甲强龙、电针华佗夹脊穴联合大鼠羊膜上皮细胞移植治疗大鼠脊髓损伤的研究
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摘要
脊髓损伤(spinal cord injury,SCI)是交通、工矿事故及运动意外中常见的中枢神经系统(central nerve system,CNS)损伤性疾病,是人类致残率最高的疾患之一,这种损伤常导致截瘫,损伤平面以下运动和感觉功能丧失,给家庭和社会造成巨大的精神压力和经济负担。自20世纪后50年至21世纪,从修复脊髓损伤的实验研究及临床观察来看,损伤脊髓修复的关键是神经纤维在脊髓内能够长距离生长,并且生长的神经纤维能穿过损伤瘢痕到达远侧端,修复脊髓上、下行神经通路,从而获得感觉和运动功能的恢复。30多年前,在唐山发生了一次罕见的大地震,造成了几十万人的伤亡,在幸存的伤员中有相当一部分是脊髓损伤患者,失去了生活自理能力。2008年5月12日在汶川,又一次发生了骇人听闻的大地震,37万余伤员中因SCI造成截瘫者占2%。目前国内、外对于脊髓损伤的治疗还是以外科手术配合激素治疗为主。据资料统计显示,脊髓损伤患者一生的治疗与康复费用平均高达75万美元以上,美国每年对SCI患者的花费超过60多亿美元。因此,寻找一种更加经济、有效的治疗方法迫在眉睫。
本研究通过中、西医相结合的方法,旨在探讨甲强龙(methylprednisolone,MP)、电针华佗夹脊穴与大鼠羊膜上皮细胞(amniotic epithelial cells,AECs)移植联合治疗对脊髓损伤大鼠功能恢复的作用。将90只成年雌性Wistar大鼠随机分成五组,每组18只。SCI对照组:采用改良的Allen s撞击法,做脊髓损伤模型,不进行治疗;甲强龙组:脊髓损伤后,用大剂量MP冲击治疗,立即静脉推注MP30mg/kg,4h后重复1次,30mg/kg,此后,静脉推注MP30mg/kg,2次/d,共3d;MP+华佗夹脊穴电针组:在MP治疗的基础上,脊髓损伤后4h,行华佗夹脊穴电针治疗。损伤平面上、下各2对取穴,左、右穴位交替使用,选用长25mm、直径0.35mm的毫针,上海产6805-II型治疗仪,设置为正、负极,疏密波,频率1～2Hz,强度0.3～1.0 mA,以针刺处肌肉轻微抖动为宜,持续15min,此后1次/d,6d为1疗程,间隔2d,进入下一个疗程,共治疗3个疗程;MP+电针+AECs移植组:在进行MP和华佗夹脊穴电针治疗的基础上,脊髓损伤后第7天,用微量注射器在SCI处中心灰质区移植AECs 5μl,浓度:1 107/μl;假手术组:只打开椎板,暴露脊髓,不造成脊髓损伤。各组术后30d行病理组织学观察和荧光红(fluorescein red,FR)逆行示踪;神经丝(neurofilament,NF)、5-羟色胺(5-hydroxytryptamine,5-HT)、降钙素基因相关肽(calcitonin gene related peptide,CGRP)和胶质原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)免疫荧光组织化学观察;神经电生理检测以及每6d行为学观察(BBB scores)。结果发现:MP+电针+AECs组脊髓损伤处GFAP的表达低于其他损伤组,可见大量的FR阳性神经纤维,通过脊髓损伤区,数量较其他损伤组明显增加,纤维走行较规则,几乎平行排列且红色荧光着色较强,并在对应的大脑皮质运动区和脊髓灰质后角见到红色FR阳性细胞;同时在SCI处可见大量Hoechst33342标记的阳性AECs(蓝色),生长良好,分布均匀。MP+电针+AECs组损伤区同时可见大量有序的NF、5-HT和CGRP阳性神经纤维,其中5-HT阳性纤维比例占假手术组5-HT阳性神经纤维的61.5%,明显高于CGRP阳性纤维占假手术组的7.6%。神经电生理检测显示MP+电针+AECs组与其他损伤组比较皮质体感诱发电位(somatosensory evoked potential,SEP)与运动诱发电位(moto evoked potential,MEP)的峰-峰值显著增加,潜伏期明显缩短,差异有统计学意义(P<0.01),其中MEP恢复程度优于SEP。综上所述,MP、电针华佗夹脊穴联合大鼠AECs移植治疗脊髓损伤能有效抑制星形胶质细胞的过度增生,恢复了神经传导通路,极大的促进了神经纤维的再生和大鼠后肢功能的恢复,且运动功能恢复优于感觉功能的恢复。此项研究创新性地应用甲强龙、电针华佗夹脊穴与羊膜上皮细胞移植联合治疗脊髓损伤,为SCI的综合治疗开辟了新思路,寻找到一种更为简便、有效的方法,同时也为临床治疗SCI提供了新的实验依据。
Spinal cord injury (SCI) is one common central nervous system(CNS) damage disease caused by traffic,movement or mining accidents,which is also the disease that renders the most paralysis,especially when it causes lots of motor and sensory dysfunctions below the SCI section , and consequently,both family and society suffer great losses and stress. Judging from experiment results and clinical observations since 1950s,the key to SCI recovery is that nerve fibers can grow in the spinal cord,and by its growth,it can penetrate the injured cicatrix,thus repairing neural pathway in the spinal cord,eventually obtaining the recovery of the motor and sensory functions. About 30 years ago , a disastrous earthquake in Tangshan,China,took away hundreds of thousands of lives,and a great many of the survivors were SCI patients,losing the ability to take care of themselves. WenChuan earthquake which occurred on May 12th,2008,also made about 2% of more than 370,000 survivors suffer from SCI. So far,the major treatment for SCI is still operation and hormone therapy. According to statistics,the average treatment fee for SCI patients is 750,000 dollars,and America spends over 6 billion on the treatment of SCI patients. Thus an economical and efficient treatment for SCI is badly needed.
