组织高表达GDNFmRNA，表达高峰在伤后72
，J’时。
3．以 ACP和 CHE组织化学染色，利用脊髓染色阳性颗粒 MG和 ED作为参照，
发现GDNF能有效缓解ACP活性增加及CHE活性减弱，证明GDNF对大鼠
脊髓损伤运动神经元具有保护作用，并且其作用高峰在伤后第10天。
4．ivlin斜极实验证实，GDW能明显增加大鼠爬坡角度，在伤后第10及ZI天
分别比SAL组增加，说明GDNF能有效改善后肢肌力，提高脊髓功能。
5．电生理结果表明，脊髓损伤后SEP及MEP波峰降低、潜伏期延长，GDNF能
缓解这一变化。
6．脊髓损伤后脊髓含水量增加、细胞内钙含量升高，GDNF治疗后脊髓含水量及
细胞内钙水平降低，但仍未达到正常水平。
本研究结果提示：
1．Alien g击模型动物通过后肢功能变化，能够反映脊髓损伤程度；
2．GDNF从表达极微（正常）至高表达，说明脊髓损伤后需大量GDNF支持，为
外源性GDNF应用治疗脊髓损伤提供了有力的理论基础，亦验证了Wrathall关
于内源性神经营养因子分泌，自发挽救神经退化、死亡这一论断，再次证实NTFs
是治疗SCI的有效成分。
3．GDNF对损伤大鼠脊髓神经元有保护作用，能够有效改善后肢的运动功能；
4．口DN’F改善脊髓功能的机制，可能与其减轻神经细胞＊广 内流有关。
ln near two decades, it has been proved that central neurons have the ability of
plasticity and regenerating, which could be enhanced by applying neurotrophics. lt
has been becoming a "hot dot" for neurotrohpics to maintain the development of
neurons and rescue the injuried neurons. Glial cell line-derived neurotrophic factor
(GDNF) is a neurotrophic polypeptide, distantly related to transforming growth
factorbeta (TGF-beta), originally isolated by virtue of its ability to induce dopamine
uptake and cell survival in cultures of embryonic ventral midbrain dopaminergic
neurons, and more recently shown to be a potent neurotrophic factor for motorneurons.
But the bioIogical activities and the expression of GDNF at injuried spinal
motorneuron in vivo presently unknown. So those was observed in this study through
a modefied Allen's spinal injury model.
The main resuIts of our research are as follows:
l. The RT-PCR showed that the expression of GDNFmRNA at spinal cord segment
elevated notablely at 24, 72 and l68 hours after SCl in contrast to normal, and the
expression peak at 72 hours postoperatively.
2. The result of Tarlov grading and Rivlin's incline plate for testing spinal function
indicated that GDNF could improve markedly motor function of hind limbs.
3. Motor evoked potential(MEP) and somatosensory evoked potential(SEP) showed
the incubation was prolonged and the amplitude was reduced at 3, 7 and l4 days
after SCI. The spinal cord signal of the MEP was more sensitive to injury than the
SEP,but the SEP retUrn earlier after injury than the MEP lt should accelerate the
recovery of the shape and incubation of the evoked potential for GPNF to be used
locally through subarachnoid.
4. EnZyme histochemistry was used to show CHE and ACP activity of spinal
3
motoneurons in spinal segnents combined With mp ~ ~ni-by Vithe
of mean grey(MG) and effective diameter(ED). The activity of CHE which was
weaken as SCI could be obviously increased at 7 and l0 days after SCI. The
activity Qf ACP could be cut dQwn for the use of GDM which had been highly
improved by reason of SCI. Exogenous GDwe protects notorneurons from SCI.
5. The cycosolic Ca2+ in the spinal cord segment increased at 24 hours and peaked at
72 hours after SCI. GDNF could slacken this tendency and there was a positive
correlation between them. Those suggest it should be a possible mechanic for
W to proteCt motomeurons from secondary SCI and improve spinal fiJnction.