Combining the treatment methods of traditional Chinese medicine and Western medicine,the present study was aimed at giving a further research on the comprehensive effect of methylprednisolone (MP),electro- acupuncture and rat amniotic epithelial cells (AECs) transplantation to recovery for SCI. In the experiment,90 adult female Wistar rats were randomly divided into five groups, with 18 in the individual group,namely-SCI control group: Improved Allen s bump equipment was employed in the present experiment,no treatment after SCI was created. MP group:MP pulse treatment after SCI. Intravenous injection MP 30mg/kg immediately after SCI,repeated the same injection 4h later,following up with two times every day,injection of MP 30mg/kg,3 days in all. MP and Huatuo spine points electro-acupuncture group: electro-acupuncture treatment on Hua Tuo jiaji acupoint 4h after SCI,based on MP group. Two paires of acupoints both above and below the SCI section,applying the left and right acupoints alternatively. The acupuncture needles were 25mm long and 0.35 mm in diameter. Therapeutic instrument (6805-II, produced in Shanghai) was set,including anode and cathode,dilatational wave, frequency(1～2Hz)and intensity(0.3～1.0 mA),kept the muscle trembled slightly still for 15min,then one time every day,with 6 days in a treatment course,after 2 days interval,the second course started,3 courses in all. MP,electro-acupuncture and AECs group:on the basis of MP and Hua Tuo jiaji acupoint electro-acupuncture , AECs 5μl (intensity=1 107/μl) were transplanted with microinjector 7d after SCI. Sham group:neural scute was opened and spinal cord was exposed without SCI. On the 30th day after the surgery,a series of tests were performed including histological testing,fluorescein red (FR) retrograde tagging , neurofilament ( NF ),5-hydroxytryptamine(5-HT),calcitonin gene related peptide(CGRP)and glial fibrillary acidic protein ( GFAP ) immunofluorescence histochemistry observation,neuro-electro-physiological tests and BBB scores (every 6d).In MP,electro-acupuncture and AECs group GFAP was discovered to have an inadequate expression compared with other SCI groups in SCI region ,a magnificent amount of FR positive nerve fibers were spotted and can be transported through SCI region,and the amount was remarkably more than other discovered in other SCI groups. The fibers were regularly arranged,almost paralleled with each other and the FR coloring appeared to be more noticeable.FR positive cells were discovered in corresponding cerebrum cortical motor area and posterior horn of gray nucleus.Meanwhile,in SCI region appeared well-grown evenly distributed transplanted AECs,labeled by Hoechst33342 (blue).In MP,electro-acupuncture and AECs group,the SCI region held numerous regularly-arranged NF,5-HT and CGRP positive nerve fibers,and the 5-HT positive nerve fibers took up 61.5% of those in sham operation group,a notable advantage over CGRP,which took up no more than 7.6%. Neuro-electrophysiological tests on both Somatosensory evoked potential (SEP) and Moto evoked potential (MEP) demonstrated that peak-to-peak value increased sharply and latency was shortened noticeably in MP ,electro-acupuncture and AECs group,and difference between this and other SCI groups is statistically significant (P<0.01). MEP recovery is better than SEP. To conclude , a combination of methylprednisolone ,electro-acupuncture Hua Tuo jiaji acupoint and AECs transplantation can restrain hyperplasy of horizontal cell and recover the pathway of nerve conduction and promote nerve fibers regeneration and hind limb function recovery for SCI rats;furthermore,motor function was prior to sensory function in the recovery. The current study bring a new idea which the combination of MP,electro-acupuncture and AECs in rat SCI treatment. It brings a new train of thought for combined therapy of SCI,and a more simple and efficient way. Meanwhile it provides a new experimental basis for clinical treatment of SCI.