且在神经营养因子（NTFs）作用下再生速度及质量都有显著增强，故神经营养因子
对神经元细胞的支持及挽救损伤神经元存活作用已成为神经科学研究的“热点”之
一。GDNF是近年(1993）发现并已克隆其基因的一种神经营养因子，属于转化生
长因子β超家族成员，是目前特异性最强的多巴胺能神经元营养因子，对离休、在
体交感、感觉神经元的存活起支持作用。它在中枢神经、周围神经及其它运动系统
组织广泛表达，尤以胚胎期及损伤后周围神经较高表达，提示对组织发育和神经损
伤修复起营养作用。近来发现GDNF对离体运动神经元亦有挽救其死亡作用，但对
损伤脊髓神经元的作用尚未见报道。
SCI后NTFs及其受体表达增加，表现自我保护作用。虽然SCI后外周靶组织
和损伤神经元能产生有限和微量NTFs，但由于轴突受损，神经元失去与靶组织的
联系，NTFs就不能再经轴突转运到神经元胞体；加之大量神经元相互竞争NTFs，
这就使不能得到足够NTFs的神经元发生程序性死亡，为外源性GDNF治疗SCI提
供了理论依据。
本研究主要方法：
1．建立了大鼠AJlen脊髓不完全损伤模型
2．经蛛网膜下腔分别给予GDNF、SAL,在不同时间作（1）脊髓功能评价：Rivlin
斜板观察大鼠爬坡角度：（2）脊髓电生理检查：包括 MEP和 SEP；（3）脊髓形
态学检查：CHE、ACP组织化学染色，以阳性颗粒的平均灰度（MG）及等效
圆直径(ED）作为指标观察神经元存活情况。
3．分别于损伤后不同时间取伤段脊髓，用半定量RT-PCR方法测定GDNFmRNA
在伤后不同时期的表达。
4．观察脊髓损伤后脊髓含水量、细胞内游离钙含量变化及局部应用GDNF对该变
化趋势的影响。
本研究主要结果如下：
l
第二 学学业殷文
l一成赠立了大鼠脊腿伤模型。
2．首次证明大鼠脊髓损伤后脊髓组织高表达GDNFmRNA，表达高峰在伤后72
，J’时。
3．以 ACP和 CHE组织化学染色，利用脊髓染色阳性颗粒 MG和 ED作为参照，
发现GDNF能有效缓解ACP活性增加及CHE活性减弱，证明GDNF对大鼠
脊髓损伤运动神经元具有保护作用，并且其作用高峰在伤后第10天。
4．ivlin斜极实验证实，GDW能明显增加大鼠爬坡角度，在伤后第10及ZI天
分别比SAL组增加，说明GDNF能有效改善后肢肌力，提高脊髓功能。
5．电生理结果表明，脊髓损伤后SEP及MEP波峰降低、潜伏期延长，GDNF能
缓解这一变化。
6．脊髓损伤后脊髓含水量增加、细胞内钙含量升高，GDNF治疗后脊髓含水量及
细胞内钙水平降低，但仍未达到正常水平。
本研究结果提示：
1．Alien g击模型动物通过后肢功能变化，能够反映脊髓损伤程度；
2．GDNF从表达极微（正常）至高表达，说明脊髓损伤后需大量GDNF支持，为
外源性GDNF应用治疗脊髓损伤提供了有力的理论基础，亦验证了Wrathall关
于内源性神经营养因子分泌，自发挽救神经退化、死亡这一论断，再次证实NTFs
是治疗SCI的有效成分。
3．GDNF对损伤大鼠脊髓神经元有保护作用，能够有效改善后肢的运动功能；
4．口DN’F改善脊髓功能的机制，可能与其减轻神经细胞＊广 内流有关。
ln near two decades, it has been proved that central neurons have the ability of
plasticity and regenerating, which could be enhanced by applying neurotrophics. lt
has been becoming a "hot dot" for neurotrohpics to maintain the development of
neurons and rescue the injuried neurons. Glial cell line-derived neurotrophic factor
(GDNF) is a neurotrophic polypeptide, distantly related to transforming growth
factorbeta (TGF-beta), originally isolated by virtue of its ability to induce dopamine
uptake and cell survival in cultures of embryonic ventral midbrain dopaminergic
neurons, and more recently shown to be a potent neurotrophic factor for motorneurons.
But the bioIogical activities and the expression of GDNF at injuried spinal
motorneuron in vivo presently unknown. So those was observed in this study through
a modefied Allen's spinal injury model.
The main resuIts of our research are as follows:
l. The RT-PCR showed that the expression of GDNFmRNA at spinal cord segment
elevated notablely at 24, 72 and l68 hours after SCl in contrast to normal, and the
expression peak at 72 hours postoperatively.
2. The result of Tarlov grading and Rivlin's incline plate for testing spinal function
indicated that GDNF could improve markedly motor function of hind limbs.
3. Motor evoked potential(MEP) and somatosensory evoked potential(SEP) showed
the incubation was prolonged and the amplitude was reduced at 3, 7 and l4 days
after SCI. The spinal cord signal of the MEP was more sensitive to injury than the
SEP,but the SEP retUrn earlier after injury than the MEP lt should accelerate the
recovery of the shape and incubation of the evoked potential for GPNF to be used
locally through subarachnoid.
4. EnZyme histochemistry was used to show CHE and ACP activity of spinal
3
motoneurons in spinal segnents combined With mp ~ ~ni-by Vithe
of mean grey(MG) and effective diameter(ED). The activity of CHE which was
weaken as SCI could be obviously increased at 7 and l0 days after SCI. The
activity Qf ACP could be cut dQwn for the use of GDM which had been highly
improved by reason of SCI. Exogenous GDwe protects notorneurons from SCI.
5. The cycosolic Ca2+ in the spinal cord segment increased at 24 hours and peaked at
72 hours after SCI. GDNF could slacken this tendency and there was a positive
correlation between them. Those suggest it should be a possible mechanic for
W to proteCt motomeurons from secondary SCI and improve spinal fiJnction.
引文
[1] 陈哲宇，陈秀青，路长林，等．人重组GDNF及其生物活性研究．生物化学与生物物理学报 1999，31(2)：207～10
[2] Nadeau KC, Azuma H, Tilney NL. Sequential cytokine dynamics in chronic rejection of rat renal allografts: Roles for cytokines RANTES and MCP-1. Proc Natl Acad Sci USA, 1995;92:8729-8733
[3] 鞠细文，曹雪涛．应用逆转录PCR技术检测细胞因子基因表达．国外医学免疫学分册 1995，4：182～6
[4] Choi Lundberg DL, Bohn MC. Ontogeny and distribution of glial cell line-derived neurotrophic factor(GDNF) mRNA in rat. Brain-Res-Dev-Brain-Res. 1995; 85(1): 80-8
[5] Schiltz CA, Benjamin J, Epstein ML. Expression of the GDNF receptors ret and GFRalphal in the developing avian enteric nervous system. J Comp Neurol 1999; 414(2): 193-211
[6] Springer JE, Mu Ⅹ, Bergmann LW, et al. Expression of GDNF mRNA in rat and human nervous tissue. Exp-Neurol. 1994; 127(2): 167-70
[7] Schaar DG, Sieber BA, Dreyfas CF, et al. Regional and cell-specific expression of GDNF in rat brain. Exp Neurol, 1993; 124(2):368-71
[8] Pochon NA, Menoud A, Tseng JL, et al. Neuronal GDNF expression in the adult rat nervous system identified by in situ hybridization. Eur-J-Neurosci. 1997; 9(3): 463-71
[9] Glazner GW, Mu Ⅹ, Springer JE. Localization of glial cell line-derived neurotrophic factor receptor alpha and c-ret mRNA in rat central nervous system. J Comp Neurol. 1998; 391(1): 42-9
[10] Bergman E, Kullberg S, Ming Y, et al. Upregulation of GFRalpha-1 and c-ret in primary sensory neurons and spinal motoneurons of aged rats. J Neurosci Res 1999; 57(2): 153-65
[11] Bar KJ, Saldanha GJ, Kennedy AJ, et al. GDNF and its receptor component Ret in injured human nerves and dorsal root ganglia. Neuroreport. 1998; 9(1): 43-7
[12] Nosrat CA, Tomac A, Lindqvist E, et al. Cellular expression of GDNF mRNA suggests multiple functions inside and outside the nervous system. Cell Tissue Res.
1996; 286(2) : 191-207
[13] Susan L, Arvind M, Phillip G, et al. Analysis of TGF-bbb , gene expression in contused rats spinal cord using quantitative RT-PCR. J Neurotrauma. 1995; 12(6) : 1003-14
[14] Bregman BS . Recovery from spinal cord injury mediated by antibodies to neurite growth inhibitors.Nature, 1995;378:498-502
[15] Lin HL,Doherty DH,Lile DJ, et al. GDNF: A glial cell-line-derived neurotrophic factor for midbrain dopaminergic neurons. Science, 1993;260: 1130-32
[16] Beck KD, Valverde J, Alexi T,et al. Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the adult brain .Nature, 1995; 373(6512) : 339-41
[17] Gash DM,Zhang ZM,Ovadia A,et al.Functional recovery in parkinsonian monkeys treated with GDNF.Nature,1996;380:252-56
[18] Henderson CE,Phillips HS,Pollock RA ,et al.GDNF:A protent survial factor for motoneurons present in the peripheral nerve and muscle. Science, 1994;266: 1062-64
[19] 马雪梅,黄秉仁,蔡良婉。胶质细胞衍生的神经营养因子与神经退行性疾病。国外医学分子生物学分册。18 (4) : 157-60
[20] Houenou LJ, Oppenheim RW, Li L,et al. Regulation of spinal motoneuron survival by GDNF during development and following injury. Cell Tissue Res 1996; 286(2) : 219-23