本研究通过中、西医相结合的方法,旨在探讨甲强龙(methylprednisolone,MP)、电针华佗夹脊穴与大鼠羊膜上皮细胞(amniotic epithelial cells,AECs)移植联合治疗对脊髓损伤大鼠功能恢复的作用。将90只成年雌性Wistar大鼠随机分成五组,每组18只。SCI对照组:采用改良的Allen s撞击法,做脊髓损伤模型,不进行治疗;甲强龙组:脊髓损伤后,用大剂量MP冲击治疗,立即静脉推注MP30mg/kg,4h后重复1次,30mg/kg,此后,静脉推注MP30mg/kg,2次/d,共3d;MP+华佗夹脊穴电针组:在MP治疗的基础上,脊髓损伤后4h,行华佗夹脊穴电针治疗。损伤平面上、下各2对取穴,左、右穴位交替使用,选用长25mm、直径0.35mm的毫针,上海产6805-II型治疗仪,设置为正、负极,疏密波,频率1～2Hz,强度0.3～1.0 mA,以针刺处肌肉轻微抖动为宜,持续15min,此后1次/d,6d为1疗程,间隔2d,进入下一个疗程,共治疗3个疗程;MP+电针+AECs移植组:在进行MP和华佗夹脊穴电针治疗的基础上,脊髓损伤后第7天,用微量注射器在SCI处中心灰质区移植AECs 5μl,浓度:1 107/μl;假手术组:只打开椎板,暴露脊髓,不造成脊髓损伤。各组术后30d行病理组织学观察和荧光红(fluorescein red,FR)逆行示踪;神经丝(neurofilament,NF)、5-羟色胺(5-hydroxytryptamine,5-HT)、降钙素基因相关肽(calcitonin gene related peptide,CGRP)和胶质原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)免疫荧光组织化学观察;神经电生理检测以及每6d行为学观察(BBB scores)。结果发现:MP+电针+AECs组脊髓损伤处GFAP的表达低于其他损伤组,可见大量的FR阳性神经纤维,通过脊髓损伤区,数量较其他损伤组明显增加,纤维走行较规则,几乎平行排列且红色荧光着色较强,并在对应的大脑皮质运动区和脊髓灰质后角见到红色FR阳性细胞;同时在SCI处可见大量Hoechst33342标记的阳性AECs(蓝色),生长良好,分布均匀。MP+电针+AECs组损伤区同时可见大量有序的NF、5-HT和CGRP阳性神经纤维,其中5-HT阳性纤维比例占假手术组5-HT阳性神经纤维的61.5%,明显高于CGRP阳性纤维占假手术组的7.6%。神经电生理检测显示MP+电针+AECs组与其他损伤组比较皮质体感诱发电位(somatosensory evoked potential,SEP)与运动诱发电位(moto evoked potential,MEP)的峰-峰值显著增加,潜伏期明显缩短,差异有统计学意义(P<0.01),其中MEP恢复程度优于SEP。综上所述,MP、电针华佗夹脊穴联合大鼠AECs移植治疗脊髓损伤能有效抑制星形胶质细胞的过度增生,恢复了神经传导通路,极大的促进了神经纤维的再生和大鼠后肢功能的恢复,且运动功能恢复优于感觉功能的恢复。此项研究创新性地应用甲强龙、电针华佗夹脊穴与羊膜上皮细胞移植联合治疗脊髓损伤,为SCI的综合治疗开辟了新思路,寻找到一种更为简便、有效的方法,同时也为临床治疗SCI提供了新的实验依据。
Spinal cord injury (SCI) is one common central nervous system(CNS) damage disease caused by traffic,movement or mining accidents,which is also the disease that renders the most paralysis,especially when it causes lots of motor and sensory dysfunctions below the SCI section , and consequently,both family and society suffer great losses and stress. Judging from experiment results and clinical observations since 1950s,the key to SCI recovery is that nerve fibers can grow in the spinal cord,and by its growth,it can penetrate the injured cicatrix,thus repairing neural pathway in the spinal cord,eventually obtaining the recovery of the motor and sensory functions. About 30 years ago , a disastrous earthquake in Tangshan,China,took away hundreds of thousands of lives,and a great many of the survivors were SCI patients,losing the ability to take care of themselves. WenChuan earthquake which occurred on May 12th,2008,also made about 2% of more than 370,000 survivors suffer from SCI. So far,the major treatment for SCI is still operation and hormone therapy. According to statistics,the average treatment fee for SCI patients is 750,000 dollars,and America spends over 6 billion on the treatment of SCI patients. Thus an economical and efficient treatment for SCI is badly needed.
Combining the treatment methods of traditional Chinese medicine and Western medicine,the present study was aimed at giving a further research on the comprehensive effect of methylprednisolone (MP),electro- acupuncture and rat amniotic epithelial cells (AECs) transplantation to recovery for SCI. In the experiment,90 adult female Wistar rats were randomly divided into five groups, with 18 in the individual group,namely-SCI control group: Improved Allen s bump equipment was employed in the present experiment,no treatment after SCI was created. MP group:MP pulse treatment after SCI. Intravenous injection MP 30mg/kg immediately after SCI,repeated the same injection 4h later,following up with two times every day,injection of MP 30mg/kg,3 days in all. MP and Huatuo spine points electro-acupuncture group: electro-acupuncture treatment on Hua Tuo jiaji acupoint 4h after SCI,based on MP group. Two paires of acupoints both above and below the SCI section,applying the left and right acupoints alternatively. The acupuncture needles were 25mm long and 0.35 mm in diameter. Therapeutic instrument (6805-II, produced in Shanghai) was set,including anode and cathode,dilatational wave, frequency(1～2Hz)and intensity(0.3～1.0 mA),kept the muscle trembled slightly still for 15min,then one time every day,with 6 days in a treatment course,after 2 days interval,the second course started,3 courses in all. MP,electro-acupuncture and AECs group:on the basis of MP and Hua Tuo jiaji acupoint electro-acupuncture , AECs 5μl (intensity=1 107/μl) were transplanted with microinjector 7d after SCI. Sham group:neural scute was opened and spinal cord was exposed without SCI. On the 30th day after the surgery,a series of tests were performed including histological testing,fluorescein red (FR) retrograde tagging , neurofilament ( NF ),5-hydroxytryptamine(5-HT),calcitonin gene related peptide(CGRP)and glial fibrillary acidic protein ( GFAP ) immunofluorescence histochemistry observation,neuro-electro-physiological tests and BBB scores (every 6d).In MP,electro-acupuncture and AECs group GFAP was discovered to have an inadequate expression compared with other SCI groups in SCI region ,a magnificent amount of FR positive nerve fibers were spotted and can be transported through SCI region,and the amount was remarkably more than other discovered in other SCI groups. The fibers were regularly arranged,almost paralleled with each other and the FR coloring appeared to be more noticeable.FR positive cells were discovered in corresponding cerebrum cortical motor area and posterior horn of gray nucleus.Meanwhile,in SCI region appeared well-grown evenly distributed transplanted AECs,labeled by Hoechst33342 (blue).In MP,electro-acupuncture and AECs group,the SCI region held numerous regularly-arranged NF,5-HT and CGRP positive nerve fibers,and the 5-HT positive nerve fibers took up 61.5% of those in sham operation group,a notable advantage over CGRP,which took up no more than 7.6%. Neuro-electrophysiological tests on both Somatosensory evoked potential (SEP) and Moto evoked potential (MEP) demonstrated that peak-to-peak value increased sharply and latency was shortened noticeably in MP ,electro-acupuncture and AECs group,and difference between this and other SCI groups is statistically significant (P<0.01). MEP recovery is better than SEP. To conclude , a combination of methylprednisolone ,electro-acupuncture Hua Tuo jiaji acupoint and AECs transplantation can restrain hyperplasy of horizontal cell and recover the pathway of nerve conduction and promote nerve fibers regeneration and hind limb function recovery for SCI rats;furthermore,motor function was prior to sensory function in the recovery. The current study bring a new idea which the combination of MP,electro-acupuncture and AECs in rat SCI treatment. It brings a new train of thought for combined therapy of SCI,and a more simple and efficient way. Meanwhile it provides a new experimental basis for clinical treatment of SCI.
引文
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