[21] Gouin A, Bloch Gallego E, Tanaka-H,et al. Transforming growth factor-beta 3, glial cell line-derived neurotrophic factor, and fibroblast growth factor-2, act in different manners to promote motoneuron survival in vitro. J Neurosci Res 1996 ; 43(4) : 454-64
[22] Sagot Y, Tan SA, Hammang JP,et al. GDNF slows loss of motoneurons but not axonal degeneration or premature death of pmn/pmn mice. J Neurosci 1996;16(7) : 2335-41
[2] Nadeau KC, Azuma H, Tilney NL. Sequential cytokine dynamics in chronic rejection of rat renal allografts: Roles for cytokines RANTES and MCP-1. Proc Natl Acad Sci USA, 1995;92:8729-8733
[3] 鞠细文，曹雪涛．应用逆转录PCR技术检测细胞因子基因表达．国外医学免疫学分册 1995，4：182～6
[4] Choi Lundberg DL, Bohn MC. Ontogeny and distribution of glial cell line-derived neurotrophic factor(GDNF) mRNA in rat. Brain-Res-Dev-Brain-Res. 1995; 85(1): 80-8
[5] Schiltz CA, Benjamin J, Epstein ML. Expression of the GDNF receptors ret and GFRalphal in the developing avian enteric nervous system. J Comp Neurol 1999; 414(2): 193-211
[6] Springer JE, Mu Ⅹ, Bergmann LW, et al. Expression of GDNF mRNA in rat and human nervous tissue. Exp-Neurol. 1994; 127(2): 167-70
[7] Schaar DG, Sieber BA, Dreyfas CF, et al. Regional and cell-specific expression of GDNF in rat brain. Exp Neurol, 1993; 124(2):368-71
[8] Pochon NA, Menoud A, Tseng JL, et al. Neuronal GDNF expression in the adult rat nervous system identified by in situ hybridization. Eur-J-Neurosci. 1997; 9(3): 463-71
[9] Glazner GW, Mu Ⅹ, Springer JE. Localization of glial cell line-derived neurotrophic factor receptor alpha and c-ret mRNA in rat central nervous system. J Comp Neurol. 1998; 391(1): 42-9
[10] Bergman E, Kullberg S, Ming Y, et al. Upregulation of GFRalpha-1 and c-ret in primary sensory neurons and spinal motoneurons of aged rats. J Neurosci Res 1999; 57(2): 153-65
[11] Bar KJ, Saldanha GJ, Kennedy AJ, et al. GDNF and its receptor component Ret in injured human nerves and dorsal root ganglia. Neuroreport. 1998; 9(1): 43-7
[12] Nosrat CA, Tomac A, Lindqvist E, et al. Cellular expression of GDNF mRNA suggests multiple functions inside and outside the nervous system. Cell Tissue Res.
1996; 286(2) : 191-207
[13] Susan L, Arvind M, Phillip G, et al. Analysis of TGF-bbb , gene expression in contused rats spinal cord using quantitative RT-PCR. J Neurotrauma. 1995; 12(6) : 1003-14
[14] Bregman BS . Recovery from spinal cord injury mediated by antibodies to neurite growth inhibitors.Nature, 1995;378:498-502
[15] Lin HL,Doherty DH,Lile DJ, et al. GDNF: A glial cell-line-derived neurotrophic factor for midbrain dopaminergic neurons. Science, 1993;260: 1130-32
[16] Beck KD, Valverde J, Alexi T,et al. Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the adult brain .Nature, 1995; 373(6512) : 339-41
[17] Gash DM,Zhang ZM,Ovadia A,et al.Functional recovery in parkinsonian monkeys treated with GDNF.Nature,1996;380:252-56
[18] Henderson CE,Phillips HS,Pollock RA ,et al.GDNF:A protent survial factor for motoneurons present in the peripheral nerve and muscle. Science, 1994;266: 1062-64
[19] 马雪梅,黄秉仁,蔡良婉。胶质细胞衍生的神经营养因子与神经退行性疾病。国外医学分子生物学分册。18 (4) : 157-60
[20] Houenou LJ, Oppenheim RW, Li L,et al. Regulation of spinal motoneuron survival by GDNF during development and following injury. Cell Tissue Res 1996; 286(2) : 219-23
[21] Gouin A, Bloch Gallego E, Tanaka-H,et al. Transforming growth factor-beta 3, glial cell line-derived neurotrophic factor, and fibroblast growth factor-2, act in different manners to promote motoneuron survival in vitro. J Neurosci Res 1996 ; 43(4) : 454-64
[22] Sagot Y, Tan SA, Hammang JP,et al. GDNF slows loss of motoneurons but not axonal degeneration or premature death of pmn/pmn mice. J Neurosci 1996;16(7) : 2335-41